Friday, June 21, 2013

Polymerase Chain reaction

It is a test tube method for rapid DNA amplification. It uses two primers that can attach to 3’ end of each strand by complementary base pairing. Primers are 20-35 nucleotides long.

The basic steps in PCR are:

  1. Denaturation of target DNA to separate two strands by heating at 900C.
  2. Cooling of denatured DNA to 40 – 600C this provides annealing of primers.
  3. Annealing of Primer to flanking 3’ end of both single stranded DNA. First temperature is increased to 2-30C above the Tm which gives greater specificity of binding of primer to complementary site and later it is decreased to 2-30C  below Tm permits  efficient amplification. This is the principle of Touchdown PCR.
  4. Extension of primer in 5’ to 3’ direction by DNA polymerase (dNTPs are added to 3’-OH of primer) in both strands. Carried at 720C.
  5. Denaturation of copied new DNA, and allowed to cyclize from step 1 though 3 to get million of copies of target DNA.

Repeated cycles result in exponential amplification of DNA. DNA polymerase from Thermus aquaticus that lives in and replicates at 70-800C is used as it is heat stable.

DNA sequences as short as 50-100 bp and as long as 10 kb can be amplified. 20 cycles provide an amplification of 220 and 30 cycles 230  This is then analyzed by gel electrophoresis, Southern hybridization or direct sequence determination.

Fig. PCR Cycles
Fig. PCR Processes
Unlike cloning, PCR is more sensitive and rapid for amplifying DNA. Traces of nucleic acid can be amplified.

PCR may also be used to amplify RNA by using the enzyme reverse transcriptase (RT-PCR).

Fig. Using cDNA for PCR

The components of PCR includes:

  • PCR buffer 
  • deionized water 
  • dNTPs
  • 1-3 mMMgCl2
  • Taq DNA polymerase 
  • Primer
  • Template DNA

Primers should be 10-24 nucleotide in length, GC content should be 40 -60%. They should not be self complementary or complementary to any other primer in the reaction mixture. The annealing temperature should be 50C lower than the melting temperature.

Variations of PCR:

Touchdown PCR (Step down PCR):

RT-PCR:   
Used   for   amplifying   DNA    from    RNA. Reverse    transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene.

Expression PCR
 
Quantitative PCR 

ASO-PCR

Multiplex    PCR:    
It    is    a    modification    of normal    PCR    in    order    to     rapidly detect deletions or duplications in a large gene. This process amplifies genomic DNA samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. Multiplex-PCR  was  first  described  in   1988   as   a   method   to   detect   deletions   in the dystrophin gene. Multiplex-PCR was used for analysis of microsatellites and SNPs.

Multiplex-PCR  consists  of   multiple   primer   sets   within   a   single   PCR   mixture   to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicons sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.

Hot Start PCR: 
is a modified form of Polymerase chain reaction (PCR) which avoids non-specific amplification of DNA by inactivating the taq polymerase at lower temperature. In this dsDNA is denatured by heating the sample at its denaturing temperature and then the temperature is suddenly reduced to 55 degree C at which primer and Taq-polymerase is added, but here the difference arises i.e. specific antibodies are used to block this Taq-polymerase at annealing temperature. Now when the temperature raises for amplification to 72 degrees, the specific antibody detaches from Taq-polymerase and the amplification with greater specificity starts.

Rapid Amplification of cDNA  Ends (RACE):  
is  used  to  obtain  the  full  length  sequence  of an RNA transcript found within a cell. RACE results in the production of a cDNA copy of the RNA sequence of interest, produced through reverse transcription, followed by PCR amplification of the cDNA copies. The amplified cDNA copies are then sequenced and from this the sequence of mRNA can be deduced or sequence of particular gene or particular site of transcript can be determined which gives the idea whether gene is present or not.

The PCR is used to.
  1. Detect infectious latent viruses which is not possible with conventional methods. To make prenatal genetic diagnoses; to detect allelic polymorphisms; to establish precise tissue types for transplants; and to study evolution, using DNA from archeological samples. For quantitative RNA analyses RT-PCR method (cDNA copies of mRNA generated by a retroviral reverse transcriptase) is used. DNA fingerprinting by means of PCR has revolutionized the analysis of evidence from crime scenes. The DNA markers analyzed for such fingerprinting are commonly short tandem repeat polymorphisms like VNTRs but smaller in size. (Verification of paternity uses the same techniques).
  2. Prenatal diagnosis and carrier detection (E.g. cystic fibrosis): It is an autosomal  recessive disease due to mutation in CFTR gene. There is 3 base deletion resulting in loss of phenylalanine from CFTR protein. Since the mutant allele is 3 base shorter than normal allele, so these two can be differentiated by the size of PCR products obtained by amplifying that portion of DNA.
  3. It can also be used in DNA sequencing. Radiolabelled or fluorescent labelled didoexynucleotide are introduced into the final stage of the reaction to generate chain terminated extension products. These are then analysized by electrophoresis.
Quantitative PCR (qPCR): 
One of the most useful PCR applications is quantitative PCR or qPCR. This allows the PCR to be used as a means of identifying the initial concentrations of DNA or cDNA template used. Previous methods used the comparison with standards. The introduction of thermal cyclers that incorporate the ability to detect the accumulation of DNA through fluorescent dyes binding to the DNA has rapidly transformed this area.

PCR is set up that includes a DNA binding dye such as SYBR green. This dye binds to the major groove do ds-DNA but not to ss-DNA and so as amplicons accumulate during the PCR process SYBR green binds the ds-DNA proportionally and fluorescence emission of dye can be detected following excitation. Thus the accumulation of DNA amplicons can be followed in real time during the reaction run. By comparing the fluorescence of known concentration of DNA the unknown concentration can be determined.

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