Polymerase Chain Reaction – PCR Steps

Polymerase chain reaction is involved replication of DNA. This is fast and reliable method in which minute copies of genetic material can be amplified millions of times. Primers and Taq polymerase are used for this purpose and Gel electropherosis helps to visualized DNA product.

PCR is a very sensitive technique to be used. This is invitro technique (reaction done in test tube not in organism) in which amplification has been done of specific genome of organism by using oligonucleotide. It gives logarithmic amplification of short DNA sequence with long double stranded DNA.

Different types of PCR used like nested Polymerase chain reaction,  Real time PCR, rtPCR. As PCR used for amplification of specific genome. Real Time PCR is also called qPCR and used to determine amount of PCR product. Nasted Polymerase chain reaction is used to design to improve the sensitivity and specificity of PCR. Two type of primers are used.Reverse transcriptase polymerase chain reaction is used to create cDNA from  RNA. It is mostly used for miRNAs.

Definition of polymerase chain reaction / what is PCR?

Polymerase Chain Reaction involved to make copy of DNA either Plant, animal or Humans . A laboratory technique  could be used for copies and this make thousands of copies of DNA. CTAB is used to Extract DNA from Plant and animals.

By using this method you can amplify any region of gene which you want. This technology is also used in forensic science especially in crime scene .a genetic marker used by forensic scientists to match crime scene DNA

Where polymerase chain reaction is used?

PCR is used in many areas of biology and medicine, including molecular biology research, medical diagnostics, and even some branches of ecology.

What is Taq Polymerase?

For DNA replication an enzyme is required which is called polymerase. A time ago polymerase enzyme was not heat stable but scientist found a heat stable enzyme which is called taq polymerase this enzyme is heat stable and used in PCR.

Scientist found T. aquaticus which lived in hot springs its DNA is most active at 70 degree that’s way its DNA is most stable and become suitable enzyme for PCR used. Human DNA and E.Coli DNA are nonfunctional at this temperature.

PCR primers

PCR primers are used to amplify the denature DNA and taq polymerase help to make DNA.

What is primer?

A short sequence of nucleotide is called primers. The sequence of DNA is determined which you want to amplified. Primers are design is such a way that they flank the target region which has to be copied. Their base pairs are complementary to the template. Sequence is opposite the strand. Two primers are used in PCR.

One primer is complementary to negative strand and second is complementary to positive strand in the presence of dNTP and DNA polymerase a complementary sequence is a synthesized. These primers are extended by DNA polymerase so that a copy is made of design sequence. After making this the same primers can be used again, not only to make another copy but also of the short copy made in the first round of synthesis this leads to the logarithmic amplification.

Template DNA:

5′ 3′

3′ 5′

Primer 1:       5′ 3′

Primer 2:      3′ 5′

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.

Three steps of PCR

The PCR is a cyclic process. PCR is method of invitro synthesis if specific DNA sequence.in this technique double stranded DNA is disrupted by high heat and PH to make single strand. This single strand serves as to template and by using polymerase enzyme double strand DNA can be made. Most polymerase required short regions of double strand nucleic acid for initiation of synthesis.

Procedure / Steps involved in PCR

In PCR ingredients are required taq polymerase, primers, template DNA and nucleotide.

These ingredients are taken in tube along co-factors needed by enzyme and are put through repeated cycles of heating and cooling that allow DNA to be synthesized.

The basic steps are

  1. Denaturation. it is necessary to raise the temperature to separate the double strand. This provides single-stranded template for the next step temperature is remain about 94 -98°C
  2. Annealing. Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA temperature should be kept  37-60°C.
  3. Extension of primers with polymerase in the presence of dNTP temperature kept about(72°C).

This cycle repeats many times which depends on the length of the DNA region being copied. From one copy you can make thousand copies but this is depending on reaction if reaction will work well.

This is not the original DNA which used as a template every time infect if one copy is formed in one round which is serve as a template in next round or cycle taq polymerase and primers are floating in the reaction and procedure goes on and hundred to thousand copies of target DNA are formed. This pattern of exponential growth is shown in the image below.

Gel Electrophoresis to visualize the results of PCR

After several rounds about 40 rounds of amplification the PCR product is examine on gel electrophoresis and by using ethidium bromide it is detected.

Principle

Movement of charge molecule is due to the electric field

Equation

V= Eq/f

E= electric field

q= the net charge on the molecule

f= friction coefficient

v= velocity of molecule

Velocity directly depends on the electric field and inversely on the friction coefficient. The applied voltages represent by E and remain constant during electrophoresis. Some reactions flow under this condition when charge is constant.

The movement of charge molecule depends on q/f. on the dependency of electric charge partials moves and separates DNA fragment according to size. DNA ladder is also including so that the size of the fragments in the PCR sample can be determined. DNA fragment of same length form band on gel which can be seen when this gel is stained through ethidium bromide and check on UV light.  After check on UV light result is look like just like the given diagram.

Different bands are formed 12,00 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, and 100 bp.

A DNA band contains many, many copies of the target DNA region, not just one or a few copies. Because DNA is microscopic, lots of copies of it must be present before we can see it by eye. This is a big part of why PCR is an important tool: it produces enough copies of a DNA sequence that we can see or manipulate that region of DNA.

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