Scribd
Upload
25 views

What Is PCR - 092001

This document summarizes polymerase chain reaction (PCR), a common laboratory technique used to amplify a specific region of DNA. It describes the basic steps of PCR, which include denaturing the double-stranded DNA template, annealing primers to the single strands, and extending the primers to synthesize complementary new strands. Key reagents for PCR are described, including Taq polymerase, primers, dNTPs, and buffer solutions. The document provides details on how Taq polymerase, isolated from a heat-resistant bacterium, is used to drive the amplification process through repeated heating and cooling cycles in a thermocycler.

Uploaded by

umarlucky1819
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
25 views

What Is PCR - 092001

This document summarizes polymerase chain reaction (PCR), a common laboratory technique used to amplify a specific region of DNA. It describes the basic steps of PCR, which include denaturing the double-stranded DNA template, annealing primers to the single strands, and extending the primers to synthesize complementary new strands. Key reagents for PCR are described, including Taq polymerase, primers, dNTPs, and buffer solutions. The document provides details on how Taq polymerase, isolated from a heat-resistant bacterium, is used to drive the amplification process through repeated heating and cooling cycles in a thermocycler.

Uploaded by

umarlucky1819
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 2

Group NO 5

ADVANCE TOPICS OF MICROSCOPY

What is PCR?
DEPARTMENT OF MICROBIOLOGY AND MOLECULAR GENETICS | SEMESTER 8 | SUBMITTED TO MAM ZILURSH | ADVANCE TOPICS OF
MICROSCOPY | UNIVERSITY OF OKARA | OKARA | PAKISTAN

SUBMITTED BY: GROUP NO 5 | MUQADDAS MAJEED 5001 | SANA LIAQAT 5002 | MUHAMMAD HAMZA 5010 | AMRIT MAHEEN 5025 | USAMA RIAZ
5037 | UMER MAQSOOD 5042

Polymerase chain reaction (PCR) is a common laboratory technique used to make many
copies (millions or billions) of a particular region of DNA. This DNA region can be anything the
experimenter is interested in.
PCR developed in 1986 by Kary Mullis. PCR is used in many areas of biology and medicine,
including molecular biology research, medical diagnostics, and even some branches of
ecology.
Reagents required for PCR:
1. Template DNA
2. Primer
3. Taq polymerase
4. DNTP’s
5. PCR buffer
6. MgCl2
7. Distilled water
Taq polymerase
Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new
strands of DNA, using existing strands as templates. The DNA polymerase typically used in
PCR is called Taq polymerase, isolated from heat tolerant bacterium (Thermus aquaticus).
T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very heat-
stable and is most active around 70°C (a temperature at which a human or E. coli DNA
polymerase would be nonfunctional
PCR primers
Like other DNA polymerases, Taq polymerase can only make DNA if it's given a primer, a short
sequence of nucleotides that provides a starting point for DNA synthesis
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 flank the target
region (region that should be copied).
Steps:
PCR tube is placed in PCR Machine or Thermocycler.
1. Denaturing
Group NO 5
ADVANCE TOPICS OF MICROSCOPY

2. Annealing
3. Extension
Denaturing stage

During this stage, template DNA and all the


other core ingredients is heated to 94-95⁰C.
The high temperature causes the hydrogen
bonds between the bases in two strands of
template DNA to break and the two strands
to separate. This results in two single
strands of DNA, which will act as templates
for the production of the new strands of
DNA. This usually takes between 15-30
seconds.

Annealing stage

During this stage the reaction is cooled to 50-65⁰C. This enables the primers to attach to a
specific location on the single-stranded template DNA by way of hydrogen bonding (the exact
temperature depends on the melting temperature of the primers you are using).Primers serve
as the starting point for DNA synthesis.

Extending stage

During this final step, the heat is increased to 72⁰C to enable the new DNA to be made by a
special Taq DNA polymerase enzyme which adds DNA bases. 72⁰C is the optimum
temperature for the Taq polymerase to build the complementary strand. It attaches to the
primer and then adds DNA bases to the single strand one-by-one in the 5’ to 3’ direction. The
result is a brand new strand of DNA and a double-stranded molecule of DNA.

THE END

You might also like