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Long Range PCR

The document discusses long range PCR, which is a technique that generates multiple copies of a target DNA segment defined by a pair of primers. It requires DNA template, primers, Taq polymerase, nucleotides, and buffer. The PCR process consists of cycles with steps of denaturation to separate DNA strands, annealing for primers to attach, and extension for DNA polymerase to fill in new strands.

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12 views

Long Range PCR

The document discusses long range PCR, which is a technique that generates multiple copies of a target DNA segment defined by a pair of primers. It requires DNA template, primers, Taq polymerase, nucleotides, and buffer. The PCR process consists of cycles with steps of denaturation to separate DNA strands, annealing for primers to attach, and extension for DNA polymerase to fill in new strands.

Uploaded by

21125809
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Long range PCR

Introduction
Polymerase chain reaction (PCR) is a commonly used method that generates multiple
copies of a target deoxyribonucleic acid (DNA) segment, delineated by a pair of
oligonucleotides or primers.

Phản ứng chuỗi polymerase (PCR) là một phương pháp thường được sử dụng để tạo
ra nhiều bản sao của đoạn axit deoxyribonucleic (DNA) mục tiêu, được mô tả bằng
một cặp oligonucleotide hoặc đoạn mồi

PCR requires several basic components.


These components are:
- DNA template, or cDNA which contains the region of the DNA fragment to be
amplifie
- Two primers, which determine the beginning and end of the region to be amplified
(see following section on primers)
-Taq polymerase, which copies the region to be amplified
- Nucleotides, from which the DNA-Polymerase for new DNA
-Buffer, which provides a suitable chemical environment for the DNA-Polymerase.
The PCR process consists of a series of twenty to thirty-five cycles. Each consists
of three steps.
1. The double-stranded DNA has to be heated to 94-960C in order to separate the
strands. This step is called denaturing; it breaks apart the hydrogen bonds that
connect the two DNA strands. Prior to the first cycle, the DNA is often
denatured for an extended time to ensure that both the template DNA and the
primers have completely separated and are now singlestrand only. Time 1-2
minutes up to 5 minutes. Also Taq-polymerase is activated by this step.
2. After separating the DNA strands, the temperature is lowered so the primers
can attach themselves to the single DNA strands. This step is called annealing.
The temperature of this stage depends on the primers and is usually 50C below
their melting temperature (45-600C). A wrong temperature during the
annealing step can result in primers not binding to the template DNA at all, or
binding at random. Time 1-2 minutes.
3. Finally, the DNA-Polymerase has to fill in the missing strands. It starts at the
annealed primer and works its way along the DNA strand. This step is called
extension. The extension temperature depends on the DNA- Polymerase. The
time for this step depends both on the DNA-Polymerase itself and on the
length of the DNA fragment to be amplified. As a ruleof-thumb, 1 minute per 1
kbp.
Types of PCR: Depending on the purpose of use, characteristics of template DNA and
target fragment size, different types of PCR can be used: regular PCR, RT-PCR, Long
range-PCr, touch down PCR...

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