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Polymerase chain reaction (PCR)

The technique used in the lab to make millions of copies of a particular section of DNA. It is simple, versatile, sensitive, specific, and reproducible assay. It was first developed in the 1980s.

History of PCR

PCR was originally developed in 1983 by American biochemist Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his pioneering work. 

  • 1953 :  Discovery of the double helix DNA structure
  • 1967: Thomas brock reports on the isolation of a extremophilic bacterium Thermophilis aquaticus
  • 1971:  Kleppe and his co-workers first described a method unsing an enzymatic assay to replicate a short DNA template with primers in vitro.
  • 1976:  Taq polymerase was isolated.
  • 1977:  Frederik Sanger and his colleagues introduced the “dideoxy” chain termination method for DNA sequencing also known as Sanger sequencing.
  • 1982:  The first cited use of microarray technology.
  • 1983:  Technique for PCR was created.
  • 1985 :  PCR technique was described in an article.
  • 1986 :  Purified Taq polymerase was first used in PCR
  • 1988 :  The  first automated PCR cycler was introduced.
  • 1989 :  Science magazine names Taq polymerase its first molecule of the year.
  • 1990 :  Amplification and detection of specific DNA
  • 1991 :  The first high fidelity DNA polymerase was characterized.
  • 1993 :  Nobel Prize to Dr. Kary Mullis, the first real-time PCR instrument was described.
  • 1995:  First complete genome of a free living organism was sequenced by Venter and his colleagues.
  • 1996:  Genome of the first eukaryotic organism was sequenced.
  • 2007 :  The first complete human genome is sequenced by Levy et al.
  • 2009:  The MIQE guidelines (Minimum information for publication of Quantitative Real- Time PCR Experiment) were published.


Background:

 In 1983, American biochemist Kary Mullis originally developed a new technique that made it possible to synthesize large quantities of DNA fragment without cloning. This technique is called polymerase chain reaction and has great practical importance and impact on biotechnology. By using this technique it is possible to generate thousands to millions copies of a particular section of DNA from very small amount of DNA. PCR is a common tool used in medical, biological research labs and even some branches of ecology.

Principle:

Polymerase chain reaction (PCR) involves the primer mediated enzymatic amplification of DNA. Which uses the enzyme DNA polymerase that synthesises new strands of DNA complementary to the template DNA. DNA polymerase adds nucleotides to the 3’ end of a custom designed oligonucleotide when it annealed (bind) to a larger template. In this manner if a synthetic oligonucleotide is annealed to a single stranded template that contain a region complementary to the oligonucleiotide. DNA polymerase can use the oligonucleotide as a primer and lengthen its 3’ end to generate an extended region of double stranded DNA.


Fig. Polymerase chain reaction
 



Requirements:

Five core ingredients.  

  1. Target  DNA/ Template  DNA/ PCR sample: Single or double stranded DNA of any origin (i.e. animal, bacterial, plant or viral) may be a PCR sample. RNA molecule are the first converted into cDNA by the enzyme reverse transcriptase prior to the PCR. As a basis, upto nanogram amount of cloned DNA, upto microgram amount of genomic DNA/ chromosomal DNA or upto 150 target DNA molecule are the best for the initial PCR testing. Quality and quantity of DNA sample are necessary for some molecular techniques.

  1. PCR primers : PCR primers are short (usually shorter the 50 nucleotides; optimum 18-25 nucleotide), single stranded, synthetically synthesized oligonucleotide. Primer may forward or reverse primers. Forward primer define 5’-3’ end and reverse primer defines 3’-5’ end. Primers that initiate the PCR reaction and designed to bind to either side of the section of DNA you want to copy.
  2. DNA nucleotide bases : DNA bases (A, C, G, and T) are the building blocks of DNA which are needed to construct the new strand of DNA.
  3. Taq polymerase : The enzyme Taq polymerase is 94Kda protein with 5’-3’ polymerase activity. It is most effectively active in 70-800C. It is thermostable with a half life at 950C for 35-40 minutes. Like is DNA replication in organism, PCR requires a DNA using existing strands as template. The DNA polymerase typically used in PCR is called Taq polymerase, which is an enzyme taken from the heat loving bacteria Thermus aquaticus. This bacteria normally lives in hot springs and can tolerate temperature above 800C. 
  4. PCR buffers  : The PCR buffer for the Taq polymerase consists of 50 ml KCl and 10 ml tris HCl of PH 8.3 at room temperature. The buffer provides the ionic strength and buffering capacity needed during the reaction.

Procedure:

This technique involves a process of heating and cooling called thermal cycling. There are three main stages which are as follows:

  • Denaturation

The template is heated to 94°C. This breaks the weak hydrogen bonds that hold DNA strand together in helix, and allowing the strands to separate creating single stranded DNA. This results in two single strands of DNA, which will act as templates for the new strands of DNA. This usually takes 15-30 seconds.

  • Annealing

The reaction is cooled to 50-70°C. which allows the primers to bind to a specific location on  the template DNA. Primers are designed to be complementary in sequence to short section of DNA on each end of the sequence to be copied. This usually takes about 10-30 seconds.

  • Extension

The temperature is then raised (heated to 72°C) to enable the new strand of DNA to be made by a special Taq polymerase enzyme which mainly adds DNA bases. The result is a brand new strand of DNA and double stranded molecule of DNA. The duration of this step depends on the length of DNA sequence being amplified.


With the one cycle, a single segment of ds DNA template is amplified into two separate pieces of ds DNA. These two pieces are then available for amplification in the next cycle. As the cycles repeated, more and more copies are generated & the number of copies of the template is increased exponentially. After PCR has been completed, a method called electrophoresis can be used to check the quantity and size of the DNA fragments produced.


Application of PCR:

1. Medical application

  • Genetic testing
  • Tissue testing
  • Cancer causing oncogene test

2. Infectious disease application

  • Determination HIV virus
  • Isolation of tuberculosis organism
  • To diagnose organism in animal population

3. Forensic application

  • Genetic finger printing
  • DNA paternity testing

4. Research application

  • DNA sequencing
  • DNA cloning
  • Hybridization of probes
  • Gene expression
  • Genetic mapping
  • Phylogenic analysis of DNA


Advantages of PCR:

  1. Small amount of DNA is required for the test.
  2. Result obtain more quickly usually within a day.
  3. Usually not necessary to use radioactive materials.
  4. PCR is much more precise to determine the size of alleles.
  5. PCR can be used to detect point mutation.


Types of PCR:

  1. Real time PCR 
  2. Quantitative real time PCR (Q-RT PCR)
  3. Reverse transcriptase PCR (RT- PCR)
  4. Multiplex PCR
  5. Nested PCR
  6. Long range PCR
  7. Single cell PCR
  8. Fast cycling PCR
  9. Methylation specific PCR (MSP)
  10. Hot start PCR
  11. High fidelity PCR
  12. In situ PCR
  13. Variable number of Tandem Repeats (VNTR) PCR
  14. Asymmetric PCR
  15. Repetitive sequence based PCR
  16. Overlap extension PCR
  17. Assemble PCR
  18. Intersequence specific PCR
  19. Ligation mediated PCR
  20. Methylation -specifin PCR
  21. Miniprimer PCR
  22. Solid phase PCR
  23. Touch down PCR

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