rDT Assignment

8/2/2019 rDT Assignment

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PCR & its Applications

Presentation To :-

Dr. Sunita KhatakPresented by:-

Sandhya Kaushik(2508463)

27-11-2010 1

Assignment


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Polymerase Chain ReactionTechnique

Developed By Kary Mullis in 1985.

Selective amplificatiion of a chosen regionof a DNA molecule.

It generates microgram copies of thedesired DNA or RNA segment, presenteven as a single copy in the initialprepration.


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Utilization for PCR

Target Secquence Two nucleotide Primers

Four Deoxynucleoside Triphosphate as

TTP(thymidine Tryphosphate),dCTP(deoxycyctidine triphosphate)dATP(deoxyadenosine triphosphates)

TTP(Thymidine triphosphate) A heat stable DNA polymerase


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Procedure for PCR Denaturation

Annealing Primer Extension


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Denaturation

Temperature between 90-98C.

First Cycle of PCR

For 2 minutes


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Annealing

At temperature 40-60C It permits that the annealing of the

primer to the complimentary secquence

in the DNA. At 3 prime end

For 1 minute


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Primer Extension

DNA polymerase Systhesizes thecomplementary strands by utilizing 3 primeOH of the Primer.

At temperature 72 C.


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PCR Machine


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PCR Primer

Should corresponding to the target region.

Size about 20 bp long.

Should not in same direction.

Due to hybridization of one or both of theprimers to non target sites on the template

DNA molecule undesired amplificationproducts.


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Annealing Temperature

Very important since the success and specificityof PCR depend on it.

Because DNA-DNA hybridization is a temp

dependent phenomena. If high paring does not take place, PCR will fail.

If low temp. not stably pair.

Specificity of PCR describe the probability of anon target sequence being amplified ;the lowerthis probability, the higher is the PCR specificity.


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Cont.

Ideal Annealing Temp. must be low enough to enablehybridization b/w primer and template but highenough to prevent amplification of non target site.

IATusually 1-2Clower than melting temp.

Melting Temp.

Tm=[4(G+C)]+[2(A+T)]

the two primer for a PCR must be design such a way

that they both have an identical Tm.


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PCR Efficiency The efficiency of amplification decline with an increase in the

length of target sequence.

Efficiency of PCR is effected by primer length; it decline if theprimer used for PCR are too long

Annealing temperature has a marked influence on PCRefficiency; a temp. higher than the ideal annealing temp reducesPCR afficiency.

Primer sequence; dimer

Target sequence; GC rich may form secondary structure in thesingle strand produces by denaturation, it could reduces PCR

efficiency. Addition of certain proteins BSA enhance PCR efficiency by

protecting the DNA polymerase by binding to PCR inhibitors.


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Variations of PCR

PCR is highly versatile technique andhave a no. types.

1.Inverse PCR

2.Anchored PCR

3.RT-PCR

4.Asymmetric PCR5.Nested PCR


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Nested PCR

Target sequence is amplified a pair ofPCR primer.

A portion of amplification product is reamplified using another pair ofPCR primer complementary to the regionof first pair of primer.


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Nested PCR


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Inverse PCR

Inverse PCR is used for the synthesis ofunknown region which are either side on theknown region.

This is done by help of primer Oligonucliotidecomplementary to the 5 prime end.

Cut the target sequence by R.E.

Restriction fragment is circularized and ligatedlow concentration of DNA.

Exponentially amplified of unknown region.


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Applications of PCR

PCR can be used to amplify specific gene present indifferent individual of a species even in diff. gamtesor somatic cell say humans sperm of an individual.This copies can be used for cloning.

PCR has been used to study DNA polymorphism ingenome of random sequence as primer for exampleRAPD which is detected band after electroforces.

PCR can be used to detect the presence of a genetranfered into organism. Amplification occur onlywhen trans gene present in the organism.

Detection can be done by using nucleic acidhybridization (colony, colony blot, southren, northrenhybridization). These approches are expensive andtake longer time and used raidoactivity but PCRdetection take a single day and not used radioactivity.


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Cont Microdissected segment of chromosome for example salivary

gland of chromosome can be used for PCR amplification todetermined the physical location of gene in chromosome.

PCR can be used to generate single strand used for DNAsequencing, thermal cycle sequencing.

PCR can be used to produced cDNA copies of mRNA this is don

by Reverse Transcriptase PCR and million of copies of DNAduplex is done by Reverse Transcriptase PCR. DNA fingerprinting is now almost exclusively based on PCR. PCR is finding an increasing application of toxonomy example is

microbial toxonomy. PCR can be used to prenatal diagnosis of genetic diseases i.e

sickle cell anemia. Reverse Trancriptase PCR can also provise information on the

activity of tumer cell and virus.


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