PCR
-Types And Applications
The Polymerase Chain Reaction (PCR) provides an extremely
sensitive means of amplifying relatively large quantities of DNA
First described in 1985, Nobel Prize for Kary Mullis in 1993
The technique was made possible by the discovery of Taq
polymerase, the DNA polymerase that is used by the bacterium
Thermus aquaticus that was discovered in hot springs
The primary materials, or reagents, used in PCR are:
- DNA nucleotides, the building blocks for the new DNA
- Template DNA, the DNA sequence that you want to amplify
- Primers, single-stranded DNAs between 20 and 50 nucleotides
long (oligonucleotides) that are complementary to a short
region on either side of the template DNA
-DNA polymerase, a heat stable enzyme that drives, or
catalyzes, the synthesis of new DNA
Steps involved:
(1) Denaturation step, the template nucleic acid is made single-
stranded by exposure to an elevated temperature (92-98~).
(2) The temperature of the reaction is then brought to between
65~ and 72~ in an annealing step by which the two primers attach to
opposite strands of the denatured template such that their 3' ends are
directed toward each other.
(3) Extension, a thermally stable DNA polymerase adds dNTPs onto
the 3' ends of the two annealed primers such that strand replication
occurs across the area between and including the primer annealing
sites.
Every cycle results in a doubling of the number of strands DNA
present
The result is a dramatic amplification of a the DNA that exists
between the primers. The amount of amplification is 2 raised to the n
power; n represents the number of cycles that are performed. After 20
cycles, this would give approximately 1 million fold amplification. After
40 cycles the amplification would be 1 x 1012.
Critical PCR parameters:
- Concentration of DNA template, nucleotides, divalent cations
(especially Mg2+) and polymerase
- Error rate of the polymerase (Taq, Vent exo, Pfu)
- Primer design
Primer selection
Critical variables are:
- primer length-specificity and the temperature of annealing
are atleast partly dependent on primer length.
- melting temperature (Tm)-
- specificity
- complementary primer sequences
- G/C content
- 3’-end sequence PCR phases in linear view
[DNA]
Cycle #
PCR phases
Exponential
◦ If 100% efficiency – exact doubling of products.
Specific and precise
Linear
◦ High variability. Reaction components are being
consumed and PCR products are starting to degrade.
Plateau
◦ End-point analysis. The reaction has stopped and if left
for long – degradation of PCR products.
Advantages & disadvantages
* The most accurate & feasible technique to determine the
amount & concentration of products.
* Rapid cycling (30 minutes to 2 hours).
* Specific & sensitive.
* Not much more expensive.
*****
* Pollution.
* Poor precision.
* Hard to get quantitative data.
Schemes of PCR
1) Standard PCR-sequence at both the ends of target sequences will be
known,two primers define define ends of the target and only that
part is amplified.
1) Inverse PCR-inside out PCR- if the gene sequence is unknown-clone
the DNA to be amplified into a vector whose primers are available-
polymerisation proceeds in inverse way to finally get the product.
1) Anchored PCR-single sided PCR-uses only one primer-at the end
poly G tail is added-new anchored primer with poly C tail is added
which synthesizes on the strand produced earlier-next step both the
primers are used.
4)Asymmetric PCR-preferentially amplify one strand of original
strand-carry out PCR as normal but with excess primer for desired
strand.LATE(Linear After The Exponential) PCR is a modification of
this type,where the limiting primer with very high Tm is used.
5)Quantitative PCR-used to measure the quantity of PCR product.
6)RT-PCR-Reverse Transcritase PCR-used to amplify,isolate or identify
a known sequence from a cellularor tissue RNA.-preceede by a reaction
using reverse transcriptase to convert RNA to cDNA.
Applications OF PCR
1) STUDY OF DNA POLYMORPHISM USING PCR
Amplify DNA from one or more genomic DNAs
Digest using one or more restriction endonucleases
Carry out electrophoresis
Photograph restriction patterns to reveal polymorphism.
It also uses random markers other than the normal markers to give
the random amplified polymorphic DNA(RAPD).
2)MOLECULAR MAPPING USING PCR
3)GENE TAGGING USING PCR-used for plant breeding and isolation
of genes.
4)CONFIRMING THE PRESENCE OF TRANSFERRED GENE
Eg:gene therapy-transfer and presence of marker gene for neomycin
resistance in blood of patients even after 60 days .
5)Human genetics using PCR:
Prenatal diagnosis using PCR-sickle cell anemia-RFLP
of PCR products or using a labelled probe-detect the
presence or absence of mutant gene .
Sexing of embryos-DNA sequences of single cells can be
studied-sex of human or livestock embryo fertilized
invitro –determine before implantation-detect sex linked
disorders-uses probes or primers specific for the
purpose-Y specific for humans.
DNA fingerprinting using PCR.
Diagnosis of pathogens,specific mutation,plant
pathogens
In Research.
Thank You
