What is Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) is used to multiply and make millions of copies of DNA (also called amplification of DNA). It is a technique used to amplify small and targeted segments of DNA to produce millions of copies of a specific gene fragment. It is an efficient and cost effective molecular technique.
Raw material used for PCR
For in vitro synthesis of DNA we need following raw material to make DNA.
(Forward and Reverse) These are short,artificial pieces of single stranded DNA usually around 2- 30 nucleotides in length. They are used to identify specific region to be copied. They attach to the beginning and the end region to be amplified.
These are enzymes which attach nucleotides with DNA thereby, copying the DNA. These enzymes are special as they can work in high temperature which is necessary for PCR procedure. Actually they are separated from bacteria Thermus aqaticus. You can buy taq polymerase here.
It is a sample DNA which contain the target sequence to amplify.
which provide suitable chemical environment for DNA Polymerase. It also act as a co-factor for the enzyme. e.g 10mM Tris at pH 8.3, 50mM KCl,1.5-2.5 mM MgCl, DMSO, Glycerol formamide etc.
Deoxynucleoside triphosphates (dNTPs)
dNTPs consist of dATPs, dCTPs, dGTPs, dTTPs. They are building blocks of new DNA strand.
PCR is carried out in a machine called Thermocycler, which can heat and cool the reaction tubes to achieve require temperature.
Steps of PCR
Polymerase Chain Reaction involves following steps
- Denaturing (at 94-96°C)
- Annealing (~68°C)
- Elongation (at 72°C)
Lets explain these steps one by one;
Denaturing: (DNA Separation) It is a separation of DNA strand. When we heat it to 94-96°C, the Hydrogen bonds between the strands break down result in releasing two strands.
Annealing: (Primer binding) In this step primer anneal or attach to the both DNA templates at 50-65°C. Primer recognize specific target DNA which we need to be copied.
Elongation: (Attachment of Nucleotides) At 72°C the Taq Polymerase attach itself to the primer and help it to collect and join new nucleotides to make a new strand of DNA.
Once the process completes, 1 DNA is converted in two copies in 1-3 min. This three step process repeat and repeat making millions of copies of DNA in 45 min.
Polymerase Chain Reaction Cycles vs Target Copies
The copies of the DNA doubled in each cycle so after “n” cycles you have 2^n (2 to the nth power). For example after 10 cycles you have 1024 copies and after 20 cycles you have 1 million copies etc.
Analysis of PCR Products
There are many techniques used to analyze PCR products.
- Agarose Gel Electrophoresis
- Labelled Probe
As name suggests Gel Electrophoresis contains a gel (made up of a polysaccharide called Agarose) The gel is placed in a tub containing a buffer (water with some salt in it). In this technique we used to separate mixture of DNA fragments or other macromolecules (e.g RNA, Proteins) according to their molecular size and charge present on them. At One end there are pocket like wells where we put DNA fragments. One end of a box is hooked to a positive end of a battery and other end is attached with a negative end as shown in the following picture.
This is how DNA fragments separated by the size. The shorter pieces travel faster than the larger ones.
Once the DNA fragments are separated. we can see and analyze the results by finding which size of DNA we’ve found. To make DNA particles visible we need to stain with a DNA binding dye and place under UV transilluminatikon. If targeted band is present it indicates otherwise don’t. You can find DNA Classroom kit here.
These are DNA/RNA fragments of variable length (100-1000 nucleotides long) is used to detect PCR based genes product (also called amplicon). By nature a probe can eleminate radioactive, fluorometric, colorometric or chemilluminiscent signals. It helps to visualize a product. Moreover, it provide specificity by highlighting specific targeted sequence (amplicons). DNA Labelled probe is available here.
Types of PCR
There are many types of PCR. Which include;
- Real time PCR
- Conventional PCR
- Nested-seminested PCR
- Multiplex PCR
- Quantitative PCR
- Touch down PCR
- Arbitrary PCR
- Inverse PCR
- Allele specific PCR
- Asymmetric PCR
- Core sample PCR
- Dial-out PCR
- Digital PCR
- Assembly PCR
- Traditional PCR
- Hot Start PCR
- In-silico PCR
- Inter sequence PCR
- Ligation-mediated PCR
- Methylation-specific PCR
- Miniprimer PCR
- Nano particle PCR
- Overlap extension PCR
- Quantitative PCR
- Solid phase PCR
- Suicide PCR
- Thermal asymmetric interplaced PCR
- Semiquantitative PCR
- Colony PCR
- After exponentional PCR
- Qualitative PCR