CHAPTER 13

NUCLEIC ACID BIOTECHNOLOGY

TECHNIQUES
Jong Wha Lee, Ph.D. April 2018
13.1 Purification and Detection of Nucleic Acids
13.2 Restriction Endonucleases
13.3 Cloning
13.4 Genetic Engineering
13.5 DNA Libraries
13.6 The Polymerase Chain Reaction
13.7 DNA Fingerprinting
13.8 DNA Sequencing
13.9 Genomics and Proteomics
13.1 Purification and Detection of
Nucleic Acids
Separation Techniques
 Gel electrophoresis is a common technique used to separate nucleic acids.

 Based on the motion of charged particle in an electric field

 Depends on the ratio of its charge to its size

 NAs, oligonucleotides

 negatively charged at neutral pH

 Approximately the same ratio of its charge to its size of the molecule

 Polymeric gels: continuous cross-

linked matrix
 Agarose for larger fragment
(thousands of
oligonucleotides): horizontal
position – submarine gel
 Polyacrylamide for smaller
fragment (hundreds of
oligonucleotides): vertical
position Well
Purification and Detection (Cont’d)
Detection Methods
 Label or tag allows visualization

 Radioactive labeling of sample used to

detect products (e.g, 32P, 35S…)
 DNA undergo reaction that incorporate
radioactive isotope into the DNA
 Autoradiography used to visualize X-ray film
image that has been exposed to
oligonucleotides that have been radiolabeled
 Luminescence: emission of light
 Fluorescent compounds + irradiation with laser
 Fluorescence
 Ethidium bromide: planar portion that
intercalate between the bases of DNA 
orange fluorescence under UV light
Summary 13-1, p. 365
13.2 Restriction Endonucleases
 Nucleases: catalyze the hydrolysis of the phosphodiester backbones
of nucleic acids
 DNase & RNase
 Endonuclease for cleavage in the middle of the chain & Exonuclease
for cleavage from the ends of the molecule

 Restriction Endonucleases (RE) - Have a crucial role in development

of recombinant DNA technology
 Bacteriophages (phages), viruses that infect bacteria, were being
studied when restriction enzymes were discovered
 Restricted growth of phage in a certain strain of the bacteria
 Growth restricting host cells contain cleavage enzyme  RE
 RE produce double-chain breaks at the unmethylated specific sequences
in DNA
Methylation of DNA
 Methylation of bases at certain sequence-specific sites in the host DNA and
not in the viral DNA  Methylation of endogenous DNA protects it from
cleavage by its own restriction endonucleases
 Unmethylated specific sequences in phage DNA are attacked by the
restriction endonucleases in the host cells.

Endogenous bacterial DNA DNA fragments from

modified by methylation infecting bacteriophage
Restriction Endonucleases (Cont’d)
 Restriction endonuclease (RE) hydrolyzes only a
specific bond of a specific sequence in DNA
 Palindromic sequences recognized by RE read the
same on both strands from left to right as from right
to left
 Palindrome(“Madam, I’m Adam” is well-known linguistic
palindromes.)
Many Restriction
Endonucleases
Produce “Sticky Ends”
 Palindromic sequences
 Sticky-end cut: EcoRI…
 Produce short, single-
stranded stretches, called
sticky ends, at the ends of
cleaved DNA
 Blunt-end cut: HaeIII…

 Name of REs: 3-letter

abbreviation of the
bacterial species
 Arrows: phosphodiester
bonds cleaved by REs
 (a)
Why are sticky
ends important?
 Sticky ends are
joined by hydrogen
bonding between
complementary (b)
bases
 The sticky ends link
DNAs from
different sources,
even inserting
eukaryotic DNA
into bacterial
genomes
Figure 13.5 Hydrolysis of DNA by restriction
endonucleases
(a) Separation of ends
(b) Resealing of ends by DNA ligase
Summary 13-2, p. 368
13.3 Cloning
 Recombinant/Chimeric DNA - DNA molecules that contain
covalently linked segments derived from 2 or more DNA sources
 Sticky Ends can be used to construct Recombinant DNA
 Restriction endonucleases
 DNA Ligase
 seals nicks in the covalent structure
 Plasmid
 small circular DNA that is not part of the main
circular DNA chromosome of the bacterium
 Extrachromosomal self-replicating genetic
elements of a bacterial cell
 Cloning
 The process of making identical copies of DNA
 Rapid growth of viruses and bacteria  obtain greater amounts of the
recombinant DNA
 Production of
Recombinant DNA
 Cloning vector: the carrier for the
gene of interest that was cloned
 plasmid, viral vector…
 The origin of replication
 A multiple cloning site
 At least one selectable marker
 Genes that confer resistance to
antibiotic
FIGURE 13.5 Production of recombinant DNA.
(1) Foreign DNA sequences can be inserted into
plasmid vectors by opening the circular plasmid
with a restriction endonuclease.
(2) The ends of the linearized plasmid DNA are
then joined with the ends of a foreign sequence,
reclosing the circle to create a chimeric plasmid.
The Cloning of a Virus

FIGURE 13.6 The cloning of a virus.

The progeny of each individual phage (bacterial virus) infects and destroys bacteria on
the petri dish, leaving clear spots known as plaques.
Each plaque indicates the presence of a clone.
The Cloning of Cells

FIGURE 13.7 The cloning of cells.

Each individual cell divides many times, producing a colony of progeny.
Each colony is a clone.
Cloning Vectors
 Cloning vector
 a small piece of DNA, taken from a virus, a plasmid, or the cell of a
higher organism, that can be stably maintained in an organism, and into
which a foreign DNA fragment can be inserted for cloning purposes
 Cloning vectors in Escherichia coli
 plasmids (the most commonly used ones)
 bacteriophages (such as phage λ)
 Cosmids
 bacterial artificial chromosomes (BACs)
 Cloning vectors in yeast
 yeast artificial chromosomes (YACs)
 Shuttle vectors
 include elements that allow them to be maintained in another organism in
addition to E. coli
Types of Cloning Vectors
 Plasmid: DNA insert of up to 15 kb in size
 pBR322 plasmid, pUC series of plasmids…
 high copy number: pUC19: 500-700 copies per cell
 phagemid: Some plasmids contain an M13 bacteriophage origin of replication
and may be used to generate ssDNA
 Bacteriophage: a maximum of 53 kb
 phage λ and M13 phage
 phage cloning vectors need to have some non-essential genes deleted (e.g., the
genes for lysogeny in phage λ
 Two kinds of phage λ vectors - insertion vector and replacement vector
 Insertion vectors contain a unique cleavage site – insertion of foreign DNA (5–11 kb)
 Replacement vectors: the cleavage sites flank a region containing genes not essential
for the lytic cycle - this region may be deleted and replaced by the DNA insert (a
larger size DNA of 8–24 kb) in the cloning process
 A lower size limit for DNA that can be packed into a phage can be used for sele
ction – vector DNA without insert may be too small, therefore only vectors with in
sert may be selected for propagation
 Viral DNA integration into host chromosomes by viral vector may cause
insertional mutagenesis
Types of Cloning Vectors (cont’d)
 Cosmid: DNA insert between 28 to 45 Kb
 plasmids that incorporate a segment of bacteriophage λ DNA that has
the cohesive end site (cos) which contains elements required for
packaging DNA into λ particles
 Bacterial artificial chromosome (BAC): DNA insert of up to 350 kb
 BACs are maintained in E. coli with a copy number of only 1 per cell
 However low-copy-number plasmids may be preferably used in certain
circumstances, for example, when the protein from the cloned gene is tox
ic to the cells
 Yeast artificial chromosome (YAC): DNA insert of up to 3,000 kb
 Human artificial chromosome: no upper limit on size for practical
purposes
 may be potentially useful as a gene transfer vectors for gene delivery
into human cells, and a tool for expression studies and determining
human chromosome function
 Plasmid pBR322
 an origin of
replication (ori)
 genes encoding
resistance to the
drugs
 ampicillin (ampr)
 tetracycline (tetr)
 restriction
endonuclease
cleavage sites

FIGURE 13.10 One of the first

widely used cloning vectors, the
plasmid pBR322.
Plasmids (Cont’d)
 As the technology to design plasmids improved,
regions were created that had many different
restriction sites in a small place
 Multiple cloning site (MCS), or polylinker

FIGURE 13.11 A vector cloning site containing multiple restriction sites.

The colored amino acids are from the lacZ gene that is part of the plasmid.
This plasmid can be used for blue/white screening.
Directional cloning Two different REs are used
to cut open the MCS and to
 Extensive multiple cut out a piece of DNA to
be cloned  DNA to be
cloning sites inserted can be
 Two different incorporated in only one
restriction enzymes orientation.

FIGURE 13.12 Cloning w/

pUC plasmids.
The pUC series of plasmids
is very popular.
 Viral
vector
 Phage vector
 Cleavage w/
same
restriction
enzyme
 Package in
phage coats
 Development
of plaques on
lawn of
bacteria

FIGURE 13.8 The cloning

of human DNA fragments
with a viral vector.
Human DNA is inserted
into viral DNA and then
cloned.
 How do we know which bacteria takes up
the desired plasmid?
 Selection: Each plasmid chosen for cloning has a selectable marker that
indicates that the growing bacteria colonies contain the plasmid of interest

transformation

FIGURE 13.9 Selecting for recombinant DNA in a bacterial plasmid.

The plasmid also contains a gene for antibiotic resistance.
When bacteria are grown in a medium that contains the antibiotic, those that have
acquired a plasmid will grow.
Bacteria without a plasmid cannot grow in this medium.
Transformation
 Transformation: the genetic alteration of a cell resulting from the
direct uptake and incorporation of exogenous genetic material from
its surroundings and taken up through the cell membrane(s)
 Natural vs. artificial transformation
 Bacterial transformation
 Exogenous genetic material may be introduced into a bacterial cell
 Conjugation: transfer of genetic material between two bacterial cells in direct
contact
 Transduction: injection of foreign DNA by a bacteriophage virus into the host
bacterium
 Transformation into nonbacterial cells, including animal and plant cells
 Introduction of foreign DNA into eukaryotic cells is often called "transfection"
Selection and Screening
 Basis for selection
 pUC plasmids contain the gene for ampicillin resistance
and the lacZ gene
 Blue/White Screening - The lacZ gene
 Multiple cloning site (MCS) of the plasmid is localized within lacZ
gene
 Inactivation of lacZ gene by DNA insert  Insertional
inactivation
 lacZ gene codes for the -subunit of -galactosidase, which
cleaves disaccharides (e.g., lactose…) or X-gal
 The -galactosidase cleaves the dye, X-gal (colorless, off-white),
causing it to turn blue
 Agar medium contains ampicillin and a dye called X-gal
 Clone Selection via
Blue/White Screening
The pUC plasmid contains a gene
for ampicillin (amp) resistance
and the lacZ gene producing the
α-subunit of -galactosidase.
Transformed cells are plated on an
agar medium containing amp and
a dye, X-gal. The lacZ gene is
found inside the multiple cloning
site of the plasmid. When the
plasmid and the DNA to be cloned
are cut with a RE and then mixed
together, 1) The DNA insert can be
incorporated, or 2) the plasmid can
recircularize w/o the insert.
When this mixture is used to
transform bacteria,
Transformation
Left: The bacteria take up a of bacteria
plasmid that has the insert. This
plasmid confers amp resistance to
the cells, but the lacZ gene is
inactivated by the presence of the
insert. These cells grow and are
the normal off-white color of
bacterial colonies.
Middle: The bacteria take up the
recircularized plasmid. This
Summary
 Cloning refers to creating a genetically identical populations.
 DNA can be combined by using restriction enzymes that create
sticky ends in the DNA. This recombinant DNA has a target
DNA sequence.
 The target DNA sequence is carried in some type of vector,
usually a bacterial plasmid or a virus.
 The target DNA sequence is inserted into host organism, and
the natural doubling time of the host organism is used to create
many copies of the target DNA sequence.
 Organisms that carry the target DNA are identified through a
process called selection. Selection often involves antibiotic
resistance.
 13.4 Genetic Engineering
 When an organism is intentionally
altered at the molecular level to exhibit
different traits, it has been genetically
engineered
 Production of large amounts of medically
and economically important proteins
 Transgenic mice
 Agriculture – greater crop yields, frost
resistance or increased resistance to
pests…
FIGURE 13.14 Two adult female Anopheles
gambiae mosquitoes (ventral view).
The one on the left is a mutant.
Scientists are attempting to produce strains of
these mutant mosquitoes, which are unable to
transmit malaria to humans, in hopes that they will
replace the malaria carriers.
Genetic Engineering (Cont’d)
 Gene therapy
 Alteration of the cells of specific tissues in a living person in a way that
alleviates the effects of a disease
 cystic fibrosis, hemophilia, Duchenne muscular dystrophy, severe
combined immune deficiency (SCID)…
 DNA recombination occurs in nature
 In vivo - Maintenance of genetic diversity, & repair of damaged DNA
 Selective breeding for the desired mutants
 Bacteria as "protein factories“
 The reproductive power of bacteria can be used to express large
quantities of a mammalian protein of interest, however, process can be
complicated
 Bacteria: no cellular apparatus for splicing introns out of RNA transcripts
to give functional mRNA
 cDNA (complementary DNA, the coding sequence) is obtained from
mRNA in a reaction catalyzed by reverse transcriptase
 Genetic Engineering
(Cont’d)
 Human proteins can be made by bacteria,
but process is not straight forward. e.g. human
insulin
 An intron is a DNA sequence that codes for
RNA that is eventually deleted in the
processing of the mRNA that directs the
synthesis of the protein
 Only the RNA transcribed from exons
appear in the mature RNA
FIGURE 13.15 Synthesis of insulin in humans.
The insulin gene is a split gene. The intervening
sequence (intron) encodes an RNA transcript that is
spliced out of the mRNA.
Only the exon portions of the gene are reflected in the
base sequence of mRNA.
Once protein synthesis takes place, the polypeptide is
folded, cut, and spliced.
The end product, active insulin, has 2 polypeptide
chains as a result.
Production of Recombinant Human Insulin

 Active human insulin production in bacteria

 Two separate batches of E. coli
Protein Expression Vectors
 Cloning vectors
 Plasmid vectors pBR322 and pUC19
 to insert foreign DNA and to amplify it

 Protein expression vectors

 pET-11

 toproduce protein from the foreign DNA, cloning

vectors are not suitable
What is an Expression Vector?
 Cloning vector (pBR322, pUC)
 The origin of replication T7 promoter/
 A multiple cloning site (MCS) Lac operator

 At least one selectable marker

 Protein expression vectors (pET-11)
 a promoter for binding a viral RNA
polymerase, T7 RNA polymerase
 T7 transcription termination site
 MCS between a promoter and a
termination site
 a ribosomal binding site of mRNA
 Gene to produce T7 RNA polymerase
under the control of the lac operon
 E. coli…
 lacI gene (regulatory gene)  produces the
repressor for lac operon
 Induction by a lactose analogue, IPTG
(isoprophylthiogalactoside)  stimulate lac
operon  produce T7 RNA polymerase
Human Proteins through Genetic
Recombination Techniques
 Insulin
 Type I diabetes mellitus
 Human growth hormone
 Genetic dwarfs
 Muscle-wasting diseases (e.g., AIDS…)
 Tissue plasminogen activator and enterokinase
 Dissolves blood clots
 Heart attack or stroke
 Erythropoietin
 Stimulates the bone marrow to produce erythrocytes (RBCs)
 Chronic kidney failure  Kidney dialysis  lose
erythropoietin  anemia
 Analytical Chemistry (chromatography)
Fusion Proteins and Fast Purifications

Promoter for T7 polymerase + start sequence ATG

+ His-tag sequence + sequence that is specific for a
proteolytic enzyme enterokinase + MCS (gene of
interest)
 Met – (His)6 – enterokinase-specific amino acid
sequence – desired protein
 Fusion proteins
 Extra amino acids at the N-terminus or C-terminus
 Incorporation of affinity chromatography ligand-binding sites into a protein to
be expressed
 His-tagged fusion protein – nickel affinity column
 Affinity chromatography: almost perfect, one-step purification of the protein
Genetic Engineering in Eukaryotes
 Plants
 Bacterial plasmid
 Safety and ethics of the
process
 Genetically modified (GM)
foods
 Bacillus thuringiensis (Bt)
plants
 Positive side: use of less
pesticide
 Negative sides
 GM crops: Potential allergic
effects
FIGURE 13.18 A transgenic tomato plant.
 Effect on nontargeted insects
Recombinant DNA methods have produced
 Potentials to create a super plants that resist defoliation by caterpillars.
breed of insect accidentally
that is immune to the effect Tomatoes with a longer shelf life are
of the toxin another result of this research.
Genome Editing
 Genome editing CRISPR/Cas9
 CRISPR/Cas9
 Site-directed zinc finger nuclease
 TAL effector nucleases
 CRISPR technology in combination w/
the Cas9 RNA-guided DNA nuclease
 Selectively cut target sequences of DNA
 targeted genome editing
 4 Key components
 CRISPR: Clustered, regularly interspaced, short palindromic repeats
 Cas9: CRISPR associated protein 9 - RNA-guided endonuclease
 sgRNA: single guide RNA – target-specific CRISPR RNA (crRNA) + trans-activ
ating CRISPR RNA (tracrRNA) – contains sequence information for insertion/d
eletion
 PAM: Protospacer adjacent motif
 Mosquito population control  malarial reduction in humans
A basic diagram of the CRISPR pathway.
A CRISPR array is transcribed and used to locate a complementary sequence
in the cell for degradation.
Genetic Engineering Summary
 13.5 DNA
Libraries
 (Genomic) DNA
library: clone total
genome of an
organism in chunks of
reasonable size
 Transformed bacteria
 cell lines
FIGURE 13.19 Steps involved in the
construction of a DNA library.
All the DNA of a given organism is
extracted and treated with a
restriction enzyme.
The DNA fragments are incorporated
into bacterial plasmids.
Specific clones can be selected.
The remaining clones are saved for
future use.
Finding an Individual Clone in a DNA
Library
 After the library has been constructed, the next
challenge is to find a single desired clone out of
hundreds of thousands, or millions
 Genomic Library Screening: Technique used to
select depends on separating and annealing
complementary strands
FIGURE 13.20 Screening a genomic library by colony
hybridization (or plaque hybridization).
Host bacteria transformed with a plasmid-based genomic
library or infected w/ a bacteriophage-based genomic
library are plated on a petri dish and incubated overnight
to allow bacterial colonies (or phage plaques) to form.
(1) A replica of the bacterial colonies (or phage plaques) is
then obtained by overlaying the plate w/ a nitrocellulose
disc. Nitrocellulose strongly binds NAs; single-stranded
NAs are bound more tightly than double-stranded NAs.
Once the nitrocellulose disc has taken up an impression
of the bacterial colonies (or phage plaques), it is removed
and the petri dish is set aside and saved.
(2) The disc is treated with 2 M NaOH, neutralized, and
dried. NaOH both lyses any bacteria (or phage particles)
and dissociates the DNA strands. When the disc is dried,
the DNA strands become immobilized on the filter.
(3) The dried disc is placed in a sealable plastic bag, and
a solution containing heat-denatured (single-stranded),
 cDNA Library
 RNA libraries  cDNA
libraries
 RNA of interest is used as
template for the synthesis
of complementary DNA
(cDNA)
 Reaction is catalyzed by
reverse transcriptase
 cDNA is incorporated into
vector, then process is
identical to the production
of genomic DNA library

FIGURE 13.22 Formation of cDNA.

Reverse transcriptase catalyzes the synthesis
of a strand of cDNA on a template of mRNA.
Summary
 A DNA library is a collection of clones of an entire
genome
 The genome is digested with restriction enzymes and the
pieces are cloned into vectors, and transformed into cell
lines
 Specific radioactive probes to a sequence of interest
are reacted to filters that have copies of the bacterial
colonies in the library
 A cDNA library is constructed by using reverse
transcriptase to make DNA from the mRNA in a cell. This
cDNA is then used to construct a library similar to a
genomic DNA library
13.6 The Polymerase Chain
Reaction
 Polymerase Chain Reaction (PCR)
 The automated procedure for amplifying DNA from
very small amounts of sample
 Cell-free procedure
 without DNA cloning - not require viral or bacterial hosts
 Any chosen DNA does not need to be separated from the
rest of the DNA in a sample before the procedure is
applied
 Oligonucleotides primer complementary to the chosen DNA
sequence prime the synthesis of only that sequence
 Applications of PCR
 Forensic applications
 Positive identification of crime victims and suspects
PCR
 Materials
 Primers
 Heat-stable Taq DNA
polymerase - from the
bacterium, Thermos
aquaticus, that lives in hot
spring
 dATP, dTTP, dGTP, dCTP
 1 cycle
 Heat to separate dsDNA:
95ºC
 Cool to anneal primers to
DNA strands: 55-60ºC
 Chain extension at 72ºC
 The amount of the
specific primed DNA
sequence is doubled in
each cycle
 20-40 cycles of DNA
polymerization
 Quantitative PCR (qPCR) Allows Sensitive
Measurement of DNA Samples
 generate time-point data to determine the original amount of DNA in the cell
 Label with fluorescent markers
 The more DNA in the sample, the earlier in the process the results can be seen

Fig 13.24 qPCR. Cycles with different amounts of initial target DNA are shown as different colors.
CSI: Forensic Uses of
DNA Testing
 Paternity test DNA fingerprint
 Q) Who is the father?
 Hint: Every band in the child’s
DNA must have a corresponding
band in either the mother or the
father’s DNA
 Band B is not seen in lane 2, 3 &
4
 Both of 2 men are not his/her
fathers.
Lane 1: control
Lane 2: mother
Lane 3 & 4: possible fathers
Lane 5: child
Summary 13-6, p. 388
13.7 DNA Fingerprinting
 DNA samples can be studied and compared by DNA
fingerprinting
 DNA is digested with restriction enzymes and then run
on an agarose gel
 When soaked in ethidium bromide, the DNA fragments
can be seen directly under UV light
 If greater sensitivity is needed or if number of
fragments would be too great to distinguish the bands,
the technique can be modified to visualize only selected
DNA sequences
 Southern blotting
 Transfer of DNA to a nitrocellulose membrane
FIGURE 13.23 The Southern blot.
Electrophoretically separated DNA fragments are transferred to a nitrocellulose sheet.
A radioactively labeled probe for a DNA sequence of interest is bound to the
nitrocellulose, and bands are visualized with an autoradiogram.
Restriction-Fragment Length Polymorphisms:
A Powerful Method for Forensic Analysis
 In organisms with two sets of chromosomes, a given gene on one
chromosome may differ slightly from the corresponding gene on the
paired chromosome

 Allele: a variant form of a given gene on the homologous

chromosomes
 Organisms are homozygous when both alleles at a gene (or locus) are
the same
 Organisms are heterozygous when the alleles are different

 Restriction-Fragment Length Polymorphisms (RFLPs)

 Restriction fragments of different sizes are obtained by treatment with
restriction endonuclease
 DNA with mutation in gene
Restriction-
Fragment Length
Polymorphism
FIGURE 13.24 The basis for
restriction-fragment length
polymorphism (RFLP).
A change of one base pair
eliminates a restriction
nuclease cleavage site.
A portion of DNA that codes DNA with
for a protein has a cleavage normal gene
site for the restriction
nuclease DdeI.
The corresponding DNA with
a mutation does not have
this cleavage site.
The difference can be
detected by electrophoresis,
followed by blotting and the
annealing of a probe specific
for this fragment.
Paternity testing
 Localization of the gene
 Cystic fibrosis (CF) RFLP markers
 Prevalent genetic disease
 The protein in the transport of Cl-
through membranes
 Defective protein
→ Cl- remains in the cells
→ Cl- takes up water by osmosis from
the surrounding mucus
→ The mucus thickens
→ Lungs: infections, particularly
pneumonia
FIGURE 13.26 Localization of the gene associated
with cystic fibrosis (CF) on human chromosome 7.
The RFLP markers are given arbitrary names.
The location of the CF gene is given relative to the
RFLP markers.
Summary
 A DNA fingerprint is created by digesting DNA with
restriction enzymes, separating the pieces on a gel, and
visualizing some of the pieces by using labeled probes

 Differences in DNA patterns between different

individuals are based on different base sequences of
their DNA

 Different restriction sites  different-length fragments

 Restriction-Fragment Length Polymorphism
13.8 Sequencing DNA
 The nature and order of monomer units determine the
properties of the whole molecule
 The method devised by Sanger and Coulson for
determining the base sequences of nucleic acids
depends on selective interruption of oligonucleotide
synthesis
 A single-stranded DNA fragment whose sequence is to
be determined is used as a template
 The synthesis is interrupted at every possible site in
DNAs depending on the presence of 2’,3’-
dideoxyribonucleoside triphosphates (ddNTPs)
Dideoxyribonucleotide
 The incorporation of the ddNTP into the growing chain
causes termination at the point of incorporation
 ddNTP lacks a 3’-hydroxyl group

2’,3’-dideoxyribonucleoside triphosphates (ddNTPs)

DNA Sequencing (Cont’d)
 The DNA to be sequenced is mixed with a short
oligonucleotide that serves as a primer for synthesis of
the complementary strand

 Gel electrophoresis is performed on each reaction

mixture, and a band corresponding to each position of
the chain termination appears

 The sequence of the newly formed strand,

complementary to the template DNA, can then be read
from the sequencing gel
The Sanger-Coulon Method for Sequencing DNA
Synthesis is
interrupted at
every possible
site.  A
mixture of
oligonucleotides
of varying
lengths is
substrates produced.
Fluorescent Labeling and
Automated Sequencing of DNA
1. Four reactions are set up, one for each base, and the primer in each
is endlabeled with one of 4 different fluorescent dyes; the dyes
serve to color-code the base-specific sequencing protocol (a unique
dye is used in each dideoxynucleotide reaction).
2. The 4 reaction mixtures are then combined and run in one lane.
3. A laser beam excites the dye in the scan area.
4. The emitted energy passes through a rotating color filter and is
detected by a fluorometer.
5. The color of the emitted light identifies the final base in the
fragment.
Summary
 DNA can be sequenced by using several techniques, the
most common being the chain termination method
 Dideoxy nucleotides are used to terminate DNA
synthesis. Multiple reactions are run with a different
dideoxy nucleotide in each reaction mix
 The reactions produce a series of DNA fragments of
different length that can be run on a gel and the
sequence determined by tracking the different length
fragments in the lanes with the four different dideoxy
nucleotides
13.9 Genomics and Proteomics
 Knowing the full DNA sequence of the human genome allows
for the investigation for the causes of disease in a way that has
not been possible until now
 Genome: the total DNA of the cell
 Genomics: the study of genomes
 Transcriptome
 the total set of transcripts in a given organism, or to the
specific subset of transcripts present in a particular cell type
 the transcriptome can vary with external environmental
conditions
 the transcriptome reflects the genes that are being
actively transcribed at any particular time
 Transcriptomics: the study of transcriptiomes
Genomics and Proteomics (cont’d)
 Genome
 Genomics
 Transcriptome
 Transcriptomics
 Proteome
 a protein version of a genome
 the entire set of proteins expressed in a given type of cells
or an organism at a given time under defined conditions
 Proteomics: the study of interactions among all the proteins in
a cell
 DNA microarray (DNA chip or gene chip)
 Allow for the analysis of an entire genome in one
experiment
The Power of Microarrays – Robotic Technology Meets Biochemistry

Fig. 13-32a, p. 389

The Power of Microarrays – Robotic Technology
Meets Biochemistry (Cont’d)
The Power of Microarrays – Robotic Technology
Meets Biochemistry (Cont’d)
Protein Array
 Microchip w/ bound proteins instead of DNA
 To test whether patient sample contains Ag(s) , microarray
w/ bound antibodies can be used
1) Add blood sample of patient
2) Add fluorescently labeled antibodies
3) Scanning
 Summary

 DNA or protein microchips is a powerful technique

being used presently, as thousands of samples of
DNA or proteins can be applied and then checked
for binding of biological samples

 The binding can be visualized by using fluorescently

labeled molecules and scanning the chip with a
computer (Figure 13.30). The pattern of fluorescent
labels then indicates which mRNA or proteins are
being expressed in the samples