18) Molecular Biology-Sb

SB
CHAPTER 18
MOLECULAR BIOLOGY
1) DNA REPLICATION
2) TELOMERE
3) INHIBITORS OF DNA REPLICATION
4) CELL CYCLE
5) DNA DAMAGE
6) REPAIR OF DNA DAMAGE
7) DISORDERS OF DNA DAMAGE
8) TRANSCRIPTION
9) POST TRANSCRIPTIONAL MODIFICATIONS
10) INHIBITORS OF TRANSCRIPTION
11) REVERSE TRANSCRIPTION
12) GENETIC CODE
13) TRANSLATION
14) INHIBITORS OF TRANSLATION
15) POST TRANSLATIONAL MODIFICATIONS
16) MUTATION
17) LAC OPERON
18) REGULATION OF GENE EXPRESSION IN EUKARYOTES
19) RECOMBINANT DNA TECHNOLOGY
20) BLOTTING
21) RFLP
22) VNTR
23) DNA FOOTPRINTING
24) CHROMOSOME WALKING
25) DNA LIBRARIES
26) PCR
27) CLONING
28) TRANSGENESIS
29) HUMAN GENOME PROJECT
30) CYTOGENETIC TECHNIQUES
31) CRISPR
32) OLD AGE GENETICS
33) GENE THERAPY
34) STEM CELLS
35) THREE PARENT IVF
36) BIOINFORMATICS AND GENOMIC RESOURCES

GENETICS
DNA REPLICATION
INTRODUCTION
1. The biological information flows from DNA to RNA and

from there to proteins
2. This is the central dogma of life
3. It is ultimately the DNA that controls every function of the

cell through protein synthesis
4. The word genome refers to the total genetic information

contained in the DNA, in each cell of the organism
5. DNA is the genetic material
6. When the cell divides, the daughter cells receive an identical

copy of genetic information from the parent cell
7. Replication is a process in which DNA copies itself to produce

daughter molecules of DNA
8. Replication is carried out with high fidelity

REPLICATION IS SEMI-CONSERVATIVE
1. The parent DNA has two DNA strands complementary to

each other
2. Both the strands undergo simultaneous replication to produce

two daughter molecules
3. Each one of the newly synthesized DNA has one half of the
parental DNA (one strand from the original) and one half of
new DNA
4. This type of replication is known as semi-conservative since

half of the original DNA is conserved in the daughter DNA
SEMI CONSERVATIVE REPLICATION
STEPS OF DNA REPLICATION IN PROKARYOTES
DNA replication is divided into the following phases:
1. Replication bubble
2. Leading and lagging strands
3. Replication fork
4. Okazaki fragments
5. Removal of RNA primer and nick sealing
REPLICATION BUBBLE
1. The initiation of DNA synthesis occurs at a site called origin

of replication
2. In case of prokaryotes, there is a single site, whereas in

eukaryotes, there are multiple sites of origin
3. These sites mostly consist of short sequences of A-T base pairs
4. A specific protein called DNA – A (20 to 50 monomers) binds

with the site of origin for replication and this causes the DNA
strands to separate
5. The two complementary strands of DNA separate at the site of

replication to form a bubble
6. Multiple replication bubbles are formed in eukaryotic DNA

molecules, which is essential for rapid replication
FORMATION OF REPLICATION BUBBLE
LEADING AND LAGGING STRANDS
1. For the synthesis of a new DNA, a short fragment of RNA

(about 5 to 50 nucleotides) is required as a primer
2. This primer is synthesized on the DNA template by a specific

RNA polymerase called primase
3. A constant synthesis and supply of RNA primers should occur

on the lagging strand of DNA
4. The leading strand has a single RNA primer
5. The replication of DNA occurs in the 5’ to 3’ direction,

simultaneously, on both the strands of DNA
6. On one strand, the leading strand, the synthesis of DNA is

continuous
7. On the other strand, the lagging strand, the synthesis of DNA

is discontinuous
8. Short pieces of DNA (15 to 20 nucleotides) are produced on

the lagging strand
LEADING AND LAGGING STRANDS
REPLICATION FORK
1. The separation of the two strands of parent DNA results in

the formation of replication fork
2. Active synthesis of DNA occurs in this region
3. The replication fork moves along the parent DNA as the

daughter DNA molecules are synthesized
4. DNA helicases are enzymes which bind to both the DNA

strands at the replication forks
5. Helicases move along the DNA helix and separate the DNA
strands
6. Single strand binding (SSB) proteins protect the single

stranded DNA from degradation by nucleases and also
keep the DNA strands separate
7. The synthesis of a new DNA strand, catalysed by DNA

polymerase III, occurs in the 5’ to 3’ direction
8. The presence of all four deoxyribonucleoside triphosphates

(dATP, dGTP, dCTP and dTTP) is required for replication
to take place
9. The incoming deoxyribonucleotides are added one after

another, to the 3’ end of the growing DNA chain
10. A molecule of pyrophosphate (PPi) is removed with the

addition of each nucleotide
DNA REPLICATION
3’
RNA PRIMER
5’ DNA POLYMERASE III

LEADING
STRAND
REPLICATION FORK
NEWLY 5’
SYNTHESIZED
DNA
3’
DNA
HELICASE
LAGGING
STRAND
3’ SSB
5’
OKAZAKI
FRAGMENTS
OKAZAKI FRAGMENTS
1. The template DNA strand (the parent) determines the base

sequence of the newly synthesized complementary DNA
2. The synthesis of two new DNA strands, takes place

simultaneously, in opposite directions:
a) One is in a direction towards the replication fork, which

is continuous
b) The other is in a direction, away from the replication fork,

which is discontinuous
3. The small fragments of discontinuously synthesized DNA

attached to RNA primers are called Okazaki fragments
4. These are produced on the lagging strand of the DNA
5. Okazaki fragments are later joined to form a continuous

strand of DNA
OKAZAKI FRAGMENT
REMOVAL OF RNA PRIMER AND NICK SEALING
1. DNA polymerase III has proof reading activity
2. It checks the incoming nucleotides and allows only the

correctly matched bases (complementary bases) to be
added to the growing DNA strand
3. DNA polymerase edits its mistakes and removes the

wrongly placed nucleotide bases
4. The synthesis of new DNA strand continues till it is in

close proximity to RNA primer
5. Now DNA polymerase I removes the RNA primer and

takes its position
6. DNA polymerase I catalyses the synthesis of a fragment

of DNA that replaces the RNA primer
7. The enzyme DNA ligase catalyses the formation of a

phosphodiester linkage between the DNA synthesized by
DNA polymerase III and the small fragments of DNA
produced by DNA polymerase I
8. This process, known as nick sealing, requires energy,

which is provided by ATP
9. As the DNA double helix separates from one side and

replication proceeds, supercoils are formed at the other side
10. The problem of supercoils is solved by a group of enzymes

known as topoisomerases
11. DNA topoisomerase cuts the single DNA strand and then
re-seals the strand
ACTION OF DNA POLYMERASE I AND DNA LIGASE
3’ 5’
NEWLY
SYNTHESIZED
DNA
5’ 3’
DNA TEMPLATE
DNA
EXCISED POLYMERASE I
RNA PRIMER
3’ 5’
5’ 3’
NICK SEALED
BY DNA LIGASE
3’ 5’ DAUGHTER DNA
5’ 3’ PARENT DNA
REPLICATION OF EUKARYOTIC DNA
Replication of DNA in eukaryotes closely resembles that of

prokaryotes.
The differences are:
1. There are multiple sites of origin of replication in eukaryotes
2. Five distinct DNA polymerases are known in eukaryotes
3. DNA polymerase alpha is responsible for the synthesis of

RNA primer
4. DNA polymerase beta is involved in the repair of DNA
5. DNA polymerase gamma participates in the replication of

mitochondrial DNA
6. DNA polymerase delta is responsible for the replication on

the leading strand of DNA and it also has proof reading ability
7. DNA polymerase epsilon is involved in DNA synthesis on the

lagging strand and proof reading function
TELOMERE
1. Replication always takes place from the 5’ to 3’ direction in the

new strand
2. A small portion (about 300 nucleotides) at the 3’ end of the

parent strand cannot not be replicated by DNA polymerase
3. This end piece of the chromosome is called telomere
4. Unless there is some mechanism to replicate telomeres, the

length of the chromosomes will go on reducing at each cell
division
5. The stability of the chromosome is thus lost
6. Shortening of the telomere end is prevented by an enzyme

telomerase
7. Telomerase contains an RNA component which provides the

template for telomeric repeat synthesis
8. Telomerase acts like reverse transcriptase
9. Telomerase recognizes the 3’ end of telomere and then a

small DNA strand is synthesized
10. In old age telomerase activity is lost; leading to chromosome

instability and cell death
11. As cancer cells have increased and persistent activity of

telomerase, the cancer cells become immortal
TELOMERE
INHIBITORS OF DNA REPLICTION – CNN DEA-56
1. Ciprofloxacin, Novobiocin and Nalidixic acid:
a) Bacteria contain a specific type-II topoisomerase, namely

gyrase
b) This enzyme cuts and reseals the circular DNA (of bacteria)
and thus overcomes the problem of supercoils
c) Bacterial gyrase is inhibited by the antibiotics ciprofloxacin,

novobiocin and nalidixic acid
d) These are widely used as antibacterial agents since they can

effectively block the replication of DNA and multiplication of
cells
e) These antibacterial agents have almost no effect on human

enzymes
2. Doxorubicin, Etoposide and Adriamycin:
Compounds that inhibit human topoisomerases are used as

anti-cancer agents e.g. doxorubicin, etoposide & adriamycin
3. 5 fluorouracil and 6 mercaptopurine:
The nucleotide analogs that inhibit DNA replication are used

as anti-cancer drugs e.g.
a) 5 fluorouracil - inhibits thymidylate synthase
b) 6 mercaptopurine - inhibits human DNA polymerase

CELL CYCLE
1. The cell cycle is a 4-stage process consisting of Gap-1 (G1),

Synthesis (S), Gap-2 (G2) and Mitosis
2. An active eukaryotic cell will undergo these steps as it grows

and divides
3. After completing the cell cycle, the cell either starts the process
again from G1 or exits through G0
PHASES OF THE CELL CYCLE
1. G1 Phase:
a) Cell increases in size
b) Cellular contents duplicated
2. S Phase:
a) DNA replication
b) Each of the 46 chromosomes is replicated by the cell
3. G2 Phase:
Cell prepares for cell division
4. M Phase:
a) Mitosis followed by cytokinesis (cell division)
b) Formation of two identical daughter cells

PHASES OF CELL CYCLE
REGULATION
1. The progression of the cell cycle is controlled by checkpoints

at different stages
2. These detect if a cell contains damaged DNA & ensure those

cells do not replicate
3. The restriction point (R) is located at G1 & is a key checkpoint
4. The vast majority of cells that pass through the R point will
end up completing the entire cycle
5. Other checkpoints are located at the transitions between G1

and S, and G2 and M
6. p53:
a) If damaged DNA is detected at any checkpoint, activation

of the checkpoint results in increased protein p53 production
b) p53 is a tumour suppressor gene that stops the progression

of thecell cycle and starts repair mechanisms for the damaged
DNA
c) If this DNA cannot be repaired, then it ensures the cell

undergoes apoptosis and can no longer replicate
CLINICAL RELEVANCE – NEOPLASIA
1. Neoplasia is a disease of unchecked cell division and its

progression, attributed to a change in activity of cell cycle
regulators
2. If a mutation occurs in a protein that regulates the cell cycle

it can lead to rapid multiplication of cancerous cells
3. When there is a defect in p53 tumour suppressor gene, it

cannot detect and bind to cells with damaged DNA to either
repair or cause apoptosis
4. This leads to unchecked replication of cells in the cell cycle

and increases the risk of neoplasm
QUESTIONS
1. Why is DNA replication called semi conservative?
2. Describe in detail DNA replication
3. Diagrammatic representation of DNA replication
4. Replication bubble
5. Replication fork
6. Okazaki pieces
7. Leading and lagging strands
8. Inhibitors of DNA replication
9. Telomere
10. Cell cycle
DNA DAMAGE
1. The DNA is constantly subjected to environmental insults and

replication errors
2. This leads to an alteration or removal of nucleotide bases

causing damage to DNA
3. There are four different types of damages to DNA:
a) Single base alteration: deamination of adenine and

hypoxanthine
b) Two base alteration: UV light induced formation of thymine

dimers
c) Chain breaks: due to ionization
d) Cross links: formed between bases of the same or opposite

strand
4. If the damage caused to DNA is not repaired, it may result in

mutation or even cell death
5. A mutation in DNA causes loss of control of cell proliferation

and leads to cancer
REPAIR OF DNA
1. Damage to DNA either by replication errors or environmental

insults can be deleterious
2. To counter this, cells developed different repair systems that

detect and rectify the DNA damage
3. Four major repair systems are involved in rectifying the DNA

damage:
a) Mismatch repair
b) Base excision repair
c) Nucleotide excision repair
d) Double strand break repair

MISMATCH REPAIR
1. A mismatch is detected in newly synthesized DNA
2. The new DNA strand is cut, and the mispaired nucleotide

and its neighbours are removed
3. The missing patch is replaced with correct nucleotides by

DNA polymerase
4. DNA ligase seals the gap in the DNA backbone
5. A defect in mismatch repair causes hereditary non-polyposis

colorectal cancer, gastric cancer, oesophageal cancer and
squamous cell cancer of head
BASE EXCISION REPAIR
1. Deamination converts a cytosine base into uracil
2. The uracil is detected and removed, leaving a baseless

nucleotide
3. The baseless nucleotide is removed, leaving a small gap in

the DNA backbone
4. The gap is filled with the right base by DNA polymerase and
sealed by DNA ligase
5. A defect in base excision repair may cause primary gastric

cancer, non-small cell lung cancer and colorectal cancer
NUCLEOTIDE EXCISION REPAIR
1. Exposure to ultra violet light may produce a thymine dimer
2. Once the dimer has been detected, the surrounding DNA is

opened to form a bubble
3. The enzyme UV specific endonuclease cuts the damaged region

out of the bubble
4. DNA polymerase replaces the excised DNA and it is sealed by

DNA ligase
5. Defect in nucleotide excision repair causes xeroderma

pigmentosum
DOUBLE STRAND BREAK REPAIR
1. Double strand breaks are common during immunoglobulin

gene rearrangement and are repaired immediately
2. Double stranded breaks may also be caused by free radicals,
radiation and chemotherapeutic agents
3. Double stranded breaks can be repaired by:
a) Homologous recombination:
In this the broken chromosome pairs up with its homologue.
The damaged region is replaced via recombination, using

sequences copied from the homologue.
b) Non-homologous end joining:
In this the chromosome is glued back together, usually with

a small mutation at the break site
4. A defect in double strand break repair causes ataxia

telangiectasia
HOMOLOGOUS RECOMBINATION
NON-HOMOLOGOUS END JOINING
DISORDERS OF DNA DAMAGE
Disorders of DNA damage are:
1. Xeroderma pigmentosum
2. Ataxia telangiectasia
3. Bloom syndrome
4. Fanconi anemia
5. Cockayne syndrome
6. Lynch syndrome
XERODERMA PIGMENTOSUM
INTRODUCTION AND CAUSE
1. XP is a rare autosomal recessive genetic disease, due to

defect in the repair of DNA damage, caused by UV rays
2. It is caused due to absence of the enzyme UV specific

endonuclease (nucleotide excision repair)
3. It is caused due to mutation in the UV specific endonuclease

(ERCC5) gene located on chromosome 13
4. XP is six times more common in Japanese people than in

other groups
5. In extreme cases all exposure to sunlight must be forbidden

and individuals with this disease are referred to as “moon
children”
CLINICAL FEATURES
The disease passes through three stages:
FIRST STAGE
1. The skin is healthy at birth

2. At the age of 6 months there is diffuse erythema, scaling
and freckle like areas of increased pigmentation
3. These changes are seen in light exposed areas of the skin
SECOND STAGE
1. It is characterized by poikiloderma - skin atrophy,

telangiectasia (spider veins), xeroderma (dry skin) and
mottled hyperpigmentation and hypopigmentation
2. Limited growth of hair on chest and legs
THIRD STAGE
1. It is characterized by the appearance of numerous

malignancies, including squamous cell carcinoma,
malignant melanoma, basal cell carcinoma and
fibrosarcoma
2. These malignancies may occur as early as 5 years of
age and are more prevalent in sun exposed areas
3. Ocular features are photophobia, conjunctivitis, corneal

ulcers and ectropion
DIAGNOSIS
1. A child presenting with severe sunburn after his first

exposure to sun may be a clue to the diagnosis of XP
2. XP can usually be conclusively diagnosed by measuring

the enzyme UV specific endonuclease from skin or blood
samples
3. Histologic findings: hyperkeratosis and increased melanin

pigment in the basal cell layer
4. DNA analysis
5. Prenatal diagnosis by amniocentesis or chorionic villus

sampling
TREATMENT
1. The most obvious and important part of treatment is

avoiding exposure to sunlight
2. This includes wearing protective clothing and using

sunscreens
3. Application of imiquimod cream in combination with
oral acitretin (retinoid)
4. Topical application of T4 endonuclease V
5. Keratosis can be treated using cryotherapy or fluorouracil
6. Excision of malignancies
7. Gene therapy
PROGNOSIS
1. Fewer than 40% of individuals with the disease survive

beyond the age 20
2. The most important causes of mortality are metastatic

malignant melanoma and squamous cell carcinoma
3. Individuals with milder disease may survive beyond middle

age
ATAXIA TELANGIECTASIA
1. Ataxia telangiectasia (AT) also referred to as Louis-Bar

syndrome is a rare, degenerative, autosomal recessive
disease causing severe disability
2. Ataxia refers to poor coordination and telangiectasia to
small dilated blood vessels, both of which are hallmarks
of the disease
3. AT is caused by mutation in the ATM gene located on

chromosome 11
4. ATM gene is responsible for managing the cell’s response

to double strand breaks in DNA
5. The protein produced by the ATM gene recognizes that there

is a break in DNA and recruit’s other proteins to fix the break
CLINICAL FEATURES
1. Growth retardation
2. Delayed onset or incomplete pubertal development and

very early menopause
3. Ataxia: difficulty with control of movement
4. Oculomotor ataxia: difficulty with coordination of head and

eye movement when shifting gaze from one place to the next
5. Dystonia: sustained & repetitive muscle contractions, resulting

in twisting and repetitive movements
6. Dysarthria: slurred, slow or distorted speech sounds
7. Drooling particularly in young children when they are tired
8. Seborrheic dermatitis, common warts and hirsutism
9. Telangiectasia (dilated blood vessels) over the sclera of the

eyes, making them appear bloodshot
10. Infections of the ears, sinuses and lungs
11. Increased incidence of cancer (lymphoma and leukemia)
12. Diabetes in adolescence or later
13. The wasted face, scattered grey hair and stooped posture
give the children an appearance of premature ageing
DIAGNOSIS
1. Increased chromosomal breakage after exposure of cell

cultures to ionizing radiation
2. DNA analysis
3. Elevated level of serum alpha feto protein
4. Low levels of immunoglobulins (especially IgA, IgG and

IgE) and low number of lymphocytes in the blood
5. CT scan and MRI
TREATMENT
1. There is no treatment known to stop the progression of the

neurologic problems
2. Chronic lung infections should be treated with appropriate

antibiotics
3. Feeding tubes can decrease the risk of aspiration in AT
patients
4. Physical therapy is useful to maintain strength and

cardiovascular health
PROGNOSIS
The life expectancy of people with AT is 25 years
BLOOM SYNDROME
1. Bloom syndrome is a rare, autosomal, recessive disorder,

caused due to mutation in the BLM gene located on
chromosome 15
2. BLM gene codes for the enzyme helicase, which plays an
important role in DNA recombination and repair
3. BLM mutation results in defects in DNA repair & genomic

instability in somatic cells, predisposing the patients to cancer
development
CLINICAL FEATURES
1. Stunted growth:
a) Growth delay is the most impressive clinical feature of Bloom

syndrome and is usually the first manifestation that causes parents
to seek medical attention
b) The length and weight are both affected, but with normal body
proportion (primordial stunted growth)
c) The child typically has reduced subcutaneous fat, yielding a

wasted appearance
2. Feeding problems:
There is lack of interest in eating; some patients have gastro-

oesophageal reflux, vomiting and diarrhoea
3. Cutaneous manifestations:
a) The characteristic rash develops later in life, mainly on the

face, following a butterfly distribution on the cheeks and nose,
mostly in the form of telagiectasia and erythema
b) Rash is exacerbated by sun exposure and may also develop

on other sun-exposed skin such as forearms and ears
c) Cheilitis, fissuring, blistering, poikiloderma and alopecia
d) Café au lait macules with adjacent hypopigmented areas

(twin spotting)
4. Facial features:
a) Long and narrow face, micrognathia and underdeveloped

malararea
b) The nose and ears of Bloom syndrome patients are often

prominent, owing to decreased amount of subcutaneous fat
5. Ocular features:
Conjunctivitis, sectoral iris pigmentation, lens opacities,

retinoblastoma, lateral strabismus and ectropion
6. Musculoskeletal abnormalities:
a) Long limbs, disproportionately large hands and feet and

progressive contracture of hands and feet
b) Quick, bird-like movements, high pitched voice,

clinodactyly and syndactyly
7. Fertility:
Male patients are infertile (primary hypogonadism) and

female patients are sub-fertile (premature menopause)
8. Immunological abnormalities:
a) Patients’ with Bloom syndrome are predisposed to
respiratory and GIT infections as a result of deficiency
of immunoglobulin A
b) Bacterial infections are more frequent than viral infections
9. Predisposition to malignancy:
a) There is a marked predisposition to early development of

cancer
b) The most common cancers include lymphomas, leukemia,

oral squamous cell carcinoma and adenocarcinoma of the
sigmoid colon
c) There is increased risk of skin cancers; basal cell carcinoma

is the most common, followed by squamous cell carcinoma;
most cancers occur in sun exposed areas
DIAGNOSIS
1. DNA analysis
2. Decreased IgA level
3. Prenatal diagnosis of at-risk pregnancies is possible by

molecular genetic testing of fetal cells obtained by
amniocentesis or chorionic villus sampling
TREATMENT
1. Management is symptomatic
2. Increased calorie density formulas and food may promote

weight gain and increased growth
3. Standard antibiotic regimens are used to treat infections
4. Intravenous immunoglobulins are given if the serum level

of immunoglobulins is low and the patient is experiencing
repeated infections
5. Skin protection, including covering exposed skin and use

of broad spectrum sunscreen is crucial to reduce sun
sensitive skin rash
6. Screening colonoscopy should be carried out regularly

from age 16
PROGNOSIS
1. The high occurrence of cancer reduces life expectancy
2. The median life expectancy is 30 years, with cancer as

the leading cause of death
FANCONI ANEMIA
1. Fanconi anemia is a rare, hereditary, autosomal recessive

DNA repair disorder characterized by progressive
pancytopenia, bone marrow failure and predisposition
to develop tumours
2. Mutation in atleast 18 genes can cause Fanconi anemia
3. The proteins encoded by these genes work together in a

common pathway called the FA pathway, that goes into
operation when DNA damage occurs
4. The FA pathway sends certain proteins to the areas of

damage so the DNA can be repaired by homologous
recombination
CLINICAL FEATURES
1. Low birth weight, developmental delay and short stature
2. Microcephaly, microphthalmia, low-set, large ears and

deafness
3. Altered skin pigmentation and café au lait spots
4. Absent or hypoplastic thumb, dysplastic ulna, clinodactyly,

absent first metacarpal, toe syndactyly and flat feet
5. High arch palate, duodenal atresia, imperforate anus and

umbilical hernia
6. Hypogenitalia and structural renal defects
7. Petechiae, bruises, fatigue and infections
8. Bone marrow failure occurs at a median age of 7 years
9. Patients may develop acute myeloid leukemia

10. Patient are highly predisposed to solid tumours of the
head, neck and ano-genital regions
DIAGNOSIS
1. DNA analysis
2. Chromosome breakage test:
a) Patient’s blood cells are treated in a test tube with

mitomycin C that cross links DNA
b) Normal cells are able to correct most of the damage

and are not severely affected, whereas FA cells show
marked chromosome breakage
3. Complete blood count reveals pancytopenia and

macrocytosis
4. Bone marrow biopsy may reveal hypocellularity, loss of

myeloid/erythroid precursors and megakaryocytes
5. Ultrasound, MRI and CT scan

TREATMENT
1. Transfusion of packed RBCs or platelets
2. Administration of granulocyte colony stimulating factor

(G-CSF)
3. The only curative treatment is stem cell transplantation
4. Radiation therapy or chemotherapy for cancer
PROGNOSIS
1. Bone marrow failure and malignancies lead to a poor

prognosis with life expectancy of 30 years
2. Stem cell transplantation may increase life expectancy
COCKAYNE SYNDROME
1. Cockayne syndrome is a rare, autosomal recessive,

multi-system disorder characterized by dwarfism,
retinopathy and photosensitivity
2. Cockayne syndrome is caused by mutations in the
ERCC 8 gene (chromosome 5) or ERCC 6 gene
(chromosome 10)
3. The proteins made by these genes are involved in

repairing damaged DNA by the nucleotide excision
repair mechanism
CLINICAL FEATURES
1. Appearance:
Microcephaly, short stature, cachexia, thin nose and

large ears give the patient a Mickey Mouse appearance
2. Skin:
a) Photosensitive eruptions with erythema and scaling
b) Affected areas show hyperpigmentation, telangiectasia

and atrophy
c) Subcutaneous lipoatrophy results in sunken eyes and

an aged progeric appearance
d) Cyanotic acral oedema of the extremities
3. Musculoskeletal system:
Long limbs with joint contractures, large hands & feet,

kyphosis and osteoporosis
4. Neurological findings:
a) Ataxia, tremors and cog wheeling
b) Mental retardation and progressive sensorineural

deafness
5. Ophthalmologic findings:
a) Salt and pepper retinal pigment, mitotic pupils and

cataract
b) Optic atrophy, corneal opacity and nystagmus
6. Dental caries
7. Hypogonadism in males & irregular menses in females
DIAGNOSIS
1. DNA analysis
2. UV-irradiated cells show decreased DNA and RNA

synthesis
3. Brain CT scanning may reveal calcifications and

cortical atrophy

TREATMENT
Management is purely supportive & includes physiotherapy,

sun protection, hearing aids & tube feeding or gastrostomy
LYNCH SYNDROME
1. Lynch syndrome also known as hereditary non-polyposis

colorectal cancer is an autosomal dominant genetic
condition associated with a high risk of cancers
2. Alteration is five genes (MLH1, MSH2, MSH6, PMS2

and EPCAM) involved in DNA mismatch repair have been
linked to Lynch syndrome
3. The increased risk of cancers is due to inherited mutations

that impair DNA mismatch repair
CLINICAL FEATURES
1. People who have Lynch syndrome have a significantly

increased risk of developing colorectal cancer
2. There is also an increased risk of developing endometrial,
gastric, breast, ovarian, small intestinal, pancreatic, prostate,
urinary tract, liver, kidney and bile duct cancers
3. The following history should raise suspicion of Lynch

syndrome:
a) Multiple cases of colorectal cancer or numerous

adenomatous polyps diagnosed in different generations
b) People younger than 50 years affected
c) The combination of syndrome-related tumours in other

organs
d) Synchronous tumours in one person
4. Polyp formation starts in the late second or early third

decade
5. Features of colorectal cancer:
a) Constipation or diarrhoea that persists for a long duration
b) Visible or occult blood in stools (black, tarry stools)
c) Iron deficiency without an identifiable cause
d) Abdominal pain, cramps and bloating
e) Fatigue, decline in appetite and unexplained weight loss
DIAGNOSIS
1. DNA analysis
2. Immunohistochemistry (IHC) testing uses monoclonal
antibodies to show which mismatch repair proteins are
present in a tissue sample
3. Microsatellite instability (MSI) is the hallmark of

defective DNA mismatch repair gene
4. Colonoscopy and barium contrast study
TREATMENT
1. Surgical care:
a) Subtotal colectomy with ileo-rectal anastomosis
b) Total colectomy
2. Surveillance sigmoidoscopy is recommended every year

following colectomy
3. To help reduce the risk of endometrial and ovarian cancer,

women over the age of 50 years who have Lynch syndrome
are recommended prophylactic hysterectomy & bilateral
salpingo-oophorectomy
4. Chemoprevention:
Sulindac, aspirin, estrogen, folic acid, calcium, beta carotene,

vit C and vit E may prevent the development of colorectal
cancer
QUESTIONS
1. DNA damage
2. Mismatch repair
3. Base excision repair
4. Nucleotide excision repair
5. Double strand break repair
6. Give the cause, clinical features, diagnosis and

treatment of xeroderma pigmentosa

treatment of ataxia telangiectasia

treatment of Bloom syndrome

treatment of Fanconi anemia

treatment of Cockayne syndrome

treatment of Lynch syndrome
TRANSCRIPTION
TRANSCRIPTION IN PROKARYOTES
INTRODUCTION
1. Transcription is a process in which RNA is synthesized

from DNA
2. Gene refers to the functional unit of the DNA that can

be transcribed
3. The genetic information stored in DNA is expressed

through RNA
4. The product formed in transcription is referred to as the

primary transcript
5. The primary RNA transcripts are inactive; they

undergo post transcriptional modifications (splicing,
additions, base modifications) to produce functionally
active RNA molecules
CODING AND NON-CODING STRANDS

1. The entire molecule of DNA is not expressed in
transcription
2. RNA’s are synthesized only for some selected regions

of DNA and for other regions of the DNA there is no
transcription at all
3. One of the two strands of DNA serves as a template and

is known as the template/non-coding strand; it produces
working copies of RNA molecules
4. The other DNA strand which does not participate in

transcription is referred to as the non-template/coding
strand
TRANSCRIPTION COMPLIMENTARY BASE RELATIONSHIP
5’ A T G C A T G G 3’ CODING
STRAND
DNA
3’ T A C G T A C C 5’ NON-CODING
STRAND
RNA5’ A U G C A U G G 3’
RNA POLYMERASE
1. A single enzyme, RNA polymerase, synthesizes all the RNA’s
in prokaryotes
2. RNA polymerase is a complex holoenzyme with 5 polypeptide

subunits: (2 alpha,1 beta, 1 beta’ and one sigma factor)
3. The enzyme without sigma factor is referred to as core enzyme
STAGES OF TRANSCRIPTION
Transcription involves three different stages:
1. Initiation
2. Elongation
3. Termination
INITIATION
1. The binding of the enzyme RNA polymerase to DNA is the

pre-requisite for transcription to start
2. The specific region on the DNA where the enzyme binds is

known as the promoter region
3. There are 2 base sequences on the coding DNA strand which

the sigma factor of RNA polymerase can recognize for the
initiation of transcription:
a) Pribnow box (TATA box):
This consists of 6 nucleotide bases (TATAAT) located on the

left side, 10 bases away (upstream) from the starting point of
transcription
b) The “-35” sequence:
This is the second recognition site in the promoter region of

DNA.
It contains a base sequence TTGACA, which is located about

35 bases away upstream (hence -35) on the left side from the
transcription site.
PROMOTER REGIONS OF DNA IN PROKARYOTES

-35 SEQUENCE PRIBNOW BOX
CODING TTGACA T A T A AT
STRAND 5’
NON-CODING
STRAND 3’ - 35 - 10
BASES BASES
START OF
TRANSCRIPTION
ELONGATION
1. As the RNA polymerase recognizes the promoter region, the
sigma factor is released and transcription proceeds
2. RNA is synthesized from 5’ end to 3’end (5’ to 3’)
3. RNA polymerase utilizes ribonucleoside triphosphates (ATP,

GTP, CTP and UTP) for the formation of RNA
4. For the addition of each nucleotide to the growing chain,

a pyrophosphate is released
5. RNA polymerase does not require a primer
6. RNA polymerase does not have endonuclease or exonuclease

activity
7. Due to lack of proof reading ability, RNA polymerase has no

ability to repair the mistakes in the RNA synthesized
8. The double helical structure of DNA unwinds as the

transcription goes on, resulting in supercoils
9. The problem of supercoils is overcome by topoisomerases

DIAGRMMATIC REPRESENTATION OF TRANSCRIPTION
5’ 3’
3’ 5’
PROMOTER TRANSCRIPTION TERMINATION
SITE SITE SITE
DNA TEMPLATE
5’ INITIATION 3’
3’ 5’
σ
σ SIGMA FACTOR
ELONGATION
5’ 3’
3’ 5’
5’
ELONGATION
5’ 3’
3’ 5’
5’
TERMINATION
5’ 3’
3’ 5’
ρ
5’
NEWLY SYNTHESIZED RNA
ρ RHO FACTOR
5’ 3’
TERMINATION
The process of transcription stops by termination signals, which
can be of 2 types: rho dependent and rho independent
RHO DEPENDENT TERMINATION
1. A specific protein, named rho factor, binds to the growing RNA,

terminates transcription and releases RNA
2. The rho factor is also responsible for the dissociation of RNA

polymerase from DNA
RHO INDEPENDENT TERMINATION
1. The termination in this case is brought about by the formation

of hairpins of newly synthesized RNA
2. This occurs due to the presence of palindromes
3. A palindrome is a word that reads alike forward and backward

(e.g. madam, rotor)
4. Due to the presence of palindromes in the base sequence of DNA

template in the termination region, the newly synthesized RNA
folds to form hairpins (due to complementary base pairing) that
causes termination of transcription
TRANSCRIPTION IN EUKARYOTES
1. The eukaryotic cells have 3 distinct RNA polymerases:
a) RNA polymerase I synthesizes ribosomal RNA’s
b) RNA polymerase II synthesizes messenger RNA’s
c) RNA polymerase III synthesizes transfer RNA’s
2. In eukaryotes, a sequence of DNA bases, known as Hogness

box (or TATA box), is located on the left side, 25 nucleotides
away, upstream, from the starting site of mRNA synthesis
3. There also exists another site of recognition between 70 to 80

nucleotides upstream from the start of transcription and this
second site is called the CAAT box
4. These two sites help the RNA polymerase to recognize the

requisite sequence on the DNA for transcription
PROMOTER REGIONS OF DNA IN EUKARYOTES
CAAT BOX HOGNESS BOX

CODING GGCCAATC ATATAA
STRAND 5’
NON-CODING
STRAND 3’ - 70 - 25
START OF
TRANSCRIPTION
POST TRANSCRIPTIONAL MODIFICATIONS
1. The RNA’s produced during transcription are called primary

transcripts
2. They undergo many alterations, which are collectively known

as post transcriptional modifications
3. This process is required to convert the RNA’s into active forms
4. A group of enzymes, called ribonucleases, are responsible for

processing the RNA’s
MESSENGER RNA
1. The primary transcript of mRNA is the hnRNA, which is

subjected to many changes before functional mRNA is
produced
2. The 5’ end of the mRNA is capped with 7 methyl guanosine;

this is called 5’ capping
3. S adenosyl methionine is the donor of the methyl group
4. This cap is required for stabilizing the structure of mRNA
5. The mRNA’s have an adenine nucleotide chain at the 3’ end;

this is called the poly A tail
6. The poly A tail stabilizes the mRNA

7. Introns are the intervening nucleotide sequences in mRNA
which do not code for proteins
8. Exons of the mRNA possess the genetic code and are

responsible for protein synthesis
9. The removal of introns is promoted by small nuclear

ribonucleoproteins (snRNPs)
10. snRNPs are formed by the association of small nuclear

RNA’s with proteins
11. snRNPs associated with hnRNA at the exon-intron

junction form spliceosomes
12. Post transcriptional modification of mRNA occurs in

the nucleus
13. The mature RNAs then enter the cytosol for translation
REMOVAL OF INTRONS
TRANSFER RNA
1. All the tRNA's undergo post transcriptional modifications
2. These include:
a) Trimming
b) Converting the existing bases into unusual ones
c) Addition of CCA nucleotides to 3’ terminal end of

tRNA’s
RIBOSOMAL RNA
The pre-ribosomal RNA’s originally synthesized are

converted to ribosomal RNA’s by a series of post
transcriptional changes
INHIBITORS OF TRANSCRIPTION - ARA important
The synthesis of RNA’s is inhibited by certain antibiotics

and toxins:
1. Actinomycin-D:
It binds with the DNA template and blocks the movement

of RNA polymerase.
It is used in cancer treatment.
2. Rifampin:
It binds with the beta subunit of RNA polymerase of

prokaryotes and inhibits its activity.
It is used for the treatment of tuberculosis and leprosy.
3. Alpha amanitin:
It binds with RNA polymerase II of eukaryotes and

inhibits transcription.
It is a toxin produced by the mushroom amanita phalloides.

REVERSE TRANSCRIPTION
1. Generally speaking, the genes are made up of DNA
2. Usually, DNA dependent RNA polymerase transfers the

information of DNA to mRNA
3. However, some of the viruses, known as retroviruses,

possess RNA as the genetic material
4. Here RNA acts as a template
5. Based on this template, the enzyme RNA dependent DNA

polymerase or reverse transcriptase makes a new DNA
strand
6. From the RNA-DNA hybrid, the RNA part is hydrolysed

by a specific RNAse-H
7. The remaining DNA acts as a template to produce double

stranded DNA
8. Thus, genetic information is transferred from RNA to DNA
9. Some of the tumour viruses possess reverse transcriptase
10. The presence of the enzyme reverse transcriptase is an

indication of retrovirus infection
11. HIV is a retrovirus

REVERSE TRANSCRIPTION
QUESTIONS
1. Initiation of transcription
2. Diagrammatic representation of transcription
3. Describe transcription in prokaryotes
4. Termination of transcription
5. RNA polymerase
6. Reverse transcription
7. Inhibitors of transcription
8. Post transcriptional modifications
GENETIC CODE
INTRODUCTION
1. The three nucleotide (triplet) base sequences in mRNA that

act as code words for amino acids in protein constitute the
genetic code
2. The three nucleotide base sequences (triplets) are known as

codons
3. The genetic code may be regarded as a dictionary of nucleotide

bases (A, C, G and U) that determines the sequence of amino
acids in proteins
4. The codons are composed of the four nucleotide bases:
a) Adenine (A)
b) Guanine (G)
c) Cytosine (C)
d) Uracil (U)
5. These four bases produce 64 different combinations (43)

of the three base codons
6. 61 codons code for the 20 amino acids found in protein
7. The 3 codons UAA, UAG and UGA do not code for amino
acids
8. They act as stop signals in protein synthesis
9. These 3 codons are known as terminator codons or

nonsense codons
10. The stop codons are also referred to as follows:
a) UAG – amber
b) UAA – ochre
c) UGA – opal
11. The codon AUG is known as initiator codon

12. Exceptions to the genetic code:
a) UGA codes for selenocysteine (the 21st amino acid) in

both bacteria and humans
b) UAG codes for pyrrolysine (the 22nd amino acid) in archaea

and bacteria
c) UGA codes for tryptophan in mitochondrial DNA of yeasts
AMBER, OCHRE AND OPAL CODONS
1. Amber codon (UAG)
UAG was the first stop codon discovered.
It was isolated by Richard Epstein and Charles Steinberg and

named after their friend Harris Bernstein (Bernstein means
“amber” in German).
2. Ochre codon (UAA)
UAA was the second stop codon to be discovered.
Reminiscent of the yellow-orange-brown colour associated with

amber, this second stop codon was given the name of “ochre”,
a orange-reddish-brown mineral pigment.
3. Opal codon (UGA)
UGA was the third and last stop codon to be discovered.
To continue matching with the theme of coloured minerals,

it came to be known as “opal”, which is a type of silica
showing a variety of colours
GENETIC CODE
CHARACTERISTICS OF GENETIC CODE
UNIVERSALITY
1. The same codons are used to code for the same amino acids in
all living organisms
2. Thus, the genetic code has been conserved during the course
of evolution and is regarded as universal
SPECIFICITY
A particular codon always codes for the same amino acid, hence
the genetic code is highly specific or unambiguous e.g. UGG is
the codon for tryptophan
NON-OVERLAPPING
1. The genetic code is read from a fixed point as a continuous base

sequence
2. It is non-overlapping, comma less and without any punctuation
3. Addition or deletion of one or two bases will radically change

the message sequence in mRNA and the protein synthesized
from such mRNA will be totally different; this is called frame
shift mutation
DEGENERATE
1. Most of the amino acids have more than one codon
2. The codon is degenerate or redundant, since there are 61 codons

available to code for only 20 amino acids e.g. glycine has four
codons
3. The codons that code for the same amino acid are called
synonyms
4. Most of the synonyms differ only in the third base of the codon
WOBBLE HYPOTHESIS
1. The codon of mRNA is recognized by the anticodon of the

tRNA
2. The usual complementary base pairing (A = U and G = C)

occurs between the first 2 bases of the codon and last 2 bases
of the anticodon
3. The third base of the codon is flexible with regard to the

complementary base
4. The wobble hypothesis was put forth by Francis Crick
5. Wobble hypothesis is a phenomenon in which a single tRNA

can recognize more than one codon
6. This is due to the fact that the third base in the codon often
fails to recognize the specific complementary base in the
anticodon
7. Wobble hypothesis explains the degeneracy of the genetic
code - the existence of multiple codons for a single amino
acid
8. Although there are 61 codons for amino acids, the number

of tRNA’s is far less (around 40), which is due to wobbling
WOBBLING
CODON BIAS
1. Many amino acids have multiple codons
2. However, one or two codons (not all of them) are preferred

and this is called codon bias
QUESTIONS
1. Characteristics of genetic code
2. Wobble hypothesis
TRANSLATION / PROTEIN SYNTHESIS
DEFINITION
Biosynthesis of a protein or a polypeptide in a living cell is

known as translation.
The sequence of amino acids in the protein synthesized is

determined by the base sequence of mRNA.
PROTEIN SYNTHESIS
COMPONENTS REQUIRED FOR TRANSLATION
AMINO ACIDS
1. Protein synthesis can occur only when all the amino acids
needed for a particular protein are available
2. If there is a deficiency in the dietary supply of any one of the

essential amino acids, then translation stops
RIBOSOMES
1. The functionally active ribosomes are the centres or factories

of protein synthesis
2. Ribosome has 2 sites: A site and P site
3. The A site is for binding aminoacyl tRNA and the P site for
binding peptidyl tRNA, during the course of translation
MESSENGER RNA
1. The specific information required for synthesis of a protein

is present on mRNA
2. The DNA passes on the genetic information in the form of

codons to the mRNA, which is then translated into a protein
sequence
TRANSFER RNA’s
1. tRNA’s carry the amino acids and hand them over to the
growing peptide chain
2. Each tRNA has a 3-nucleotide base sequence known as the

anticodon, which is responsible to recognize the codon of
mRNA for protein synthesis
ENERGY SOURCES
Both ATP and GTP are required for the supply of energy
in protein synthesis
PROTEIN FACTORS
1. The process of translation involves a number of protein factors
2. These are needed for the initiation, elongation and termination

ACTIVATION OF AMINO ACIDS
1. Amino acids are activated and attached to tRNA’s
2. The enzyme required is aminoacyl tRNA synthetase
Amino acid + tRNA + ATP
Amino acyl tRNA synthetase
Amino acyl tRNA + AMP + PPi
STAGES OF TRANSLATION
Translation takes place in 3 stages:
1. Initiation
2. Elongation
3. Termination
INITIATION
1. The initiation factors IF1, IF2 and IF3 bind with the
ribosome
2. The ribosome binding site in the mRNA (near the 5’ end)

is called the Shine-Dalgarno sequence (GGAGG); it is
located 10 nucleotides upstream of the initiator codon
3. The rRNA has a base sequence complementary to

Shine-Dalgarno sequence and this helps in binding of
mRNA to rRNA
4. The codon AUG is responsible for the initiation of

protein synthesis and it codes for methionine
5. The tRNA carrying methionine attaches to the P site

of the ribosome
BINDING OF CODON (mRNA) AND ANTICODON (tRNA)
CODON
INITIATION
MET
P SITE A SITE
AUG ACC UGG GAC
5’ 3’
ELONGATION
1. The initiation tRNA occupies the P site of the ribosome
2. The incoming next amino acid is deposited at the A site

of the ribosome
3. This requires elongation factors and energy is provided by

GTP
4. The enzyme peptidyl transferase catalyses the formation of

peptide bond between the two amino acids
5. Now, the tRNA occupying the P site does not have any amino
acid attached and the tRNA occupying the A site has 2 amino
acids attached joined by peptide bond
6. The ribosome then moves to the next codon (3 bases) in the

mRNA
7. This process called translocation, involves the movement of

the growing peptide chain from the A site to the P site of the
ribosome
8. Translocation requires elongation factor (EF) and GTP
9. The new amino acid then comes in and attaches to the A site
of the ribosome, which is free
10. The elongation process is repeated again and again with the
addition of one amino acid each time, till the signal of
termination is reached
ELONGATION
ATTACHMENT OF
NH NH NEW AMINO ACID
MET THR TO A SITE
COOH COOH
P SITE A SITE
AUG ACC UGG GAC

5’ 3’
NH
MET
CO PEPTIDYL
THR TRANSFERASE
NH
COOH PEPTIDE BOND

FORMATION
P SITE A SITE
AUG ACC UGG GAC

5’ 3’
TERMINATION
1. One of the three codons UAA, UAG and UGA act as stop
signal and terminate translation
2. The terminator codons do not have specific tRNA’s to bind

with them
3. As the terminator codon occupies the A site of the ribosome,

the release factors (RF1, RF2, RF3) bind with the codon
4. These factors release the newly synthesized protein
5. The releasing factors are also responsible for the dissociation

of ribosomes from mRNA
6. As the protein synthesis is completed, the N terminal amino

acid, methionine is removed by hydrolysis
TRANSLOCATION
MET
THR
TRANSLOCATION OF
GROWING PEPTIDE
CHAIN TO P SITE
P SITE A SITE
AUG ACC UGG GAC

5’ 3’
MET
THR TRP
ATTACHMENT OF
NEW AMINO ACID
TO A SITE
P SITE A SITE
AUG ACC UGG GAC

5’ 3’
INHIBITORS OF TRANSLATION – STREP-CD
1. Streptomycin:
a) It inhibits initiation of protein synthesis
b) It causes misreading of the mRNA and interferes with

the normal pairing between codon and anticodon
2. Tetracycline:
It inhibits binding of the aminoacyl tRNA to the ribosome
3. Ricin:
a) Ricin is highly toxic and found naturally in castor beans
b) Ricin blocks protein synthesis by inhibiting assembly of

amino acids into proteins
c) There are fears that ricin may be used as a biological

weapon in warfare
4. Erythromycin:
It inhibits translocation by binding with the bacterial

ribosome
5. Puromycin:
a) It has structural resemblance to aminoacyl tRNA
b) Puromycin enters the A site and gets incorporated into

the growing peptide chain and causes its release
6. Chloramphenicol:
It acts as a competitive inhibitor of the enzyme peptidyl

transferase and thus interferes with elongation of the
peptide chain
7. Diphtheria toxin:
It prevents translocation in eukaryotes by inactivating

elongation factor (eEF2)
POST TRANSLATIONAL MODIFICATIONS
1. The proteins synthesized in translation are not functional
2. Many changes take place in the proteins after the

completion of protein synthesis
3. These modifications are known as post translational

modifications
4. Post translational modifications are:
a) Proteolytic degradation
b) Phosphorylation
c) Hydroxylation
d) Glycosylation
e) Carboxylation
PROTEOLYTIC DEGRADATION
1. Many proteins are synthesized as precursors, which are

much bigger in size than the functional proteins
2. Some portion of the precursor molecules are removed by

proteolysis to liberate active protein e.g.
a) Conversion of proinsulin to insulin
b) Conversion of trypsinogen to trypsin
PHOSPHORYLATION
1. The hydroxyl group containing amino acids of proteins,

namely serine & threonine are subjected to phosphorylation
2. The phosphorylation may either increase or decrease the

activity of proteins
3. The enzyme required for phosphorylation is protein kinase
HYDROXYLATION
1. During the formation of collagen, the amino acids proline

and lysine are, respectively, converted to hydroxyproline
and hydroxylysine
2. This hydroxylation occurs in the endoplasmic reticulum

and requires vitamin C
GLYCOSYLATION
1. The attachment of carbohydrates is essential for some

proteins to perform their functions
2. The complex carbohydrates are attached to the amino acids

serine & threonine, leading to the synthesis of glycoproteins
CARBOXYLATION
1. Vitamin K dependent carboxylation of glutamic acid residues

in clotting factors is a post-translational modification
2. Vitamin K brings about post-translational modifications of

blood clotting factors II (prothrombin), VII, IX and X
COMPARISON OF TRANSLATION IN EUKARYOTES &
PROKARYOTES
Feature Eukaryotes Prokaryotes
1. DNA Open Circular
2. Ribosome 80 S 70 S
3. tRNA 31 22
4. Initiating Methionine Formyl

amino acid methionine
5. Effect of Not Inhibited

tetracycline & affected
chloromycetin
6. Start signal Only one Multiple

QUESTIONS
1. Diagrammatic representation of translation
2. Post translational changes
3. Inhibitors of translation
4. Initiation of translation
5. Elongation of translation
6. Translocation
7. Termination of translation
8. Components required for translation
9. Comparison of translation in eukaryotes & prokaryotes
10. Describe translation in prokaryotes

MUTATION
DEFINITION
1. Mutation refers to a change in DNA structure of a gene
2. The substances (chemicals) which can induce mutations

are called mutagens
3. The chemical changes that occur in DNA on mutation are

reflected in replication, transcription and translation
TYPES OF MUTATIONS
Mutations are of two major types:
1. Point mutations
2. Frame shift mutation
POINT MUTATIONS
The replacement of one base pair by another result’s in

point mutation.
Point mutations are of two types:
1. Transition:
In this case, a purine (or a pyrimidine) is replaced by another
2. Transversion:
In this case, a purine is replaced by a pyrimidine or vice versa
CONSEQUENCES OF POINT MUTATIONS
The change in a single base sequence in point mutation may

cause one of the following:
1. Silent mutation
2. Missense mutation
3. Nonsense mutation
POINT MUTATION CONSEQUENCES
UCU
(Codon for serine)
Silent
mutation
UCA
(Codon for serine)
Mis-sense Nonsense
mutation mutation
ACA UAA
(Codon for threonine) (Termination codon)
SILENT MUTATION
1. The codon of mRNA containing the changed base, may code

for the same amino acid
2. Example - UCA codes for serine and change in the third base
(UCU) still codes for serine
3. This is due to degeneracy of the genetic code
4. Thus, there is no detectable effect in silent mutation
MISSENSE MUATION
1. In this case, the changed base may code for a different amino
acid
2. Example - UCA codes for serine while ACA codes for

threonine
3. The mistaken or missense amino acid may be acceptable,

partially acceptable or unacceptable with regard to function
of protein molecule
4. Sickle cell anaemia is caused due to missense mutation

NONSENSE MUTATION
1. Sometimes the codon with the altered base may become a

termination or nonsense codon
2. Example - change in the second base of the serine codon

(UCA) may result in formation of nonsense codon UAA
3. The altered codon acts as a stop signal & causes termination

FRAME SHIFT MUTATION
These occur when one or more base pairs are inserted or

deleted from the DNA respectively, causing insertion or
deletion mutation
CONSEQUENCES OF FRAME-SHIFT MUTATION
1. The insertion or deletion of a base in a gene results in an

altered reading frame of the mRNA (hence the name frame
shift)
2. The machinery of the mRNA (containing codons) does not

recognize that a base was missing or a new base was added
3. Since there are no punctuations in the reading of the codons,

translation continues
4. The result is that the protein synthesized will have several

altered amino acids or may be prematurely terminated
FRAME SHIFT MUTATION
NORMAL mRNA AUG GCC UCU UGC
PROTEIN MET ALA SER CYS
DELETION mRNA AUG GCC CUU GCA

OF ONE U
PROTEIN MET ALA LEU ALA
GARBLED
MUTATIONS AND CANCER
Mutations are permanent alterations in DNA structure,

which have been implicated in the pathogenesis of cancer
QUESTIONS
1. Define mutation & describe in detail types of mutation
2. Point mutation and its consequences
3. Frame shift mutation

REGULATION OF GENE EXPRESSION
INTRODUCTION
1. DNA is the chemical vehicle of heredity
2. DNA is composed of functional units called genes
3. The term genome denotes the total genetic information

contained in a cell
4. The cistron is the smallest unit of genetic expression – it

is the fragment of DNA coding for a subunit of a protein
molecule
5. There are two types of gene regulation:
a) Positive regulation – the gene regulation is said to be

positive when its expression is increased by a regulatory
element (positive regulator)
b) Negative regulation - the gene expression is said to be

negative when its expression is decreased by a regulatory
element (negative regulator)
LAC OPERON
INTRODUCTION
1. The operon is the coordinated unit of genetic expression

in bacteria
2. The concept of operon was introduced by Jacob and Monod,

based on their observations on the regulation of lactose
metabolism in E. coli
3. This is known as lac operon
STRUCTURE OF LAC OPERON
1. The lac operon consists of:
a) Regulator gene (I)
b) Operator gene (O)
c) Three structural genes (Z, Y, A)
2. There is also a promoter site (P), next to the operator gene,

where the enzyme RNA polymerase binds
3. The structural genes code for the following enzymes:
a) Z – beta galactosidase
b) Y – galactoside permease
c) A – galactoside acetylase
4. Beta galactosidase hydrolyses lactose to galactose & glucose
5. Galactoside permease transports lactose into the cell

6. The function of galactoside acetylase is unknown
7. The structural genes Z, Y and A transcribe into a single large

mRNA with 3 independent translation units for the synthesis
of 3 distinct enzymes
8. An mRNA coding for more than one protein is called

polycistronic mRNA
REPRESSION OF LAC OPERON
1. The regulator gene (I) is expressed at a constant rate leading

to the synthesis of lac repressor
2. Lac repressor is a tetrameric (4 subunits) regulatory protein,

with a molecular weight of 150,000
3. Lac repressor specifically binds with the operator gene (O)
4. This prevents the binding of the enzyme RNA polymerase to

the promoter site (P), thereby blocking the transcription of
structural genes (Z, Y and A)
5. This is what happens in the absence of lactose in E. coli
6. The repressor molecule can act as a negative regulator of

gene expression
STRUCTURE OF LAC OPERON
I P O Z Y A
REGULATOR PROMOTER OPERATOR

GENE SITE GENE
STRUCTURAL GENES
REPRESSION OF LAC OPERON
I P Z Y A
mRNA
REPRESSOR
REPRESSOR TETRAMER
SUBUNITS
DEREPRESSION OF LAC OPERON
1. In the presence of lactose (inducer), a small amount of it

can enter E. coli cells
2. The repressor molecules have a high affinity for lactose
3. The lactose molecules bind and induce a conformational

change in the repressor
4. The result is that the repressor gets inactivated & therefore

cannot bind to the operator gene (O)
5. The RNA polymerase attaches to the promoter site and

transcription proceeds, leading to the formation of
polycistronic mRNA (for genes Z, Y and A), and finally
the three enzymes
6. Thus, lactose induces the synthesis of the three enzymes
7. Lactose acts by inactivating the repressor molecule, hence

this process is known as derepression of lac operon
DEREPRESSION OF LAC OPERON
CAP - cAMP
I P O Z Y A
RNAP
mRNA POLYCISTRONIC mRNA
β GALACTOSIDASE PERMEASE ACETYLASE
LACTOSE
REPRESSOR
INACTIVE REPRESSOR
CAP - cAMP CATABOLITE GENE ACTIVATOR

PROTEIN BOUND TO cAMP
RNAP RNA POLYMERASE

CATABOLITE GENE ACTIVATOR PROTEIN
1. The cells of E. coli utilize glucose in preference to lactose

when both of them are present
2. After the depletion of glucose in the medium, utilization

of lactose starts
3. Thus, glucose interferes with the induction of lac operon
4. The attachment of RNA polymerase to the promoter site

requires the presence of a catabolite gene activator protein
(CAP) bound to cyclic AMP
5. The presence of glucose lowers the intracellular

concentration of cyclic AMP
6. Due to the diminished levels of cyclic AMP, the formation

of CAP-cAMP is lowered and the transcription is almost
negligible in the presence of glucose
7. Thus, glucose interferes with the expression of lac operon

by depleting the cyclic AMP levels
8. The presence of CAP-cAMP is essential for transcription

of structural genes of lac operon
9. Thus, CAP-cAMP acts as a positive regulator for gene

expression
GENE REGULATION IN EUKARYOTES
The following are the important methods of gene regulation

in eukaryotes:
1. Gene amplification
2. Gene rearrangement
3. Role of enhancers and silencers
4. Transcription proper
5. Processing of mRNA
6. Transport of mRNA
7. Degradation of mRNA
8. Translation proper
9. Epigenetic regulation of gene expression
GENE AMPLIFICATION
1. In this mechanism, the expression of a gene is increased

several-fold
2. Example - in fruit fly (drosophila) the amplification of genes

coding for egg shell proteins is observed during the course of
oogenesis
3. The occurrence of gene amplification has also been reported

in humans
4. Methotrexate is an anti-cancer drug which inhibits the enzyme
dihydro folate reductase
5. The malignant cells develop drug resistance to long term

administration of methotrexate by amplifying the gene coding
for dihydrofolate reductase
GENE AMPLIFICATION
GENE REARRANGEMENT
1. The body possesses an enormous capacity to synthesize a

wide range of antibodies
2. The human body can produce about 10 billion antibodies in

response to antigenic stimulation
3. This is explained on the basis of gene rearrangement
4. The structure of a typical immunoglobulin molecule consists

of two light (L) and two heavy (H) chains
5. Each one of these chains (L or H) contains an N terminal variable

(V) and C terminal constant (C) region
6. Each light chain can be synthesized by three distinct DNA

segments, namely:
a) Variable (Vl)
b) Joining (Jl)
c) Constant (Cl)
7. The genome contains:
a) 500 Vl segments
b) 6 Jl segments
c) 20 Cl segments
8. During the course of differentiation of B lymphocytes, one Vl

segment is brought closer to Jl and Cl segments
9. This occurs on the same chromosome
10. The rearranged DNA (with Vl, Jl and Cl fragments) is then

transcribed to produce a single mRNA for the synthesis of
a specific light chain of the antibody
11. By innumerable combinations of Vl, Jl and Cl segments, the

body’s immune system can generate millions of antigen
specific immunoglobulin molecules
12. The formation of heavy (H) chains of immunoglobulins also

occurs by rearrangement of 4 distinct genes:
a) Variable (Vh)
b) Diversity (D)
c) Joining (Jh)
d) Constant (Ch)
GENE REARRANGEMENT FOR SYNTHESIS OF LIGHT CHAIN OF
IMMUNOGLOBULIN
V (500) J (6) C (20)
ORIGINAL DNA
REARRANGED DNA
V100 J3 C10
PRIMARY TRANSCRIPT
mRNA
PROTEIN (LIGHT CHAIN OF Ig)

ROLE OF ENHANCERS AND SILENCERS
1. Certain DNA segments promote the synthesis of RNA, which

are known as enhancers
2. Some genes diminish the transcription process and are known

as silencers
3. The regulatory proteins bind to specific DNA segments and

control transcription
TRANSCRIPTION PROPER
Various transcription factors assemble in a specific sequence

to form a pre-initiation complex (PIC), which is a potential
site for eukaryotic gene regulation
PROCESSING OF mRNA
1. The RNA synthesized in transcription undergoes modifications

resulting in a functional RNA
2. The changes include:
a) Intron-exon splicing
b) Polyadenylation
c) 5’capping
TRANSPORT OF mRNA
The transport of mRNA from the nucleus into the cytosol as part
of the information pathway is a step for the regulation of gene
expression
DEGRADATION OF mRNA
1. The expression of genes is indirectly influenced by the stability

of mRNA
2. Certain hormones regulate the synthesis and degradation of

some RNA’s
3. Example - estradiol prolongs the half-life of vitellogenin mRNA

from a few hours to about 20 hours
TRANSLATION PROPER
1. Gene expression can also be regulated at the level of translation
2. The genes encoding transferrin, ferritin and haemosiderin, the

transport & storage proteins of iron are subjected to regulation
at the translational level
3. Certain iron binding proteins bind to special structures called

iron response elements and either stimulate or inhibit the gene
4. This occurs in response to fluctuations in the level of iron in the

cell
EPIGENETIC REGULATION OF GENE EXPRESSION
1. The term epigenetics is used to describe the changes in

characteristics of a cell or an organism that are not due to
changes in the nucleotide sequence of the DNA
2. Epigenetics regulates gene expression by modulating

chromatin structure via histone modification or modification
of DNA via methylation
3. For instance, acetylation of histones leads to activation of

gene expression, while DNA methylation is associated with
a reduction in gene expression
4. Epigenetics is heritable due to coordination between histone

modification and methylation of DNA
QUESTIONS
1. Describe lac operon
2. Repression of lac operon
3. Derepression of lac operon
4. Structure of lac operon
5. Catabolite gene activator protein
6. Gene regulation in eukaryotes
7. Gene rearrangement
8. Gene amplification
RECOMBINANT DNA TECHNOLOGY
DEFINITION
The genetic manipulations in the laboratory, involving isolation

and cloning (making identical copies) of a specific DNA are
collectively referred to as recombinant DNA technology or
genetic engineering.
Recombinant DNA technique was developed by Paul Berg,

Herbert Boyer and Stanley Cohey.
STAGES OF RECOMBINANT DNA TECHNOLOGY
RCD is divided into four stages:
1. Isolation of a specific fragment
2. Selection of vector
3. Preparation of chimeric DNA
4. Cloning of chimeric DNA
ISOLATION OF A SPECIFIC DNA FRAGMENT
1. The genomic DNA is huge in size
2. Isolation of a specific fragment of DNA is achieved by cleaving

or splicing brought about by a group of enzymes known as
restriction endonuclease
3. The presence of these enzymes in bacteria restrict the growth of
bacterial viruses, hence the name restriction endonuclease
4. The nomenclature of restriction endonucleases is done as

follows:
a) The first letter of the enzyme denotes the genus
b) The next two letters denote the species of the bacteria
c) A subunit is added to identify the strain of the bacteria
d) The final number indicates order of discovery of the enzyme
5. Examples: EcoRI is from Escherichia coli and HaeIII is from

Haemophilus aegypticus
6. Each restriction endonuclease can specifically recognize a short

sequence (4 to 7 base pairs) of DNA for cleaving
7. The spliced DNA fragments may have staggered cuts to produce

sticky ends (cohesive ends) or blunt ends
8. DNA fragments with sticky ends are particularly useful in

recombinant DNA technology
DNA SPLICING BY RESTRICTION ENDONUCLEASES
G G A T C C
C C T A G G
STICKY ENDS
BamHI
(from Bacillus amyloliquefaciens)
G G A T C C
C C T A G G
G T T A A C
C A A T T G
BLUNT ENDS
HpaI
(from Haemophilus
parainfluenzae)
G T T A A C
C A A T T G
SELECTION OF VECTOR
1. A vector is a carrier of DNA molecule to which the fragment

of DNA of interest (human gene) is attached for cloning
2. The essential properties of a vector include:
a) Ability for autonomous replication
b) The presence of a specific nucleotide sequence recognized

by a restriction endonuclease
c) Gene for identification
3. Three types of vectors are commonly used:
a) Plasmids
b) Bacteriophages
c) Cosmids
PLASMIDS
1. Some of the bacteria contain small, independent, circular,

double stranded DNA molecules known as plasmids
2. Plasmids provide resistance to the bacteria against antibiotics
3. Plasmids DNA replicate independently and they can be easily

separated from host bacteria
4. Plasmids are the most commonly used vectors and they can
accept DNA fragments of 6 to 10 kilo base (kb) in length
(1 kb = 1000 nucleotide long base sequence)
BACTERIOPHAGES
1. Bacteriophages are linear DNA molecules, containing several

restriction enzyme sites
2. Bacteriophages can accept foreign DNA fragments of 10 to 20

kilo bases in length
COSMIDS
1. Cosmids are specialized plasmids containing DNA sequence,

namely CO sites
2. Cosmids can accept much longer DNA fragments of 20 to 50

kilo bases in length
PREPARATION OF CHIMERIC DNA
1. The main purpose of genetic engineering is to insert a DNA of

interest into a vector DNA so that the DNA fragment replicates
along with the vector
2. The hybrid combination of two fragments of DNA is referred

to as recombinant DNA or chimeric DNA
3. Chimera is a monster from Greek mythology that has a lion’s

head, a goat’s body and a serpent’s tail
4. This may be compared to Narsimha in Indian mythology
5. A fragment of target DNA (from human) and the vector DNA

(from plasmid) produced by a specific restriction endonuclease,
possessing sticky ends, can be used for the preparation of
chimeric DNA
6. The human DNA and vector DNA with complementary
sequences are annealed
7. The enzyme DNA ligase seals the DNA fragments to produce

recombinant DNA
8. For the ligation of two DNA fragments, sometimes a synthetic

DNA called a linker is used
9. The linker helps in the recognition of sequence and effective

binding between the human DNA and vector DNA
PRODUCTION OF A RECOMBINANT DNA MOLECULE
PLASMID DNA
RESTRICTION HUMAN DNA
ENDONUCLEASE RESTRICTION
ENDONUCLEASE
HUMAN DNA
PLASMID DNA (WITH STICKY ENDS)
(WITH STICKY ENDS) ANNEAL
DNA LIGASE
RECOMBINANT DNA
CLONING OF THE CHIMERIC DNA
1. A clone means a large population of identical copies
2. The chimeric DNA contained in a plasmid (vector) can be

introduced into bacterial cells by a process called transformation
3. The replicating bacterial cell permits the amplification of the

chimeric DNA of the vector
4. In this way cloning results in the production of a large number

of identical target DNA molecules
5. The cloned target DNA is released from the vector by cleavage

(using appropriate restriction endonucleases), isolated,
characterized and used for various purposes
CLONING OF A RECOMBINANT DNA
RECOMBINANT DNA
(IN A PLASMID)
BACTERIAL CELL TRANSFORMATION

AMPLIFICATION
BACTERIA LYSED AND

PLASMIDS ISOLATED
RESTRICTION ENDONUCLEASE
ORIGINAL PLASMID DNA FRAGMENT OF INTEREST
APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY
1. Production of proteins:
a) Using recombinant DNA technique, several proteins have

been produced in abundance for therapeutic purposes
b) These include insulin, growth hormone, erythropoietin,

interferon, follicle stimulating hormone, blood clotting factor
VIII, granulocyte colony stimulating factor, tissue plasminogen
activator, glucocerebrosidase and superoxide dismutase
c) The first licensed drug generated using recombinant DNA

technique was insulin
2. Molecular analysis of diseases:
Recombinant DNA technology has helped in the understanding

of diseases such as sickle cell anaemia, thalassemia and
hypercholesterolemia
3. Laboratory diagnostic applications:
By using recombinant DNA technology, the diagnosis of many

diseases such as AIDS has become simple and rapid
4. Prenatal diagnosis of diseases:
DNA collected from amniotic fluid can be used for predicting

the risk of developing genetic diseases such as sickle cell
anaemia by using recombinant DNA technology
5. Applications in forensic medicine:
Recombinant DNA technology helps in forensic medicine in

identification of criminals and to settle the disputes of
parenthood of children (to identify the father)
6. Gene therapy:
Gene therapy and recombinant DNA technology can be used to

cure genetic diseases such as adenosine deaminase deficiency,
sickle cell anaemia and thalassemia
7. Industrial applications:
Enzymes synthesized by recombinant DNA technology are used

to produce sugars, cheese and detergents
8. Agricultural applications:
Genetically engineered plants have been developed to resist

drought and disease, besides the higher crop yield e.g. BT cotton
9. Transgenesis:
Transgenesis refers to the transfer of genes into the fertilized

ovum which will be found in the somatic as well as germ cells
and passed on to the successive generations
10. Evolution:
Recombinant DNA technique is of great use in bridging several

missing links in evolution.
This is done by amplifying the DNA (by PCR) from the

archaeological samples of extinct animals
QUESTIONS
1. Describe the steps in preparation of recombinant DNA
2. Applications of recombinant DNA technology
3. Vectors required for recombinant DNA technique
BLOTTING TECHNIQUES
1. The analytical techniques used for the identification of a specific

DNA, RNA or a protein from thousands of each are collectively
referred to as blotting techniques
2. Blotting techniques are:

a) Southern blot: Detection of DNA
b) Northern blot: Detection of RNA
c) Dot blot: Modification of Southern and Northern blot
d) Western blot: Detection of protein
e) Eastern blot: Detection of specific post-translational modifications
of proteins
f) Far eastern blot: Detection of lipid linked oligosaccharides
SOUTHERN BLOT TECHNIQUE
This technique involves the following steps:
1. Extraction of DNA from the cells
2. Splicing the DNA into fragments by restriction endonucleases
3. Separation of the fragments of different sizes by agarose gel

electrophoresis
4. Transfer of DNA fragments from agarose gel to a sheet of

nitrocellulose paper by blotting
5. The radioactively labeled DNA probes are now added to the

nitrocellulose paper
6. Labeled DNA hybrid complexes are formed which can be
identified on exposure to X-ray film; this is called
autoradiography
APPLICATIONS
1. It is used for identifying mutant genes such as sickle cell

anemia, cystic fibrosis, Duchenne muscular dystrophy &
phenylketonuria
2. Presence of viral DNA (hepatitis virus B and C) can be

identified by this method
3. It is used to detect minute quantities of DNA, which is

useful for forensic purpose (e.g. determining paternity,
identifying thieves and rapists)
4. It is used for determination of RFLP associated with a

pathological condition
5. It is used for the confirmation of DNA cloning results
SOUTHERN BLOTTING
DNA MOLECULE
RESTRICTION ENDONUCLEASE
DNA FRAGMENTS
ELECTROPHORESIS
AGAROSE GEL
BLOTTING
NITROCELLULOSE PAPER
HYBRIDIZATION WITH DNA PROBE
RADIOAUTOGRAPH WITH HYBRID DNA’s
NORTHERN BLOT
1. Extraction of RNA from the cells
2. Separation of the fragments of different sizes by agarose gel

electrophoresis
3. Transfer of RNA fragments from agarose gel to a sheet of

diazo-benzyloxy-methyl paper (DBM paper) or
4. The radioactively labeled cDNA probes (complementary

DNA) are now added to the DBM paper
5. The hybrids formed between cDNA and RNA can be
autoradiography
APPLICATIONS
1. Northern blotting provides insight into the function of a gene
2. It is used to observe a particular gene’s expression pattern

between organs, developmental stages, pathogen infection
and over the course of treatment
3. It is used to show over-expression of oncogenes and down

regulation of tumour suppressor genes in cancerous cells
when compared to normal cells
4. It is used to size and quantitate specific RNA molecules
NORTHERN BLOTTING
RNA MOLECULE
RNA FRAGMENTS
ELECTROPHORESIS
AGAROSE GEL
BLOTTING
DBM PAPER
HYBRIDIZATION WITH cDNA PROBE
RADIOAUTOGRAPH
DOT BLOT
1. Dot blotting is a modification of Southern & Northern blotting

techniques
2. In this approach the nucleic acids (DNA or RNA) are directly

spotted onto the filters and not subjected to electrophoresis
3. The hybridization procedure is the same as in the original

blotting techniques
APPLICATION
Dot blotting technique is particularly useful in obtaining
quantitative data for the evaluation of gene expression
WESTERN BLOT
1. Extraction of protein from the cells
2. Separation of the fragments of different sizes by

polyacrylamide gel electrophoresis
3. Transfer of protein fragments from gel to a sheet of

4. The radioactively labeled antibody probes are now added

to the nitrocellulose paper
5. The hybrids formed between protein and antibody can be

autoradiography
APPLICATIONS
1. This is a technique for the identification of a specific protein

in a tissue, reflecting the expression of a particular gene
2. Diagnosis of HIV:
In Western blotting, antibodies against different components

of the virus are analyzed, so it is considered to be the
confirmatory test for diagnosis of HIV
3. Diagnosis of Lyme disease (an infectious disease cause by

the Borrelia bacterium which is spread by ticks)
4. Diagnosis of hepatitis B infection
5. Diagnosis of Creutzfeldt-Jakob disease (prion disease)
6. Western blotting test is used in the analysis of biomarkers

such as hormones, growth factors and cytokines
WESTERN BLOTTING
PROTEIN
PROTEIN FRAGMENTS
ELECTROPHORESIS
POLYACRYLAMIDE GEL
BLOTTING
NITROCELLULOSE PAPER
HYBRIDIZATION WITH ANTIBODY PROBE
RADIOAUTOGRAPH
EASTERN BLOT
1. The eastern blot is used for detection of specific post-

translational modifications of proteins
2. Proteins are separated by gel electrophoresis before being

transferred to a blotting matrix
3. Post-translational modifications are detected by specific

antibodies
FAR EASTERN BLOT
1. The far eastern blot is for detection of lipid linked

oligosaccharides
2. High performance thin layer chromatography is first used

to separate the lipids by physical & chemical characteristics,
then transferred to a blotting matrix
3. The oligosaccharides are detected by specific antibodies
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
1. The human genome is heterogeneous; containing slight variations

in base sequences (usually every hundred base pairs) that differ
from individual to individual but may not generally affect
structure and function
2. These differences in DNA structure are known as restriction

fragment length polymorphisms (RFLPs)
3. RFLPs can be examined by cleaving DNA into fragments by
restriction endonucleases
APPLICATIONS
1. RFLPs in the diagnosis of diseases:
a) When the RFLP is within or close to the locus of the gene

that causes a particular disease, it is possible to trace the
defective gene by analysis of RFLP in the DNA
b) RFLPs can detect single gene based diseases with 95%

accuracy
2. Genetic defects:
RFLPs can be used to detect genetic defects in foetal tissue e.g.
a) Sickle cell anemia (chromosome 11)
b) Huntington’s disease (chromosome 4)
c) Cystic fibrosis (chromosome 7)
d) Alzheimer’s disease (chromosome 21)
3. Forensic medicine:
In forensic medicine RFLPs serve as DNA fingerprints for the

identification of criminals or in settling the disputes of
parenthood (identification of the father)
4. Tumours:
a) RFLPs are used to distinguish hyperplasia (increased production

& growth of normal cells) from neoplasia (growth of tumour cells)
b) An identical DNA marker in all tumour cells would indicate a
monoclonal origin
VNTR
1. Variable number of tandem repeats (VNTR) are short sequences

of DNA located at scattered sites
2. VNTR are present in chromosomes
3. The number of these repeat units varies from person to person,

but is unique for a particular person; therefore, it serves as a
molecular fingerprint
4. VNTR is also known as DNA profile
5. Probability of similarity between two persons is only 1 in

3x1010 persons
6. DNA probes have been developed, so they hybridize to a

number of these loci to produce individual specific fingerprints
7. The major drawback of VNTR is that they are not evenly

distributed throughout the genome
8. VNTR tend to be localized in the telomeric regions, at the

end of the chromosomes
APPLICATIONS
1. Forensic medicine:
a) VNTR is used to pinpoint the culprit of the crime, and also

disputes of parenthood
b) DNA can be isolated from stains on clothing made of blood

or semen stained several years before
c) Sperm nucleic acid can also be separated from vaginal swabs

of rape victims
2. Genetic diseases:
a) VNTR are also useful for the detection of certain genetic

diseases e.g. Huntington’s chorea and fragile X syndrome
b) In Huntington’s chorea, the VNTR exceed 40 repeat units
DNA FOOTPRINTING
1. DNAs which are protein bound are resistant to digestion by

DNAase enzyme
2. When a sequencing reaction is performed using such DNA,

a protected area, representing the ‘footprint’ of the bound
protein, will be detected
3. This is known as DNA footprinting
CHROMOSOME WALKING
1. Chromosome walking is a method to isolate and clone target

DNA from a long segment of DNA
2. In chromosome walking, a fragment representing one end of

a long piece of DNA is used to isolate another fragment that
overlaps, but extends the first
3. This process is continued till the target DNA is isolated
4. Cystic fibrosis gene was the first to be isolated solely by

chromosome walking
DNA LIBRARIES
1. The collection of genetic fragments constitutes DNA libraries
2. There are 2 types of DNA libraries – genomic library and

complementary DNA library
3. A genomic library is prepared by the partial digestion of total
DNA of a genome by the use of restriction endonuclease
4. The genomic library of man has one million DNA fragments
5. The complementary (cDNA) library contains only copies of

DNA sequences that are present in the mRNA molecules
6. It is extremely difficult to choose a DNA sequence of interest

from the millions of DNA fragments present in DNA libraries
7. A group of molecules, known as DNA probes are used to search

the libraries for specific genes
8. The probes are usually single stranded pieces of DNA’s

(sometimes RNA’s), labeled with radioisotopes such as P32
9. The probes containing complementary base sequence are used

to identify specific DNA fragments of DNA libraries
10. The southern blot and northern blot techniques respectively,

identify DNA and RNA by use of probes
11. DNA probes are now readily available for many genetic
disorders e.g. cystic fibrosis, Duchenne muscular dystrophy
and Tay Sach’s disease
QUESTIONS
1. Blotting techniques
2. Southern blotting
3. Northern blotting
4. Western blotting
5. RFLP
6. VNTR
7. DNA footprinting
8. Chromosome walking
9. DNA libraries
POLYMERASE CHAIN REACTION (PCR)
DEFINITION
1. PCR is a test tube method for amplifying a selected DNA

fragment
2. PCR permits the synthesis of thousands of copies of specific
DNA sequence in a few hours
3. This method was invented in 1984 by Kary Mullis, for which

he won the Nobel Prize in 1993
FLANKING SEQUENCES
1. For PCR technique, it is essential to know the nucleotide

sequence of short segments (25 to 30 nucleotides), known
as flanking sequences, at each end of target DNA
2. Two oligonucleotides complementary to the flanking

sequences of each DNA strand are first synthesized
3. These oligonucleotides acting as primers are responsible

for the specificity of the target DNA
STEPS OF PCR
1. The double stranded target DNA is heated to separate into single

strands
2. This is called denaturation of DNA

3. The single DNA strands are cooled and allowed to anneal with
the two primers (one for each strand) complementary to the
flanking sequences
4. On the addition of the enzyme DNA polymerase and the

substrates deoxy ribonucleoside triphosphates, the synthesis
of new DNA strands occurs
5. These strands are complementary to the target DNA
6. As the first cycle of DNA replication is complete, the double

stranded DNA’s are denatured by heating
7. Each strand is allowed to bind to the complementary primers
8. The cycle of DNA amplification is repeated again and again
9. 25 cycles of PCR result in the amplification of target DNA by

a million-fold with high specificity
10. A heat stable DNA polymerase, known as Taq polymerase,

which is obtained from the bacterium Thermus Aquaticus is
used in PCR
11. Taq polymerase is not denatured by heat upto 80 degrees

celsius
POLYMERASE CHAIN REACTION

ADVANTAGES OF PCR
1. PCR technique is very fast and the target DNA can be amplified
a million times in 25 cycles
2. PCR is extremely specific in the amplification of target DNA

molecule
3. PCR is highly sensitive and can detect the presence of even a
single molecule of DNA
APPLICATIONS OF PCR
1. Rapid diagnosis of retroviral infections:
PCR is used for the diagnosis and monitoring of retroviral

infections e.g. HIV infection
2. Prenatal diagnosis of inherited diseases:
PCR is employed in the prenatal diagnosis of inherited

diseases by using chorionic villus samples or cells from
amniocentesis.
Thus, diseases like sickle cell anemia, beta thalassemia and

phenylketonuria can be detected by PCR in these samples.
3. PCR in forensic medicine:
A single molecule of DNA from any source (blood stains,

hair or semen) of an individual is adequate for amplification
by PCR.
Thus, PCR is very important for identification of criminals.
4. PCR is used to settle the disputes of parenthood

(identification of the father)
5. PCR in comparative studies of genomes:
As a technique which can amplify even minute quantities of

DNA from any source (hair, mummified tissue, bone or
fossilized material), PCR is very important in the study of
evolutionary biology, paleontology and archaeology
6. Diagnosis of bacterial infections:
PCR is used for the diagnosis of bacterial infections e.g.

tuberculosis
7. Diagnosis of cancers:
a) Cervical cancer caused by HPV can be detected by PCR
b) Follicular lymphoma caused due to chromosomal

translocation can be detected by PCR
8. PCR in sex determination of embryos:
a) Sex of human embryos fertilized in vitro can be determined

by PCR
b) PCR is also useful to detect sex linked disorders in fertilized

embryos
9. PCR in DNA sequencing:
As the PCR technique is much simpler and quicker to amplify

DNA, it is conveniently used for DNA sequencing
TYPES OF PCR
1. Reverse transcriptase PCR (RT-PCR):
a) It is the method used to amplify, isolate and identify a known

sequence from a cell or tissue RNA library
b) The PCR is preceded by reverse transcription (to convert the
RNA to cDNA)
c) The enzyme used is Thermus thermophiles polymerase

(Tth polymerase)
d) Presence of HIV RNA in blood can be detected by this

method as early as 4 weeks after infection
2. Real time PCR:
a) By this method, quantitation of the viral load in a sample

can be calculated e.g. viral load in HIV or HBV
b) So, the treatment modalities can be planned and the

response to treatment can be assessed
3. Multiplex PCR:
By targeting multiple genes at once, additional information

may be elicited from a single test run that otherwise would
require several times the reagents and time to perform
QUESTIONS
1. Describe the technique of PCR
2. Advantages of PCR
2. Applications of PCR
3. Types of PCR
CLONING
DEFINITION OF CLONING
1. The term cloning has two broad meanings
2. When a gene of a higher organism is introduced into a bacterial

DNA, it is called cloning of the gene or bacterial cloning
3. When a cell from an animal is grown to an exact duplicate of

that animal it is called cloning of an animal or somatic cloning
4. It made big news when Ian Wilmut and Keith Campbell of

Scotland cloned a sheep named Dolly in July 1996
5. Dolly died naturally in 2002, not due to any complications of

cloning
6. Today the sheep, tomorrow it could by the shepherd, so this

raised a number of moral, ethical and legal issues
TECHNIQUE
1. Dolly, the first ever mammal clone, was developed by Wilmut

and Campbell in 1996
2. It is a sheep (female lamb) with a mother and no father

3. The technique primarily involves nuclear transfer and the
phenomenon of totipotency
4. The character of a cell to develop into different cells, tissues,

organs and finally an organism is referred to as totipotency or
pluripotency
5. Totipotency is the basic character of embryonic cells
6. As the embryo develops, the cells specialize to finally give

the whole organism
7. As such the cells of an adult lack totipotency
8. Totipotency was induced into adult cells for developing Dolly
9. The mammary gland cells from a donor ewe were isolated
10. They were subjected to total nutrient deprivation (starvation)

for five days
11. By this process, the mammary cells abandon their normal

growth cycle, enter a dormant stage and regain totipotency
character
12. An ovum (egg cell) was taken from another ewe, and its
nucleus was removed to form an enucleated ovum
13. The dormant mammary gland cell and the enucleated ovum
were fused by pulse electricity
14. The mammary cell cover membrane was broken, allowing the
ovum to envelope the nucleus
15. The fused cell, as it had gained totipotency, multiplied and
developed into an embryo
16. This embryo was then implanted into another ewe which
served as the surrogate (foster) mother
17. Five months later Dolly was born
CLONING
MAMMARY GLAND CELL OVUM WITH NUCLEUS
FIVE DAYS NUTRIENTS

DEPRIVATION
DORMANT TOTIPOTENT CELL ENUCLEATED OVUM
FUSE AND ACTIVATE
FUSED CELL
(MAMMARY CELL NUCLEUS WITH OVUM ENVELOPE)
IN VITRO
EMBRYO CULTURE
EMBRYO
IMPLANT
FOSTER MOTHER
DOLLY
APPLICATIONS OF CLONING
1. Animals with genetically desirable traits could be bred more

efficiently e.g. cows yielding more milk
2. By November 1998, the first goats were born, who were
genetically engineered to produce milk containing anti-thrombin
III
3. Any human protein could be introduced into the makeup of goat

or cow and the desired protein can be obtained through milk
4. Eggs have been genetically manipulated to produce interferon

and insulin in egg white
5. Cloning is successfully employed in agriculture to propagate

plants such as rubber, banana and orchids
6. If a good yielding rubber is available, it is cloned, so that

thousands of progenies of the same quality could be produced
within a short time
7. Species threatening to become extinct could be reproduced

easily
8. In the movie “Jurassic Park” scientists produced dinosaurs;

it is quite improbable in actual life, but not impossible
DISADVANTAGES OF CLONING
1. Cloning will never replace selective breeding
2. Cloning halts any further progress

3. Cloning produces animals/plants with the same characteristics;
new characteristics cannot be developed
4. The cloned animal and parent are not exactly identical because:
a) DNA in adult cell differs from the DNA in foetal cell by the
accumulated damages of a lifetime
b) Any animal is not just a product of its genes, but also of its
environment, both in utero and after birth; this is especially so
when higher organisms are concerned
TRANSGENESIS
1. Transgenesis refers to the phenomenon of introduction of

exogenous DNA into the genome to create and maintain
a stable heritable character
2. The foreign DNA that is introduced is called transgene
3. The animal whose genome is altered by adding one or more

transgenes is said to be transgenic
4. The transgenes behave like other genes present in the animals’

genome and are passed on to the offspring
5. Thus, transgenic animals are genetically engineered or

genetically modified with a new heritable character
6. Transgenesis is a powerful tool for studying the gene

expression and developmental processes in higher organisms
besides the improvement in their genetic characteristics
7. Transgenic animals serve as good models for understanding

human diseases
8. Several proteins introduced by transgenic animals are

important for medical and pharmaceutical applications
9. Thus, transgenic farm animals are a part of the lucrative

biotechnology industry with great benefits to mankind
HUMAN GENOME PROJECT
INTRODUCTION
1. The US Department of Energy and National Institute of Health

started this project in 1990
2. James Watson was the first head of this project and Francis
Collins succeeded him
3. The project included scientists from 16 countries
4. In 1997 Celera genomics headed by Craig Venter independently

embarked on a similar project
5. The project was to decode and sequence the entire human DNA
6. The DNA sequences were identified by shot-gun sequencing

method, which involved breaking the DNA into small pieces
and sequencing each of them
7. The date 26th June 2000 will be remembered as one of the most
important dates in the history of mankind because on this day
Francis Collins and Craig Venter jointly announced the
working draft of the human genome sequence
8. The HGP is one of the greatest achievements of humanity
9. The detailed results were later published in February 2001 in

Nature and Science
10. The draft represents about 90% of the entire human genome;
the remaining 10% of the genome sequences are at the very
ends of the chromosomes (telomeres)
INSIGHTS LEARNT FROM HGP
1. The human genome contains 3164 million nucleotide bases

2. The average gene consists of 3000 bases but the sizes vary greatly
and the largest known human gene is for dystrophin (2.4 million
bases)
3. The total number of genes in the body is 20,500
4. The order of 99% of nucleotide bases is the same in all humans
5. Less than 2% of genome encodes for the production of proteins
6. Repetitive sequences that do not code for proteins (junk DNA)

make up 50% of human genome
7. The human genomes gene rich urban centers are predominantly

composed of the DNA building blocks G and C and the gene
poor deserts are rich in the building blocks A and T
8. Genes appear to be concentrated in random areas along the

genome, with vast expanses of non-coding DNA (junk DNA)
in between
9. Chromosome 1 has the most genes (2968) and Y chromosome

has the lowest (231)
APPLICATIONS OF HGP
1. The draft sequence is already having an impact on finding genes

associated with diseases
2. Over 30 genes have been pin-pointed and associated with breast
cancer, muscle diseases, deafness and blindness
3. Finding the DNA sequences underlying common diseases such

as cardiovascular diseases, diabetes mellitus, arthritis and cancer
is being aided by the human gene variation maps generated by
HGP
4. This also provides focused targets for the development of

effective new therapies
5. With whole genome sequences and new technologies,

researchers can approach questions systematically and on a
greater scale
6. Researchers can study all the genes in a genome or all the

transcripts in a particular tissue/organ/tumour
7. Researchers can also study how thousands of genes work

together in inter-connected networks to orchestrate the
chemistry of life
SELECTED LIST OF GENES ALONG WITH THEIR

RESPECTIVE CHROMOSOMES
1. Uroporphyrinogen decarboxylase - 1
2. Ornithine decarboxylase - 2
3. Transferrin - 3
4. Fibrinogen - 4
5. HMG CoA reductase - 5
6. Major histocompatibility locus (MHC) - 6
7. Leptin - 7
8. Carbonic anhydrase - 8
9. Interferon - 9
10. Uroporphyrinogen III synthase - 10
11. Parathyroid hormone - 11
12. Glyceraldehyde 3 phosphate dehydrogenase - 12
13. Breast cancer (BRCA2) - 13
14. Alpha-1 anti trypsin - 14
15. Cytochrome P450 - 15
16. Alpha chain of haemoglobin - 16
17. Growth hormone - 17
18. Pre-albumin - 18
19. Beta chain of HCG - 19
20. Adenosine deaminase - 20
21. Superoxide dismutase - 21
22. Lambda chain of immunoglobulin - 22
23. Glucose 6 phosphate dehydrogenase - X
24. Sex determining gene – Y
QUESTIONS
1. Describe the technique of cloning. Add a note on the

applications of cloning.
2. Advantages and disadvantages of cloning
3. Transgenesis
4. Human genome project
CYTOGENETIC TECHNIQUES
INTRODUCTION
1. The word chromosome (coloured body) is a combination of two
Greek words, ‘chroma’ means colour and ‘somes’ means body
2. Chromosomes have 2 arms:
a) p arm is the short arm, p stands for ‘petite’
b) q arm is the long arm, q is the letter next to p
SAMPLES FOR CHROMOSOME ANALYSIS
1. Prenatal (fetal) chromosome
2. Amniocytes by amniocentesis
3. Chorionic villi by chorionic villus sampling – allows early

detection of anomalies < 10 weeks
4. Fetal blood by percutaneous umbilical cord sampling

(PUBS) in the late second trimester
CLASSIFICATION OF CYTOGENETIC TECHNIQUES
1. Chromosome banding
2. FISH
3. Microarrays
4. Array based comparative genomic hybridization (CGH)
5. Single nucleotide polymorphism (SNP) array
CHROMOSOME BANDING
1. Cytogenetic analysis is most commonly carried out on cells in
mitosis
2. Halting mitosis in metaphase is essential, because chromosomes

are at their most condensed state during this stage of mitosis
3. The banding pattern of a metaphase chromosome is easily

recognizable and is ideal for karyotyping
4. Stains used are Giemsa, Acridine orange & Quinacrine mustard
5. Important banding techniques are G banding, R banding, C

banding and Q banding
6. Disadvantages of conventional banding techniques:
a) Deletions smaller than several million base pairs are not

routinely detectable by standard G banding techniques
b) Chromosomal abnormalities with indistinct or novel banding

can be difficult or impossible to interpret
c) To carry out cytogenetic analysis, cells must be dividing,

which is not always possible to obtain (e.g. in autopsy or
tumour material that has already been fixed)
FLUORESCENT IN SITU HYBRIDIZATION (FISH)
INTRODUCTION
1. FISH is done for detection of specific genetic information in

a morphologically intact tissue, cell or chromosome using
fluorescent probes
2. To metaphase spread of chromosomes on a glass slide,

fluorescent probe is added
3. Fluorescence microscopy can be used to find out where the

fluorescent probe is bound to the chromosomes
ADVANTAGES OF FISH
1. FISH permits determination of the number and location of
specific DNA sequences in human cells
2. FISH can be performed on metaphase chromosomes, as with

G-banding, but can also be performed on cells not actively
progressing through mitosis
3. FISH performed on non-dividing cells is referred to as

interphase FISH or nuclear FISH
DISADVANTAGES OF FISH
1. FISH requires a pre-selection of an informative molecular

probe prior to analysis
2. So, a prior knowledge of the anomaly is needed
USES OF FISH
1. Detection of numeric abnormalities of chromosomes

(aneuploidy)
2. The demonstration of subtle microdeletions
3. Detection of complex translocations not detectable by

routine karyotyping
4. For analysis of gene amplifications, e.g. HER2 / NEU

in breast cancer or N-MYC amplification in neuroblastoma
5. For mapping newly isolated genes of interest to their

chromosomal loci
MICROARRAYS
1. There are three types of microarrays:
a) DNA microarray or DNA chip
b) RNA microarray
c) Protein microarray
2. DNA Microarray:
a) To the DNA chip containing known oligonucleotide sequence,

fluorescently labelled unknown oligonucleotides are added
b) The computerized algorithms can decode the unknown

oligonucleotide by detecting the location of fluorescent
hybridization pattern on the chip
c) This technique is used to analyze a DNA sample for the

presence of gene variations or mutations (genotyping)
3. RNA Microarray:
a) To the known array of oligonucleotide, fluorescently labelled

cDNA prepared from unknown mRNA is added
b) Computerized algorithms can then rapidly decode the cDNA

sequence based on fluorescent hybridization pattern on the chip
c) This technique is used for gene expression studies
4. Protein microarray:
a) Immobilized human antibodies are placed on a glass slide and
fluorescently tagged target protein is added
b) By antigen-antibody reaction the tagged protein is detected
c) This technique is used in the study of proteomics
ARRAY BASED COMPARATIVE GENOMIC

HYBRIDIZATION
INTRODUCTION
1. CGH is a hybridization technique done on a microarray or DNA

chip, hence the name array based
2. Here two genomes are compared, hence the name comparative

genomic hybridization
3. CGH is used for the diagnosis of diseases of unknown etiology

such as cancer, autism and mental retardation
PROCEDURE
1. In array CGH the test DNA and a reference (normal) DNA are
labelled with two different fluorescent dyes Cy5 (red) and
Cy3 (green)
2. The samples are added to a DNA chip spotted with the entire
human genome at regularly spaced intervals
3. If the contributions of both samples are equal for a given

chromosomal region then all samples on the array will
fluoresce yellow (the result of an equal admixture of green
and red dyes)
4. If the test sample shows an excess of DNA at any given

chromosomal region (such as resulting from an amplification),
there will be a corresponding excess of signal from the dye
with which the sample was labelled
5. If the test sample shows deletion, there will be an excess of

the signal used for labelling the reference sample (green)
USES
1. To detect gene amplification
2. To detect gene mutation
3. To detect genetic variation
SNP ARRAY
1. SNPs represent the position in the genome where some

individuals have one nucleotide (e.g. G) while others
have a different nucleotide (e.g. C)
2. It is estimated that the human genome contains at least 3

million SNPs
3. SNP arrays are used to find out SNPs that are distributed
across the genome
4. SNPs are highly useful as DNA markers since there is no need

for gel electrophoresis and this saves a lot of time and labour
5. Uses:
a) Used in genome wide association studies to identify disease

susceptibility genes
b) To identify genomic deletions and duplications
c) To detect regions of the genome that have an excess of

homozygous genotypes & absence of heterozygous genotypes
QUESTIONS
1. Chromosome banding
2. FISH
3. Microarrays
4. Array based comparative genomic hybridization (CGH)
5. Single nucleotide polymorphism (SNP) array
CRISPR
INTRDODUCTION
1. CRISPR (pronounced ‘crisper’) stands for clustered regularly

interspaced short palindromic repeats, which are the hallmark
of a bacterial defense system that forms the basis for CRISPR
-Cas9 genome editing technology
2. In the field of genetic engineering CRISPR-Cas9 can be

programmed to target specific stretches of genetic code and
to edit DNA at precise locations
3. With CRISPR-Cas9, researchers can permanently modify genes

in living cells and organisms and in the future, may make it
possible to correct mutations at precise locations in the human
genome in order to treat genetic diseases
DISCOVERY OF CRISPR
1. CRISPRs were first discovered in archaea (and later in bacteria)

by Francisco Mojica, a scientist at the University of Alicante
in Spain
2. He proposed that CRISPRs serve as part of the bacterial immune

system, defending against invading viruses
3. They consist of repeating sequences of genetic code, interrupted

by ‘spacer’ sequences – remnants of genetic code from past
invaders
4. The system serves as a genetic memory that helps the cell detect
and destroy invaders (bacteriophages) when they return
5. In January 2013, the Zhang lab published the first method to
engineer CRISPR to edit the genome in human cells
CRISPR TECHNIQUE
1. CRISPR ‘spacer’ sequences are transcribed into short RNA

sequences (CRISPR RNAs) capable of guiding the system
to matching sequences of DNA
2. When the target DNA is found, Cas9 – one of the enzymes

produced by the CRISPR system binds to the DNA and cuts
it, shutting the target gene off
3. Using modified versions of Cas9, researchers can activate

gene expression instead of cutting the DNA
4. This technique allows researchers to study the genes function
5. CRISPR-Cas9 can be used to target and modify ‘typos’ in the

three billion letter sequence of the human genome in an effort
to treat genetic diseases
ADVANTAGES OF CRISPR
1. CRISPR-Cas9 is proving to be an efficient and customizable
alternative to other existing genome editing tools
2. Since the CRISPR-Cas9 system itself is capable of cutting DNA

strands, CRSPRs do not need to be paired with separate cleaving
enzymes as other tools do
3. CRISPRs can also easily be matched with tailor made ‘guide’

RNA (gRNA) sequences designed to lead them to their DNA
targets
4. Thousands of such gRNA sequences have already been created

and are available to researchers
5. CRISPR-Cas9 can also be used to target multiple genes

simultaneously, which sets it apart from other gene editing
tools
CRISPR-Cpf1
1. CRISPR-Cpf1 enzyme is smaller than Cas9, making it easier

to deliver to cells
2. CRISPR-Cpf1cuts DNA in a different manner to Cas9, which

helps in precise insertion (allowing researchers to integrate a
piece of DNA more efficiently and accurately)
3. Cpf1 cuts far away from the recognition site, meaning that even
if the targeted gene becomes mutated at the cut site, it can likely
still be re-cut, allowing multiple opportunities for correct
editing to occur
4. Cpf1 system provides new flexibility in choosing target sites
USES OF CRISPR
1. CRISPR gene editing allows scientists to quickly create cell
and animal models, which researchers can use to accelerate
research into diseases such as cancer and mental illness
2. CRISPR-Cas9 in vivo approach (delivery of CRISPR-Cas9
either systemically or into specific target organs in the body)
may be used to treat diseases such as GSD-Ia, Duchenne
muscular dystrophy and cystic fibrosis
3. Gene editing approach aims to treat beta thalassemia and
sickle cell anemia, by increasing fetal haemoglobin
4. CRISPR-Cas9 will drive the next generation of immuno-
oncology cell therapy
5. CRISPR-Cas9 enables regenerative medicine (use of stem
cells to repair or replace tissue or organ function)
6. CRISPR is now being developed as a rapid diagnostic tool
e.g. diagnosis of zika and dengue virus infections
CRISPR
OLD AGE GENETICS
1. The concept is that “genes determine how one ages”
2. Environment determines 70-80% of how we age and genetics

contributes remaining 20-30% of the same
3. Environmental factors are lifestyle habits like diet, exercise,

smoking and alcohol
4. Not only how long one lives, but how healthy one is during
one’s lifetime is determined atleast partially by genes
5. Molecular variations or single nucleotide polymorphisms

(SNPs) in numerous genes are responsible for this
phenomenon
6. Three gene systems determine lifespan:
a) Insulin signaling:
Insulin greatly influences the body’s ability to grow and

utilize energy.
Elderly people can live longer if they consume fewer calories.
b) Antioxidants:
Antioxidants mitigate the dangerous cell killing activities of

free radicals and prolong life
c) DNA repair mechanisms:
DNA repair systems help the body’s fight against harmful

agents in the environment and help to sustain healthy lifespan
7. Specific variations in certain genes are also found to influence
healthy lifespan; some of these genes include telomerase,
inflammatory cytokines and mitochondrial DNA
GENE THERAPY
DEFINITION
Gene therapy is intracellular delivery of genes to generate

a therapeutic effect by correcting an existing abnormality
PROCEDURE
1. Isolate the healthy gene along with the sequence controlling its
expression
2. Incorporate this gene into a carrier or vector as an expression

cassette
3. Finally deliver the vector to the target cells
HOW THE GENES ARE INTRODUCED
1. Ex-vivo strategy
Patient’s cells are cultured in the lab, new genes are infused into
the cells and modified cells are administered back to the patient
2. In situ strategy
The expression cassette is injected to the patient either intra-

venously or directly to the tissue
3. In vivo strategy
The vector is administered directly to the cell e.g. cystic fibrosis

gene to the respiratory tract cells
THE VECTORS
Vectors used for gene delivery are:
1. Retroviruses
2. Adenoviruses
3. Plasmid liposome complex
4. Gene gun
RETROVIRUSES
1. Retroviruses are RNA viruses that replicate through a DNA

intermediate
2. Commonly used one is the Moloney Murine Leukemia Virus

(MMLV)
3. The gag, pol and env genes are deleted from the wild type
retrovirus, rendering it incapable of replication inside the
human body
4. Then the human gene is inserted into the virus
5. This is introduced into a culture containing packaging cells

having gag, pol and env genes
6. These cells provide the necessary proteins to pack the virus

7. The replication-deficient, but infective, retroviral vectors
carrying the human gene, come out of the cultured cells
8. These are introduced into the patients
9. The virus enters into the target cell via specific receptor
10. In the cytoplasm of the human cells, the reverse transcriptase

carried by the vector converts the RNA to pro-viral DNA, which
is integrated into the target cell DNA
11. The normal human gene can now express
12. Advantages
a) Retrovirus is modified and replication deficient; so, the infection

with viral particle is limited to one cycle and is very safe
b) Retroviruses can infect a wide variety of human cells; this strategy

is very suitable for treatment of all diseases produced by single gene
mutations
13. Disadvantages
a) Retrovirus requires dividing cells as the targets and allows only

low titers of virus to be generated
b) Insertional mutagenesis is a theoretical possibility

ADENOVIRUSES
1. Adenoviruses are DNA viruses
2. The virus carrying the human gene reaches the nucleus of target
cells
3. It is not integrated, but remains as epi-chromosomal
4. Advantage
Adenovirus offers high titers and easier ability to infect wide

variety of cells
5. Disadvantage
The expression is usually transient, the useful effect varying

from a few weeks to months only
PLASMID LIPOSOME COMPLEX
1. It is a non-viral vector system
2. Liposomes are artificial lipid bilayers, which could be

incorporated with plasmids carrying the normal human DNA
3. The complexes can enter into the target cells by fusing with
the plasma membrane
4. Cationic liposomes (positively charged) can form complexes

spontaneously with DNA (negatively charged)
5. Advantages
The vector can carry human gene of larger size, do not replicate
and evoke only very weak immune responses
6. Disadvantage
Most of the complexes are destroyed inside the host cell and
so the efficiency of gene transfer is less
GENE GUN
1. Tungsten particles are coated with plasmid DNA and accelerated

by helium pressure discharge
2. This enables particles to penetrate the target tissues
3. Advantage
It is quick and could be used in almost all tissues
4. Disadvantage
Cellular damage and transient gene expression
ACCOMPLISHMENTS
1. Severe combined immunodeficiency:
Adenosine deaminase enzyme on chromosome 13 and 20

transferred into lymphocytes by retrovirus
2. Duchenne muscular dystrophy (DMD):
Dystrophin gene on short arm of X chromosome transferred

by retrovirus
3. Cystic fibrosis (CF):
CFTR gene on chromosome 7 transferred to bronchial epithelium

by adenovirus
4. Familial hypercholesterolemia:
LDL receptor gene on chromosome 19 transferred to hepatocytes

by retrovirus
5. Haemophilia:
A and B genes for factors VIII and IX transferred to fibroblasts

by retrovirus
6. Cancer:
Activation of p53 gene (tumour suppressor gene) by liposome
7. Leber’s hereditary optic neuropathy:
Introducing the gene for the enzyme using an adenoviral vector

directly into the retina
OBSTACLES TO SUCCESS
1. Inconsistent results
2. Lack of ideal vector
3. Lack of targeting ability in non-viral vectors
4. Death of the patient during the course of gene therapy for

ornithine transcarbamoylase deficiency was reported
5. Reactivation of retroviral vector due to illegitimate combination

of the inserted gene leading to leukemia in the patient has posed
a setback in this field
THE FUTURE
1. The potential of gene therapy is enormous
2. It is now theoretically possible to cure all the genetic diseases
3. However it may take several decades to get it available for

common clinical use
STEM CELLS
1. Stem cells are defined as cells with the capacity for self-renewal
and having potential to differentiate into progenitors of different
lineages which ultimately give rise to mature tissues
2. Stem cells have the ability to divide for an indefinite period
3. They can give rise to a variety of specialized cell types
4. This phenomenon is known as developmental plasticity
5. Stem cells can be isolated from embryos, umbilical cord as well

as from adult bone marrow
6. Plasticity is defined as the ability of stem cells from one germinal

layer to give rise to tissues of another germinal layer
7. Plasticity is more for embryonic stem cells
8. Stem cells have the unique capacity to produce unaltered daughter

cells (renewal) and also to generate specialized cells (potency)
9. Stem cells may be capable of producing all type of cells of the

organism (totipotent) or able to generate cells of the three germ
layers (pluripotent)
10. Active research is being done to utilize stem cells in the
treatment of the following diseases:
Stroke, Alzheimer’s disease, Parkinsonism, wound healing,

myocardial infarction, spinal cord injury, diabetes mellitus
and cancer
THREE PARENT IVF BABIES
1. The three parent IVF technique can prevent some inherited

incurable diseases from being passed down the maternal line,
affecting around 1 in 6500 children worldwide
2. However this is seen by some as a step towards creating

designer babies
3. The technique involves mitochondrial donation, which is

known as ‘three parent’ in vitro fertilization, because the
babies would have the DNA from a mother, a father and
from a female donor
4. The process combines the DNA of three persons into one

embryo in order to allow women carrying mitochondrial
disorders (due to defective mtDNA) to have healthy children
5. The babies that result from this technique can only attribute
around 0.1 percent of their DNA to the third party, so ‘parent’
is a bit of a strong word
6. The donor provides only her mitochondria which gives the

new born a hope for life without suffering
BIOINFORMATICS AND GENOMIC RESOURCES
The databases that helps in contemporary molecular,

biochemical, epidemiological & clinical research are:
1. UniProt KB
2. GenBank
3. PDB
4. Tagged SNPs
5. HapMap
6. ENCODE
7. Entrez gene
8. GWAS
UNIPROT KB
1. The UniProt knowledge base, is jointly sponsored by the Swiss

Institute of Bioinformatics and the European Bioinformatics
Institute
2. UniProt KBs stated objective is to provide the scientific

community with a comprehensive, high quality and freely
accessible resource of protein sequence and structural
information
3. It is organized into two sections, Swiss-Prot and TrEMBL

4. Swiss-Prot:
a) Swiss-Prot contains entries whose assigned functions, domain

structure and post-translational modifications have been verified
by manual curation
b) Swiss-Prot contains slightly more than 500,000 entries
5. TrEMBL:
a) TrEMBL, on the other hand, contains empirically determined

and genome derived protein sequences whose potential functions
have been assigned automatically solely on the basis of
computer algorithms
b) TrEMBL includes more than 80 million entries
GENBANK
1. GenBank is the genetic sequence database
2. The goal of GenBank of the National Institute of Health (NIH),

is to collect and store all known biological nucleotide sequences
and their translations in a searchable form
PDB
1. The RCSB Protein Data Base (PDB) is a repository of the

three-dimensional structures of proteins, polynucleotides
and biological macromolecules
2. The PDB presently contains over 95,000 three dimensional

structures for proteins
TAGGED SNPs
1. When sets of SNPs localized to the same chromosome are

inherited together in blocks, the pattern of SNPs in each block
is termed as haplotype
2. TagSNPs, is a subset of SNPs in a given block sufficient to

provide a unique marker for a given haplotype
3. Selected regions are then subject to more detailed study to

identify the specific genetic variations that contribute to a
specific disease or physiologic response
HAPMAP
1. HapMap (Haplotype Map Project) is a comprehensive effort to

identify SNPs associated with common human diseases and
differential responses to pharmaceuticals
2. The long-term goal of the project is to provide earlier & more

accurate diagnosis of potential risk factors that lead to more
effective patient management
ENCODE
1. National Human Genome Research Institute (NHGRI) initiated

the ENCODE (Encyclopedia of DNA Elements) Project
2. ENCODE is a collaborative effort that combines laboratory and

computational approaches to identify every functional element
in the human genome
3. These include mapping sites of DNA methylation and assessing

local histone methylation
ENTREZ GENE
1. Entrez Gene is a data base maintained by the National Center

for Biotechnology Information (NCBI)
2. This provides a variety of information about individual human

genes
3. The information includes the sequences of the genome in and

around the gene, exon-intron boundaries, the sequence of the
mRNAs produced from the gene and any known phenotypes
associated with a mutation of the gene
GWAS
1. In GWAS (Genome Wide Association Studies) large cohorts

of patients with and without a disease are examined across the
entire genome for genetic variations or polymorphisms that
are over-represented in patients with disease
2. This identifies regions of the genome that contain a variant

gene or genes that confer disease susceptibility
3. The casual variant within the region is then provisionally

identified using candidate gene approach
4. By this approach, in the previously identified region, genes

are selected based on how tightly they are associated with the
disease and whether their biologic function seems likely to be
involved in the disease under study
QUESTIONS
1. Gene therapy
2. Stem cells
3. Three parent IVF babies
4. CRISPR and its applications
5. Bioinformatics & genomic resources
6. Old age genetics

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3) INHIBITORS OF DNA REPLICATION

6) REPAIR OF DNA DAMAGE

7) DISORDERS OF DNA DAMAGE

9) POST TRANSCRIPTIONAL MODIFICATIONS

10) INHIBITORS OF TRANSCRIPTION

11) REVERSE TRANSCRIPTION

12) GENETIC CODE

14) INHIBITORS OF TRANSLATION

15) POST TRANSLATIONAL MODIFICATIONS

18) REGULATION OF GENE EXPRESSION IN EUKARYOTES

19) RECOMBINANT DNA TECHNOLOGY

23) DNA FOOTPRINTING

24) CHROMOSOME WALKING

25) DNA LIBRARIES

29) HUMAN GENOME PROJECT

30) CYTOGENETIC TECHNIQUES

32) OLD AGE GENETICS

33) GENE THERAPY

34) STEM CELLS

35) THREE PARENT IVF

36) BIOINFORMATICS AND GENOMIC RESOURCES

1. The biological information flows from DNA to RNA and

2. This is the central dogma of life

3. It is ultimately the DNA that controls every function of the

4. The word genome refers to the total genetic information

5. DNA is the genetic material

6. When the cell divides, the daughter cells receive an identical

7. Replication is a process in which DNA copies itself to produce

8. Replication is carried out with high fidelity

1. The parent DNA has two DNA strands complementary to

2. Both the strands undergo simultaneous replication to produce

4. This type of replication is known as semi-conservative since

DNA replication is divided into the following phases:

2. Leading and lagging strands

5. Removal of RNA primer and nick sealing

1. The initiation of DNA synthesis occurs at a site called origin

2. In case of prokaryotes, there is a single site, whereas in

3. These sites mostly consist of short sequences of A-T base pairs

4. A specific protein called DNA – A (20 to 50 monomers) binds

5. The two complementary strands of DNA separate at the site of

6. Multiple replication bubbles are formed in eukaryotic DNA

1. For the synthesis of a new DNA, a short fragment of RNA

2. This primer is synthesized on the DNA template by a specific

3. A constant synthesis and supply of RNA primers should occur

4. The leading strand has a single RNA primer

5. The replication of DNA occurs in the 5’ to 3’ direction,

6. On one strand, the leading strand, the synthesis of DNA is

7. On the other strand, the lagging strand, the synthesis of DNA

8. Short pieces of DNA (15 to 20 nucleotides) are produced on

1. The separation of the two strands of parent DNA results in

2. Active synthesis of DNA occurs in this region

3. The replication fork moves along the parent DNA as the

4. DNA helicases are enzymes which bind to both the DNA

6. Single strand binding (SSB) proteins protect the single

7. The synthesis of a new DNA strand, catalysed by DNA

8. The presence of all four deoxyribonucleoside triphosphates

9. The incoming deoxyribonucleotides are added one after

10. A molecule of pyrophosphate (PPi) is removed with the

5’ DNA POLYMERASE III

1. The template DNA strand (the parent) determines the base

2. The synthesis of two new DNA strands, takes place

a) One is in a direction towards the replication fork, which