GEL ELECTROPHORESIS

PCR & DNA SEQUENCING

SAKHAWAT HUSSAIN LECTURER BIOLOGY USWA GIRLS
PS&C SKARDU
Separation of DNA Fragments by Gel
Electrophoresis
DNA fragments must be separated and
purified prior to many rDNA procedures.
A convenient method for both separation
and purification is gel electrophoresis.
DNA molecules have a uniform charge to
mass ratio. In an electric field they run
towards the anode and are separated
based on size (length) when
electrophoresed through a gel sieving
network made of polyacrylamide or
agarose (Fig. 5.19a). DNA bands can be
visualized by radiolabeling the DNA or by
noncovalent binding of the fluorescent
dye known as ethidium bromide (Fig.
5.19b). The region of the gel containing
the band can be excised and the DNA
fragment obtained by extraction with a
buffer.
 Polymerase Chain Reaction (Part 1)
The PCR is a method for amplifying a
DNA sequence region located between
two primers (Fig. 5.20). Amplification is
specific and highly sensitive, allowing a
target sequence to be specifically
amplified starting from a complex
mixture of DNA. In Cycle 1, double-
stranded DNA containing the target
sequence is first denatured by heating
to >90˚C, PCR primers are annealed by
reducing the temperature to ~ 50-60˚C,
and then the primers are elongated by a
DNA polymerase. The process is
repeated over many cycles (next slide).
 Polymerase Chain Reaction (Part 2)
In later cycles, most of the
DNA synthesized corresponds
exclusively to the sequence
region between the primers.
The yield of amplified DNA
increases exponentially based
on the cycle number, n (yield is
proportional to 2n). A heat-
resistant DNA polymerase,
typically Taq polymerase from
the Yellowstone Archaen
organism, Thermus aquaticus,
is used as the DNA
polymerase to prevent its
denaturation due to heating.
 Cloning of PCR-amplified DNA
PCR fragments can be readily cloning into a vector by incorporating
restriction enzyme sites into the ends of the primers used in amplification
(Fig. 5.21). The added sequences do not interfere with polymerization
reactions.
 DNA Sequencing (Part 1)
The classical method for DNA sequencing is
dideoxy chain-termination sequencing (Sanger
sequencing). The basic approach involves 1)
enzymatic synthesis of a set of specifically
labeled (at each base) daughter strands from
the molecule being sequenced that differ by
one nucleotide in length, and 2) separation of
the fragments by electrophoresis. The
sequence then is read from the positions of
consecutive fragments on the gel. Termination
at each base is accomplished using
dideoxyribonucleoside triphosphates (ddNTPs)
(figure). These nucleotides are incorporated
into a growing DNA chain, but block further
elongation because they lack a 3'-hydroxyl
group.
 Sequencing by the Sanger Dideoxynucleotide
Chain Termination Method

1. Prepare replication template

unknown sequence known sequence
DNA template 5’ TAGGCGA GATCTG 3’
3’ 5’
denature,
add synthetic primer,
promote annealing

5’ TAGGCGA GATCTG 3’
CTAGAC
3’ 5’

2. Add components for in vitro replication

tube with large number of annealed DNA
and primer complexes from step 1

distribute into 4 separate tubes

to each tube, add:

- reaction buffer (salts, pH, etc.)
- 4 dNTP precursors
(1 radioactively labeled)
- DNA polymerase
ddCTP ddTTP ddGTP ddATP 1 dideoxynucleotide

ddATP
H
 Sequencing by the Sanger Dideoxynucleotide
Chain Termination Method, continued

3. Replication reactions

5’ TAGGCGA GATCTG 3’
CTAGAC
3’ 5’

CTAGAC 5’
ddCTP { CTAGAC 5’
CTAGAC 5’

ddTTP { CTAGAC5’
CTAGAC 5’
ddGTP { CTAGAC5’

ddATP { CTAGAC5’

4. Electrophoresis and visualization of replication products

ddC ddT ddG ddA

rxn rxn rxn rxn

-
3’ 5’
A T
T A
C G
C G
G C
C G
T A
+ 5’ 3’
 Automated DNA Sequencing
Use 4 ddNTPs, each Individual products are
labeled with different differentiable by size and
fluorochrome, in single attached fluorochrome.
reaction.

As bands migrate during electrophoresis, a detector senses

the different fluorochromes and feeds the information to a
computer for data analysis.