Transcript Biology 4.15 PCR

Polymerase Chain Reaction (PCR)

Aims:

Must be able to outline the polymerase chain
reaction (PCR).

Should be able to explain the importance of
PCR.

Could be able to describe the stages of PCR in
datail.
DNA Amplification

Using the technique called polymerase chain reaction (PCR), researchers
are able to create vast quantities of DNA identical to trace samples. This
process is also known as DNA amplification.

Many procedures in DNA technology require substantial amounts of DNA to
work with, for example;


DNA sequencing

DNA profiling/fingerprinting

Gene cloning

Transformation

Making artificial genes
Samples from some sources, including those shown here, may be difficult to
obtain in any quantity.
Steps in the PCR Process

Separate Strands
The laboratory
process called the
Separate the target DNA
strands by heating at 98°C for 5
minutes
polymerase chain
reaction or PCR
involves the following
steps 1-3 each cycle:
Add Reaction Mix
Add primers (short RNA strands
that provide a starting sequence
for DNA replication),
nucleotides (A, T, G and C) and
DNA polymerase enzyme.
Incubate
Cool to 60°C and incubate for a few
minutes. During this time, primers
attach to single-stranded DNA. DNA
polymerase synthesizes
complementary strands.
Repeat for about 25
cycles
Repeat cycle of heating
and cooling until enough
copies of the target DNA
have been produced.
Polymerase Chain Reaction

Original
DNASample
Although only three
cycles of replication are
shown here, following
cycles replicate DNA at
Cycle 1
an exponential rate and
can make literally billions
of copies in only a few
hours.

Cycle 2
The process of PCR is
detailed in the following
slide sequence
of steps 1-5.
Cycle 3
PCR
cycles
No. of target
DNA strands
1
2
2
4
3
8
4
16
5
32
6
64
7
128
8
256
9
512
10
1024
11
2048
12
4096
13
8192
14
16 384
15
32 768
16
65 536
17
131 072
18
262 144
19
524 288
20
1 048 576
21
2 097 152
22
4 194 304
23
8 388 608
24
16 777 216
25
33 554 432
The Process of PCR 1
A DNA sample called the
target DNA is obtained
DNA is denatured (DNA strands
are separated) by heating the
sample for 5 minutes at 98C
Primers (short strands of mRNA)
are annealed (bonded) to the DNA
Primer annealed
The Process of PCR 2
Nucleotides
The sample is cooled to 60°C.
A thermally stable DNA
polymerase enzyme binds to
the primers on each side of the
exposed DNA strand.
This enzyme synthesizes a
complementary strand of DNA
using free nucleotides.
After one cycle, there are now
two copies of the original
sample.
Nucleotides
Activity

Answer the questions from pages 231 and
251/2 in the Biozone books.