Polymerase Chain Reaction (PCR) FULL NOTES
Polymerase chain reaction/History / Types /application
RECOMBINANT DNA TECHNOLOGY
By Mr. VIKAS LODHI
POLYMERASE CHAIN REACTION (PCR) AND ITS APPLICATIONS
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By Topic Name
INTRODUCTION
Polymerase chain reaction (PCR) is a widely employed technique in molecular biology to amplify single or a few copies of DNA, generating millions of copies of a particular DNA sequence. The polymerase chain reaction results in the selective amplification of a target region of a DNA or RNA molecule. PCR has been extensively exploited in cloning, target detection, sequencing etc. The method consists of thermal cycles of repeated heating followed by cooling of the reaction mixture to achieve melting and primer hybridization to enable enzymatic replication of the DNA.
HISTORY
By 1971, a “repair synthesis" process was reported which was an artificial system containing primers and templates that can allow DNA polymerase to copy target gene. The DNA polymerases initially employed for in vitro experiments were unable to withstand these high temperatures. In 1976, Chien et al discovered a novel DNA polymerase from the extreme thermophile Thermus aquaticus which naturally dwell in hot water spring (122 to 176 °F). The enzyme was named as Taq DNA polymerase which is stable upto 95°C. In 1985, Kary Mullis invented a process Polymerase Chain Reaction (PCR) using the thermo-stable Taq polymerase for which he was awarded Nobel Prize in 1993.
PCR Thermo cycler
Basic Protocol for Polymerase Chain Reaction:
Components and reagents:
A basic PCR set up requires the following essential components and reagents :
1. Template DNA containing the DNA region (target) to be amplified.
2. Primers that are complementary to the 5' ends of each of the sense (Forward primer)
and anti-sense strand of the DNA target (Reverse primer).
3. Taq polymerase or other thermostable, high fidelity DNA polymerase (Pfu polymerase
isolated from Pyrococcus furiosus).
4. Deoxyribonucleotide triphosphates (dNTPs), which are the building-blocks for a newly
synthesized DNA strand.
5. Buffer solutions to provide a suitable chemical condition for optimum activity and
stability of the DNA polymerases.
6. Divalent cations (eg. magnesium or manganese ions). They act as a co-factor for Taq
polymerase which increases its polymerase activity. Generally Mg2+ is used, but Mn2+
can be applied to achieve PCR-mediated DNA mutagenesis. This is because higher Mn2+
concentration leads to higher error rate during DNA synthesis.
Procedure:
Typically, PCR is designed of 20-40 repeated thermal cycles, with each cycle
consisting of 3 discrete temperature steps: denaturation, annealing and extension. The thermal cycles are often proceeded by a temperature at a high range (>90°C), and followed by final product extension or brief storage at 4 degree celsius. In PCR cycles, the temperatures and the duration of each cycle is determined based on various parameters like the type of DNA polymerase used, the melting temperature (Tm) of the primers, concentration of divalent ions and dNTPs in the reaction etc. The various steps involved are:-
a) Initial Denaturation b) Denaturation c) Annealing d) Extension e) Final extension
The sequential steps of PCR
Key factor affecting the polymerase chain reaction: Designing of Primer:
The specificity of the PCR depends crucially upon the primers. The following factors are important in choosing effective primers.
a) Primers should be 17 to 30 nucleotides in length. However for certain studies the RAPD primer length might be of 10-12 nucleotides.
b) Ideally GC content of the primers is 50% .For primers with a low GC content, it is desirable to choose a long primer so as to avoid a low melting temperature.
c) The basic formula to calculate melting temperature is
Tm = 4°C x (number of G’s and C’s in the primer) + 2°C x (number of A’s and T’s
in the primer)
Two primers must have a similar Tm value. In case of several primer candidates, we have to choose primers which have the higher Tm value among them.
d) Sequences with long runs (i.e. more than three or four) of a single nucleotide should be avoided.
e) Primers with significant secondary structures (self –hair pin, loop formation) are undesirable.
f) There should be no base complementarities between the two primers.
Applications:
1. Infectious disease diagnosis, progression and response to therapy
2.Diagnosis of genetic diseases
3.Genetic counselling
4.Forensic sciences
5.Research in Molecular Biology
Variations in PCR:
1.Reverse Transcription PCR (RT-PCR):
In reverse transcription polymerase chain reaction (RT-PCR), first a RNA strand (template) is reverse transcribed into its complementary DNA copy using reverse
transcriptase, and subsequently cDNA is amplified using PCR.Various types of Reverse
transcriptase enzyme, isolated from Avian myeloblastosis virus (AMV),Moloney murine
leukemia virus (MMLV or MuLV) are generally used to produce a DNA copy from RNA
template. Random primers, an oligo (dT) primer or sequence-specific primers are used to amplify cDNA. Alternatively, some thermostable DNA polymerases (e.g., Tth DNA polymerase isolated fromThermus thermophilus) having reverse transcriptase activity,
which requires manganese (Mn2+)as a cofactor for activation instead of magnesium.
Basic PCR follows this initial reverse transcription step for amplification of the target
sequence. RT-PCR is widely used in the diagnosis of genetic disorders and semi
quantitatively in the calculation of specific expression level of particular RNA molecules
within a cell or tissue. RT-PCR also helps in obtaining eukaryotic exon sequences from
mature mRNAs.
2.Real Time PCR or Quantitative PCR (qPCR):
3.Hot Start PCR:
4.Colony PCR:
5.Nested PCR:
6.Touchdown PCR:
7.Inverse PCR:
8.In-situ or Slide PCR:
9.Multiplex PCR:
10.Assembly PCR:
11.Solid Phase PCR:
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