Dna Analysis - 1.2

DNA ANALYSIS -1.
Aparna P, Ad hoc Faculty CSED,NITC

 There are different techniques for analysing DNA
1. copying DNA
DNA 2.cutting & pasting DNA
ANALYSIS 3. measuring DNA
4. probing DNA
 DNA Amplification/DNA copying
 DNA amplification allow us to

1.DNA  Studied - sequenced
 manipulated – mutagenized or engineered
Copying/Ampli-  expressed -generation of protein
fication Major Techniques used in DNA Amplification

1.PCR (Polymerase chain reaction)
2. Cloning
 Polymerase chain reaction (PCR) is a common laboratory
technique used to make many copies (millions or billions!) of a
particular region of DNA.
1.1 PCR
It might be a gene whose function a researcher wants to
understand, or a genetic marker used by forensic scientists to match
crime scene DNA with suspects.
 The goal of PCR is to make enough of the target DNA region that
it can be analyzed or used in some other way.
For instance, DNA amplified by PCR may be sent

for sequencing, visualized by gel electrophoresis, or cloned into a
Goal of PCR plasmid for further experiments.
• PCR is used in areas of biology and medicine, like molecular

biology research, medical diagnostics, and even some branches of
ecology.
Components
- PCR requires a DNA polymerase enzyme that makes new strands of
DNA, using existing strands as templates.
1. Taq
The DNA polymerase typically used in PCR is
polymerase called Taq polymerase ( from a heat-tolerant bacterium
(Thermus aquaticus) from which it was isolated)
Enzyme
 Like other DNA polymerases, Taq polymerase can only make DNA
if it's given a primer, a short sequence of nucleotides that provides
2.PCR primers a starting point for DNA synthesis.
 In a PCR reaction, the experimenter determines the region of DNA

that will be copied, or amplified, by the primers that they choose.
 PCR primers are short pieces of single-stranded DNA, usually
around 20 nucleotides in length.
 Two primers are used in each PCR reaction, and they are designed
so that they attached to the region that should be copied.
PCR  i.e, they are given sequences that will make them bind to
opposite strands of the template DNA, just at the edges of the
region to be copied.
 The primers bind to the template by complementary base pairing.
 When the primers are bound to the template, they can be
extended by the polymerase, and the region that lies between
them will get copied.

 Both primers, when bound, point “inward” – that is, in the 5’ to 3’
direction towards the region to be copied.
i.e, synthesize of DNA done only in the 5’ to 3’ direction.
Directionality
• When the primers are extended, the region that lies between
them will thus be copied.
 The key ingredients of a PCR reaction are
- template DNA
- Primers
Ingredients of - Taq polymerase

- nucleotides : new DNA from the end of the primer, single units of
a PCR reaction the bases A, T, G, and C, which are essentially "building blocks" for
new DNA
• The ingredients are assembled in a tube, along with cofactors
needed by the enzyme, and are put through repeated cycles of
heating and cooling that allow DNA to be synthesized.
The basic steps are:
1.Denaturation (96°C): Heat the reaction strongly to separate, or

denature, the DNA strands.
This provides single-stranded template for the next step.
2.Annealing/Priming (55 - 65°C): Cool the reaction so the primers

Steps in PCR can bind to their complementary sequences on the single-stranded
template DNA.
3.Extension (72°C): Raise the reaction temperatures

so Taq polymerase extends the primers, synthesizing new strands of
DNA.
This cycle repeats 25 - 35 times in a typical PCR reaction,
which generally takes 2 - 4 hours, depending on the
length of the DNA region being copied.
If the reaction is efficient (works well), the target region

can go from just one or a few copies to billions.
 That’s because it’s not just the original DNA that’s used as a
template each time. Instead, the new DNA that’s made in one
round can serve as a template in the next round of DNA synthesis.
 There are many copies of the primers and many molecules

of Taq polymerase floating around in the reaction, so the number
of DNA molecules can roughly double in each round of cycling

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DNA ANALYSIS -1.

Aparna P, Ad hoc Faculty CSED,NITC

 DNA amplification allow us to

fication Major Techniques used in DNA Amplification

For instance, DNA amplified by PCR may be sent

• PCR is used in areas of biology and medicine, like molecular

 In a PCR reaction, the experimenter determines the region of DNA

Ingredients of - Taq polymerase

1.Denaturation (96°C): Heat the reaction strongly to separate, or

2.Annealing/Priming (55 - 65°C): Cool the reaction so the primers

3.Extension (72°C): Raise the reaction temperatures

If the reaction is efficient (works well), the target region

 There are many copies of the primers and many molecules

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