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Types and Applications

The polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA through repeated cycles of heating and cooling. It involves using primers to target the region of interest and a thermostable DNA polymerase to replicate the DNA. Each cycle doubles the amount of target DNA, resulting in exponential amplification. After 20-40 cycles, PCR can produce over a million copies of the target region. It has many applications, including DNA analysis, gene cloning, disease diagnosis, forensics, and molecular evolution studies.

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27 views

Types and Applications

The polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA through repeated cycles of heating and cooling. It involves using primers to target the region of interest and a thermostable DNA polymerase to replicate the DNA. Each cycle doubles the amount of target DNA, resulting in exponential amplification. After 20-40 cycles, PCR can produce over a million copies of the target region. It has many applications, including DNA analysis, gene cloning, disease diagnosis, forensics, and molecular evolution studies.

Uploaded by

Meera AK
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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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

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