BIOCHEMISTRY

Topic: Introduction to Molecular Medicine

1 Lecture by: Dr. Bravo

MOLECULAR MEDICINE Symmetric Cut with Blunt ends

 It is the application of Molecular Biology, Biotechnology and
Molecular Genetics to the understanding of human health, and
diagnosis and treatment of disease
 It aims to understand how health is maintained and the origins and
mechanisms of human diseases
 Cut exactly at the midline of the palindromic DNA sequences
 The cut produces clean ends = no overhangs  ‘blunt ends’
Example:

RESTRICTION ENDONUCLEASES (RE)

 Restriction endonucleases (RE) are enzymes derived from bacteria
which cleave DNA
 Unlike other endonucleases, restriction endonucleases act on very Asymmetric Cut with Sticky ends
specific DNA sequences called palindromes  Point of cleavage is off-midline
 Produces either a 5’ overhang or 3’ overhang  ‘sticky ends’
Key Points Regarding Restriction Endonucleases:
o Enzymes derived from bacteria Examples:
o Highly specific Asymmetric Cut with 5’ overhang
o Recognize and cleave DNA at specific sequences
o Act on DNA sequences that are palindromic

What is a Palindrome?
 Palindromes are DNA sequences which are the same when read
forward and backward Asymmetric Cut with 3’ overhang
Example: 5’-GATC-3’ – template strand
3’-CTAG-5’ – complementary strand
Explanation of example above:
DNA has a certain polarity. Normally, it should be read from 3’ to
5’ direction. But given above, we read the template strand as
GATC (when read from 5’ to 3’ direction). So if there is a Uses of Restriction Endonucleases
complementary strand, it would be read not as CTAG but still as 1. DNA fingerprinting
GATC which is also that same as the template strand 2. Formation of Recombinant DNA
3. DNA Propagation (Cloning)

1. DNA Fingerprinting

Table above:
These are examples of RE and their specific target sequences. Like for EcoRI,
which comes from the bacteria E. coli, it will only recognize and cut the
sequence GAATTC. So when it is presented with a DNA sequence that has
GAATTC, it will cut it. But in cases where the GAATTC sequence is not
present, it will only ignore it.
Picture Above: Brief explanation of how DNA fingerprinting is done
Whenever RE cut their respective palindromic sequences, there are 3 possible Let’s say that the above is a short DNA sequence with its corresponding RE,
outcomes: cutting the specific sites represented by the purple arrow. Using that RE, the
1. Symmetric cut with Blunt ends DNA sequence can be cut into 4 pieces, the sizes of which depend on the
2. Asymmetric cut with 5’ overhang location of the RE cutting sites. These can be arranged from largest to
3. Asymmetric cut with 3’ overhang smallest using Gel Electrophoresis

5’ or 3’ overhangs are called Sticky ends because they will readily stick or anneal
with their partner by base pairing

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Topic: Introduction to Molecular Medicine
2 Lecture by: Dr. Bravo

Gel Electrophoresis 2. Formation of Recombinant DNA

 It is commonly used to separate and analyze the nucleic acids based
on their size
 It is called such because it uses a gel, which is a porous material
o Small-sized DNA molecules can easily pass through the
pores compared to bigger-sized molecules
o “DNA of the same size travel together”
 Molecular cloning or Recombinant DNA is the process by which a
single gene, or segment of DNA, is isolated and amplified
 It is a molecule made up from the combination of DNA fragments
from two or more different organisms
 This is accomplished by recombining DNA fragments in vitro and then
propagating the DNA fragments of interest

Lecture Explanation:
One possibility with the use of RE, particularly those that produce
‘sticky ends’, we can create recombinant DNA (rDNA). In layman, this
is what they call Genetic Engineering wherein we piece foreign DNA
together

Picture Above: Principle of Gel Electrophoresis

When fragments of nucleic acid are loaded on the gel, an electric field (EF) is
applied
o DNA is negatively charged, so upon application of EF, the
fragments are forced to migrate towards the positive side
o Rate of migration is dependent on size
Small fragments  travel faster and farther
Bigger fragments  travel longer and slower

As seen on the picture above, the small fragments travelled farther, while
the big fragments travelled slower and therefore will not move much away
from the point of application Picture Above: Explanation how Recombinant DNA is formed
Stain is applied to visualize the DNA. A banding pattern unique for this DNA (B) a piece of foreign DNA is cleaved using RE, such as EcoRI, producing DNA
will be produced; thus it’s called a fingerprint fragments with sticky ends
o Banding pattern will not be visible until after stain (e.g. ethidium (A) a plasmid is an extrachromosomal circular DNA found in bacteria. If this
bromide) is applied plasmid has the same RE cutting sites as that of EcoRI, then it can be opened
up by EcoRI. After cutting it will also produce sticky ends
NOTE:
o Distance of bands is determined by the size of DNA fragments (B) Sticky end + (A) Sticky end  Reanneal producing rDNA
produced
o Number of bands is dependent on number of cutting sites of the At this point we only produced 2 copies of it. We can produce multiple copies
RE on the DNA sequence of Recombinant DNA by utilizing the process known as DNA Propagation or
Cloning.
Summary of DNA fingerprinting:
1. Cut DNA sequence with RE to produce fragments Summary of producing Recombinant DNA:
2. Subject to Gel electrophoresis 1. Cleave foreign DNA you wish with an RE, producing sticky ends
a. Apply solution of DNA to gel at the negative side 2. Cleave plasmid with RE producing also sticky ends. Plasmid must
(point of application) have antibiotic resistance gene, RE cutting sites and origin of
b. Apply electric current to force DNA fragments to replication
migrate towards positive end 3. Reanneal the sticky ends of the foreign DNA and plasmid. You
3. Stain with dye to visualize banding patterns made by the DNA now have rDNA which you can reproduce via different DNA
fragments Propagation techniques or cloning
a. Nearest the point of application are bigger fragments
(did not travel much)
b. Farthest from the point of application are the smaller
fragments

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Topic: Introduction to Molecular Medicine
3 Lecture by: Dr. Bravo

3. DNA Propagation (Cloning) Picture on lower left: Schematic diagram of DNA Propagation (Cloning)
Cloning – is the production of large quantities of identical DNA molecules
1. Recombinant DNA (rDNA) production
a. Cell-based DNA propagation o Piece of foreign DNA was cleaved using RE EcoRI which produced
b. Polymerase Chain Reaction (PCR) sticky ends
o Plasmid (vector) was also cleaved using EcoRI producing also
Cell-based DNA propagation sticky ends. Remember that the plasmid should contain:
 In this method of DNA propagation, a vector is required to clone  Antibiotic resistance gene
either cDNAs or copies of genes  RE cutting site
 A vector is where we insert the foreign DNA  Origin of replication
 Classes of vectors: o The sticky ends are reannealed producing rDNA. Next step is to
plasmids, lambda, yeast artificial chromosome (YAC) produce many copies of this rDNA
(We will focus more on plasmids since it is the most basic of all)
End product: rDNA containing the foreign DNA and the plasmid
Plasmid Vectors with antibiotic resistance gene
 Are the most basic of all vectors
2. Transformation (Introduce the rDNA to the host microorganism)
 They serve to carry a piece of foreign DNA into the internal
o Host microorganism: E. coli
environment of a host cell
o E. coli is treated with calcium chloride to render their cell
 These plasmid are autonomously replicating closed circular
membrane permeable to the rDNA
DNA distinct from the genome of the microorganism
 (+) Some E. coli are successful in taking up the rDNA
 In order for a plasmid to be useful as a vector, it should
 (-) Some E. coli refused to take up the rDNA
contain the following DNA sequences:
1. Origin of Replication (OriC) – these allows the
We are interested in the (+) E. coli, so we select these
recombinant plasmid to undergo replication
microorganisms that has been successfully transformed
process by utilizing the host microorganism’s
DNA polymerase
3. Selection (Select the microorganism that has been successfully transformed)
2. RE cuttings sites – allows insertion and ligation
o Transfer into an agar media that contains an antibiotic
of a piece of foreign DNA that is going to be
Purpose of Antibiotic: to destroy bacteria
clones
o Remember that the microorganisms that has been successfully
3. Antibiotic Resistance Gene – allows selection of
transformed contain the antibiotic resistance gene of the
successfully transformed microorganism host
plasmid. So when placed in agar with antibiotic:
 (+) E. coli will survive and flourish
Maximum size of DNA that can be inserted into the plasmid is 10,000
 (-) E. coli will be killed and weeded out
base pairs
End product: colonies of E. coli containing the rDNA
Key Points to Remember Regarding Plasmid:
 Circular DNAs found in bacteria 4. Propagation and purification (Isolate and purify your rDNA from the E. coli)
 Autonomous replication separate from the host genome
(contains OriC)
Summary of Cell-based DNA propagation (Cloning):
 Contains RE cutting site
1. Produce rDNA
 Contains antibiotic resistance gene that allows selection
2. Introduce rDNA into microorganism
 Limitation: Can only clone DNA less than 10,000 base pairs
3. Select microorganism which successfully took up the rDNA by
in length
utilizing the antibiotic resistance gene property
4. Isolate and purify rDNA from microorganism
Steps in Cell-based DNA propagation (Cloning):

What to do with the Propagated Recombinant DNA?

 Establish a DNA library
 Express the Recombinant DNA into a Recombinant Protein
 Re-introduce the Recombinant DNA into another organism
1. Recombinant DNA production
2. Transformation Establish a DNA library
3. Selection
4. Propagation and purification o DNA library is a population of identical vectors that each contains
a different DNA insert
o To construct a DNA library, the target DNA (e.g. human genomic
DNA) is digested with a RE that gives a desired average insert size,
ranging from 100 bp to more than a megabase (for very large
insert sizes, the DNA is typically incompletely cut with a RE). The
cleaved DNA is then mixed with the appropriate vector, cut with
\ the same RE, in the presence of ligase. This creates a large
collection of vectors with different DNA inserts
o Different kinds of libraries are made using insert DNA from
different sources. The simplest are derived from total genomic
DNA, cleaved with a RE, these are called genomic libraries. This
type of library is most useful when generating DNA for
sequencing a genome

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Topic: Introduction to Molecular Medicine
4 Lecture by: Dr. Bravo

o To enrich for encoding sequences in the library, a cDNA library is o Basis for Gene Therapy
created. Instead of starting with genomic DNA, mRNAs are
converted into DNA sequences. The process that allows this is
called reverse transcription and is performed by a special DNA
polymerase (reverse transcriptase) that can make DNA from an
RNA template. From this point on, construction of the library
follows the same strategy as does construction of a genomic
library– the cDNA products and vector are treated with the same
RE, and the resulting fragments are then ligated into the vector

Genomic library vs. cDNA library

Genomic library cDNA library
A collection of all fragments (exons, Only contain DNA molecules that are
introns, junk DNA, promoter site, other synthesized from mRNAs. It is a
regulatory sequences) representing all collection of DNAs representing the
DNA in the chromosomes of a cell expressed gene (expressed exon) in cell
or tissue type 1. Extracted stem cells from the bone marrow of the patient are grown and
cultured in a media to have its non-functional gene replaced with a functional
Express the Recombinant DNA into a Recombinant Protein one
o Recombinant DNA may be expressed by re-insertion into an 2. A viral vector, which now carries a functional gene generated from the
expression vector stem cells, is introduced into a host cell, which propagates and purified
o Expression vector contains a gene which can be transcribed successfully
under controllable conditions 3. The cells now containing the functional gene is re-introduced back to the
o Example: Lac operon bone marrow of the patient. That is, the functional gene replaces the
defective gene of the patient

Polymerase Chain Reaction (PCR)

Lac operon – consists of lac-z gene which is under the control
of promoter
 Lac-z gene codes for lactase (ß-galactosidase)
 When grown in media with lactose, the lac-z gene
is expressed to produce ß-galactosidase
 PCR is an in-vitro enzymatic method of amplifying a target sequence
of DNA up to 600 base pairs (size of DNA fragment that can be
copied)
 In layman’s term, PCR is simply producing copies of DNA, similar to
reproducing a page in a book several times
 It requires a DNA polymerase enzyme called Taq polymerase which
can withstand high temperature because PCR is done near boiling
temperature
 It is done in 30 cycles of 3 steps
 More than 1 billion DNA copies are produced

Key Points to Remember Regarding PCR:

 Method of amplifying a target sequence of DNA up to 600
bp
Lac-z gene can be cleaved off using RE and replaced with a
recombinant DNA (e.g. insulin gene isolated from a human  Requires DNA polymerase enzyme to remain stable at high
being)  place in a host microorganism  grown in media temperature (Taq polymerase)
that contains lactose, the recombinant DNA will be expressed  In-vitro
producing the desired protein  Done in 30 cycles of 3 steps
 >1 Billion DNA copies are produced
How useful is this technology?
In the olden times, when you want insulin, insulin was derived 3 Steps in PCR that is performed in several cycles:
from cows and pigs, where the pancreas of dead animals are 1. Denaturation of the DNA
gathered and insulin is isolated. Nowadays, insulin genes are 2. Primer Annealing
isolated from human samples (not from cadavers) and is 3. Primer Extension by Taq polymerase
expressed via microorganisms to produce human insulin
1. Denaturation of the DNA
Re-introduce the Recombinant DNA into another organism
o Example is green fluorescent protein gene from jellyfish is
isolated, cloned and introduced into the genome of mice during
the embryonic stage. When the fully-grown mice is exposed to UV Undergo 92-94oC
light, they turn green

o Objective is to separate the dsDNA into separate single strands

via denaturation through application of heat
o When we increase the temperature (around 92-94oC) the two
DNA strands will be separated. Hydrogen bonds between the two
strands of DNA are broken

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Topic: Introduction to Molecular Medicine
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2. Primer Annealing Tabulation of Selected Monogenic Disorder Which May Be Detected Using PCR
Diseases Gene Affected
Primer
Adenosine Deaminase Deficiency Adenosine deaminase
Lesch-Nyhan Syndrome HGPRT
Alpha1-Antitrypsin Deficiency Alpha1-Antitrypsin
Fabry’s Disease Alpha-galactosidase
Cystic Fibrosis Transmembrane
Cystic Fibrosis
Conductance Protein (CFTR)
Gaucher’s Disease Glucocerebroside
Sandhoff-Jatzkewitz Disease Hexosaminidase A and B
Tay-Sach’s Disease Hexosaminidase A
o Synthetic primers (preformed and are not produced by primase) Familial Hypercholesterolemia LDL receptor
bind to the regions that you wish to amplify G6PD Deficiency Glucose-6-Phosphate dehydrogenase
o Temperature is lowered to about 40-60oC Maple Syrup urine Disease Alpha-keto acid decarboxylase
Phenylketonuria Phenylalanine Hydroxylase
3. Primer Extension by Taq Polymerase OTC Deficiency Ornithine Transcarbamoylase
Retinoblastoma (Rb) Rb gene product
Point mutation in Beta globin gene resulting
Sickle Cell anemia in improper folding of protein
Mutation in Beta-globin gene that results in
Beta-Thalassemia loss of synthesis of protein
Fragile X pre-mutation FMR1 gene
Huntington’s Disease Huntingtin gene

PCR in the Diagnosis of Diseases:

 Expansion mutations of trinucleotide repeats such as in Huntington’s
disease can be diagnosed via PCR
o Your primers will be extended by the Taq polymerase synthesizing  Huntington’s Disease involves CAG expansion mutations of the
your desired DNA sequence
huntingtin gene just like what happens in Fragile X Syndrome
o Temperature is carried at 70oC
Facts about Huntington’s disease
This cycle is repeated a number of times. Growth of DNA is exponential: o Expansion mutation of Huntingtin gene
Cycle 2 – 4 copies, Cycle 3 – 8 copies, etc. o Autosomal dominant
o Take note that the size of fragments produced are not always of o Affects basal nuclei
the same size or the correct size o Characterized by chorea, athethosis, etc.
o There are usually 30 cycles in performing PCR, taking about 4
hours
o In the video: At the end of cycle 3, we are now producing
fragments of the correct size. At the end of cycle 5, there are
already more of the DNA of the correct size
o At the end of cycle 30, 1 billion copies are produced

Summary of Polymerase Chain Reaction (PCR):

1. Denaturation of DNA. Separate dsDNA into ssDNAs via
denaturation (temperature: 92-94oC)
2. Primer Annealing. Introduce synthetic preformed primase
(temperature: 40-60oC)
3. Primer Extension. Extend primers via synthetic heat resistant
DNA polymerase which is Taq polymerase. (temperature: 70oC)

PCR medical applications:

 Investigate a potentially defective DNA sequence
 Diagnosis of diseases in which a specific mutational site is in question
(monogenic disorders)
 Primers spanning the site are used
 Amplified DNA can be de sequenced or compared with normal
reference control

Picture Above: Huntingtin gene expansion can be detected by PCR

On the left is the normal huntingtin gene, with normal CAG repeats. While
on the right is the mutated huntingtin gene, with expanded CAG repeats. We
can amplify these DNA sequences using PCR thus producing 1 billion copies
of the Normal and Expanded DNA sequences. Since they have different sizes
(Expanded CAG repeats being bigger), we can differentiate both through gel
electrophoresis. It can be noted that the normal gene travelled faster while
the expanded gene travelled less and is found near the point of application

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Case: Jay, age 50, developed symptoms of Huntington’s disease at age 40. His
wife, Marie, appears normal. Will their offsprings, Jessica, age 10, and Martin,
age 7, develop Huntington’s as well?
 Most of the time, DNA polymerase uses normal nucleotides (dNTPs)
and DNA molecules grow normally
 Occasionally, DNA polymerase uses a dideoxynucleotide (ddNTPs),
which terminates further growth in that molecule
 Random insertion of ddNTP leaves (optimally) at least a few chains
terminated at every occurrence

How is Sanger’s Method done:

To illustrate this, for example we have 4 test tubes (for 4
different reactions). Each test tube contains DNA
templates, primers, DNA polymerase, dNTPs and ddNTPs.
In the figure on the right, the test tube has a chain
terminator for A which is ddATP

Let’s focus first on this one. So what happens in the test

tube is that:
1. DNA polymerase synthesizes the DNA chain using the ssDNA as
template

Case Analysis: Based on Dr. Bravo’s lecture

Marie has 2 copies of the huntingtin gene, one slightly longer than the other,
but nonetheless, both copies are normal
Jay has 2 copies of huntingtin gene, one is normal and the other one has 2. When the chain terminator is incorporated, further growth is
expansion mutation. The mutated gene is the one responsible for the stopped and we produce a fragment that ends in an A
disease. There is a 50% chance that the offsprings will get the mutant variant
of the huntingtin gene (since it is autosomal dominant)
Jessica has 2 normal copies of the huntingtin gene, one coming from the
mother and the other from the father 3. There are however instances wherein the “correct A” is incorporated
Martin has 1 normal copy of the huntingtin gene coming from the mother and the DNA grows normally until another chain terminator is
and the other one is a mutated gene coming from his father. So Martin will incorporated. We again produce another fragment (which is of
definitely have Huntington’s disease when he reaches 40 yrs. old different length) that also ends in A

DNA SEQUENCING
Sanger’s method of DNA sequencing
 It is based on DNA polymerase reaction
 It is also known as Chain Termination Method
 It requires the following:
o DNA template (ssDNA)
o Primer
o DNA polymerase 4. In the end, we have several DNA fragments of different length in one
o dNTPs (substrates) test tube and you are sure that all of them ends in A
o dideoxynucleotide (chain terminator) 5. These DNA fragments can be arranged according to size using gel
 Sanger’s method run four separate reactions electrophoresis. Here we can only partially deduce the sequence of
 Each reaction mixture contains DNA template, primer, DNA the DNA since all of it ends in A
polymerase
 Each reaction mixture contains substrates (dATP, dGTP, dCTP, dTTP)
 Each reaction also contains a small amount of one
dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP

What is the difference between substrate dNTP and chain terminator ddNTP?

Question: How do we determine the full sequence?

We can determine the full sequence by using the other 3 test tubes that
has the chain terminators for the nucleotides G T and C

 Deoxynucleotide (usual substrate) – 3’-hydroxy is an important

attachment site for phosphodiester bond formation Continued next page……
 Dideoxynucleotide does not have 3’-hydroxy such that if it is the
one incorporated into the chain, strand synthesis will stop there
therefore polymerization process is terminated

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Topic: Introduction to Molecular Medicine
7 Lecture by: Dr. Bravo

For this lecture we will only discuss Southern Blot since it is the original
blotting technique and is the basis of the other three

Southern Blot
 Formerly called a DNA blot
 Developed by Dr. Edward Southern of Edinburgh University in 1970
 It is the analysis of DNA utilizing complementary radioactive DNA
probe
 It is a method to isolate and identify a DNA sequence of interest in a
complex mixture

How is Southern blot done?

We wish to identify the DNA sequence in the green fragment
1. Cut the DNA into manageable fragments using Restriction
Picture above: Whole process of DNA sequencing Endonuclease
1. The requirements are placed in 4 separate test tubes
a) Each test tube contains DNA template, primers, DNA polymerase
and regular substrates (dNTPs)
b) In one test tube, there is a chain terminator for A, another one
for G, another one for T and another one for C
2. Arrange the DNA fragments according to size using gel
2. Let’s go to test tube with chain terminator for A
electrophoresis
a) DNA grows. If it is the Normal A that is inserted, it continues to
grow, but if it is the chain terminator that is inserted, it stops
b) This happens to several templates. So in the end you have DNA
fragments with varying size, all of them ending in A
c) The same thing also happens with Test tube G, Test tube T and
Test tube C

3. You can arrange the fragments according to size using gel electrophoresis

Highlighted here is the one we’re interested in but in reality we do

not know that it is there. How do we identify it? This is where a
probe comes in

3. Transfer the DNA from the gel into a piece of nitrocellulose by simply
blotting

Here the smallest fragment, you know that it ends with G, so the first
nucleotide is probably a G. The next is T. The third smallest is an A and so on.
Thus, the sequence is revealed

Nowadays, sequencing is already an automated process which is still based

on the Sanger’s Method. Instead of reading it, a laser beam scans it and the 4. That nitrocellulose DNA blot is then heat-baked
information is sent on a computer which then prints out the sequence

BLOTTING TECHNIQUES
 Blotting techniques are methods to separate molecules from
complex mixture and identify molecule of interest using probe
 There are four blotting techniques:
1. Southern Blot
2. Northern Blot
3. Western Blot
4. Southwestern Blot

Tabulation of the Four Traditional Blotting Techniques

Blotting Technique Analysis of Gel for separation Probe
Southern DNA Agarose Radioactive DNA The purpose of baking is that it causes the DNA to attach
Northern RNA Agarose Radioactive DNA permanently on the nitrocellulose paper. It also causes the DNA
Western Protein SDS polyacrylamide Radioactive antibody to denature (dsDNA split into ssDNA) as shown in the schematic
Southwestern DNA Agarose Radioactive antibody side view. The denaturation of DNA is useful later for it to accept
the probe

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How is Southern Blot done continued…..

5. Nitrocellulose paper is then soaked into a solution that contains the
radioactive probe Detection of Mutation in Sickle Cell (RFLP):
1. DNA is isolated and digested with the restriction enzyme Mst II which
recognizes the sequence GAG.

DNA probe

As shown above, the normal cell will have 3 cleavage site. While in the
Radioactive probe – is a piece of DNA that is complementary to sickle cell, since GTG is not recognized, it will only have 2 cleavage site
the DNA you are trying to find
So when they are mixed together, it will bind to its match. At this 2. After RE digestion, the DNA fragment of interest would look like this:
point, the fragments are still invisible. This is where x-ray
photography comes in

6. This is a radioactive x-ray picture. Everything is clear except for this

one (the DNA you are trying to locate, it is visualized because the
radioactive probe is attached to it)

DNA fragment for the normal cell is shorter (1.2 kb) while DNA
fragment with mutation is longer (1.4 kb)

3. Arrange the DNA fragments by size via gel electrophoresis

4. Blot it into the nitrocellulose paper then heat-bake it. Soak in a
solution that contains the probe complementary to the DNA
fragment of interest. Probe will bind but we still cannot visualize it
yet at this point

Summary of Southern Blot:

1. Isolate and cut DNA into fragments
2. Arrange DNA fragments by size via gel electrophoresis
3. Blot transfer DNA from gel to nitrocellulose paper
4. Bake, denature paper and hybridize with probe
5. X-ray photography to reveal the DNA of interest

Applications of Southern Blot

 Gene discovery and mapping, evolution and development studies,
diagnostics and forensics 5. Perform x-ray photography. Everything else disappears except for the
 Analyze restriction digestion fragmentation of a person’s DNA DNA with radioactive probes. You can now see that the migration
(fingerprinting) pattern of mutated would be different from the pattern of normal
 Definitive test to ensure that a particular section of DNA of known
genetic sequence has been successfully incorporated into the
genome of the host organism

For this lecture we will focus more on the application of Southern

Blot on diagnostics and forensics

Applications of Southern Blot: Detection of Mutation in Sickle Cell (RFLP)

 RFLP – Restriction Fragment Length Polymorphism
o RF: Generated fragments using RE digestion
o LP: Compare the DNA fragments according to size

There are diseases that result from point-mutation like in Sickle Cell wherein
there is a mutation from GAG to GTG (from Glu to Val). This can be detected
using Southern blot

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Case: Jay, age 50, developed symptoms of Sickle cell disease at age 4. His wife, Detection of Full Mutation in FXS:
Marie, appears normal. Will their off springs, Jessica, age 10, and Martin, age 7, 1. DNA is isolated and digested with RE which recognizes the DNA sequence
be carriers? Or will they develop Sickle cell? for FMR1 gene

2. DNA fragments are arranged according to size using gel electrophoresis

3. Blot, bake and soak it with solution which contains the radioactive probe

Case Analysis: Based on Dr. Bravo’s lecture

Marie, both copies of genes are normal showing only 1 band. The 2 copies
are superimposed against each other 4. Undergo X-ray photography to be able to visualize the patterns
Jay, both copies of genes have point mutation. Both copies are large and also
superimposed against each other. So definitely the children will inherit the
mutated gene copies of the father
Martin and Jessica both has one normal copy from the mother and one
mutated copy from the father.

What is the difference between the children and the father?

The difference between the children and the father is that Jay (father) has a
Homozygous mutation while the children has Heterozygous mutation
Case: Jay and his wife, Marie, appear normal. Their offsprings, Jessica, age 30, and
Homozygous mutation – symptoms are much more worse because they Martin age 27, have mild learning disability while growing up. Will the offsprings of
possess 2 copies of mutated genes. They are therefore affected by the Jessica and Martin, with their respective spouses, carry the mutation?
disease
Heterozygous mutation – may or may not have the symptoms of the disease
as they have 1 mutated copy and 1 normal copy of gene. They are therefore
carriers of the disease. Whether the patient will manifest the disease
depends on whether the disease is X-linked or Autosomal; Autosomal
dominant or Autosomal recessive

Applications of Southern Blot: Detection of Full Mutation in Fragile X Syndrome

Facts about Fragile X
o 2nd most common cause of mental retardation
o X-linked recessive
o Anticipation (symptoms become worse with successive
generations)
o Expansion mutation of FMR1 gene (located in X
chromosome)
o Expansion mutation occurs during oogenesis but not
Case Analysis: Based on Dr. Bravo’s lecture
during spermatogenesis
Jay, since men only have 1 copy of FMR1 gene (from X chromosome), he has 1
band and for this case it is small
How do we know if FMR1 gene is on its Full mutation or Pre-mutation? Marie, has 2 copies, 1 short and 1 long. The long gene is already on its pre-
 Pre-mutation has 600 base pairs or 200 trinucleotide repeats mutation stage. This means there is a danger if she passes this onto her offsprings
 Full mutation has >600 base pairs because it could go into expansion mutation
Martin, he has 1 copy and it was inherited from the mother. It is longer than the
mother’s because it has undergone expansion mutation
Southern Blot is superior than PCR when it comes to detecting of expansion
Sarah, has 2 copies, 1 short and 1 long. The long gene was inherited from the
mutations which are considered full mutation because it can detect >600
father which is just the same in terms of length (no expansion mutation
base pairs of mutation unlike in PCR, it only detects <600 base pairs which is happened). She does not have FXS because it is just in its pre-mutation. If she’ll
only good for detecting pre-mutations have an offspring and she passes it on, her offspring could possibly have FXS
Jessica, has the same case as Martin
Bryan, has 1 copy of FMR1 gene. In this case it was inherited from the mother and
it has undergone expansion mutation. He has a full-blown FXS

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 BIOCHEMISTRY
Topic: Introduction to Molecular Medicine
10 Lecture by: Dr. Bravo

Applications of Southern Blot: DNA fingerprinting DNA Fingerprinting: Forensic Case

 Analyze restriction digestion fragmentation of a person’s DNA
(fingerprinting)
In DNA fingerprinting, we analyze fragments called VNTR, the
junk DNA

 VNTR sequences are analyzed for its fragmentation pattern

VNTR (Variable Number of Tandem Repeats)
o Junk DNA (noncoding, not transcribed)
o Tandem repeats (repeating as in ‘ATATATAT’); variable
number (varies in number across individual or within
own DNA)
o May be used for forensic and paternity testing
o Unique for each person and can therefore be used as
basis for determining identity and individuality 
useful for forensic cases
o A copy of VNTR of an individual comes from the
mother and father  great for establishing paternity

How is DNA fingerprinting done? Case Analysis: Rape Case

1. First we cut the DNAs using RE. DNA fragments will be produced of The Female cells (evidence) has the same DNA banding pattern as the Victim
varying sizes so it shows that it is really hers
Suspect 1 is the one who committed the rape because the DNA banding
pattern is the same as the Sperm DNA (evidence)

DNA Fingerprinting: Establishing Paternity

2. Arrange the fragments by size using gel electrophoresis. Banding

pattern that is invisible will be produced

Case Analysis: Establishing Paternity

3. Transfer into paper by blotting, heat-bake and then soak it in
radioactive probes for the VNTR to be utilized
4. Take an X-ray photography of the paper. A banding pattern that is
unique for the individual is then made
Daughter 1 – have bands that match both the mother and father 
legitimate child of the mother and father
Daughter 2 – have bands that match for the mother but does not have bands
for the father  daughter from another father
Son 1 – a legitimate child of the mother and father
Son 2 – have no bands that match the father and mother  Adopted son

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 BIOCHEMISTRY
Topic: Introduction to Molecular Medicine
11 Lecture by: Dr. Bravo

DNA MICROARRAY Application of DNA Microarray:

 Overcomes the limitations of Southern and Northern blot: analyzes  Identification of sequence (gene/gene mutation)
a single gene at a time  Determination of expression level (abundance) of genes
 Humans have around 30,000 genes  Single Nucleotide Polymorphisms (SNPs)
 Most diseases are result of interplay of several and not just a single  Drug discovery and toxicology research
gene (except for monogenic disorders)  Disease diagnosis
We would want to analyze all the 30,000 genes simultaneously
because the diseases that are common like DM, hypertension and KEY POINTS TO REMEMBER
cancer results from the interplay of the mutation of several genes
 Restriction Endonuclease are sequence specific endonuclease
 DNA recombinant technology became possible because of RE
 It is somewhat like Southern or Northern Blot but you do it a
thousand times on one single DNA microchip
 DNA can be propagated by:
 DNA microchip is a revolutionary new tool used in Molecular Biology
o Cell-based propagation/Cloning
and Medicine
o PCR
 The chip contains thousands of short, synthetic, single-stranded DNA
 PCR – is a repeated cycle of:
sequences (genes)
1. Denaturation
2. Primer Annealing
How is DNA Microarray done?
3. Primer Extension
1. Extract mRNA (blue)
o mRNA represents the expression of a gene
 DNA can be sequenced via Sanger’s Method
o If the gene is active, it would produce mRNA
 Sanger’s Method is also known as chain termination method
o But mRNA is unstable, so we convert it first to cDNA (light
o It is based on DNA polymerase reaction
blue) which is more stable. Conversion from RNA to DNA
is done through reverse transcription
 Southern Blot is DNA analysis using radioactive DNA probe
 Application of Southern Blot includes:
o Detection of mutation
o DNA fingerprinting

 DNA microarray analyzes thousands of gene sequences

simultaneously on a single chip
 It overcomes the limitation of traditional biotechnology tools
2. Conjugate the cDNA with the fluorescent dye so that it can be
visualized later on

References:
 PPT
 AMR Cayco Trans on Introduction to
Molecular Medicine
 Biochemistry Manual (2019)
 Dr. Bravo Recordings

3. All those cDNA are mixed on a microchip and they will be hybridized
to their respective templates
4. Scanned by laser beam
5. The first and second spot contain a radioactive dye and will appear as
a green light in the microchip. Spots with no radioactive dye will
appear dark
6. Green spots represent active genes and dark spots are inactive

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