polymerase chain reaction 1227174436086668 8

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PCR


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Contents

Standard Polymerase Chain Reaction (PCR)

PCR Reaction Components

Thermal Cycling Profile for Standard PCR

PCR Phases PCR Products

Variants of PCR

Polymerase Chain Reaction: Uses

PCR Protocols: http://www.protocolonline.org/prot/Molecular_Biology/PCR/


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Polymerase chain reaction

Polymerase chain reaction was invented by Kary Mullis and hiscolleagues in the 1983. Nobel prize 1993. It has become the mostwidely used nucleic acid amplification technology and gold standardfor amplification processes in diagnosis and research.

PCR is a technique forin vitro amplification of specific DNAsequences via the temperature mediated DNA polymerase enzyme bysimultaneous primer extension of complementary strands of DNA.

PCR is a test tube system for DNA replication that allows a "target"DNA sequence to be selectively amplified, several million-fold in just

a few hours.


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Standard Polymerase Chain Reaction

http//:nobelprize.org/nobel_prizes/chemistry/laureates/1993/illpres/pcr.gif


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Template: previously isolated and purified.

Two primers: to flank the target sequence.

Four deoxynucleosides triphosphate (dNTPs): toprovide energy and nucleosides for the synthesis ofDNA.

Buffer system containing magnesium.

DNA polymerase


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Template: The recommended amount of template for standard PCR is:

The maximum amount human genomic DNA should be up to 500 ng.

1-10 ng bacterial DNA. 0.1-1 ng plasmid DNA.

Primers: Primer concentration between 0.1 and 0.6 QM are generally optimal.

Higher primer concentrations may promote mispriming and accumulation of

non specific product. Lower primer concentrations may be exhausted beforethe reaction is completed, resulting in lower yield of desired product.

Deoxynucleosides Triphosphate (dNTPs) Concentration: Balanced solution of allfour dNTPs minimize polymerase error rate. Imbalanced dNTP mixture willreduce Taq DNA polymerase fidelity.The final concentration of dNTPs shouldbe 50-500 Q M (each dNTP). They are usually included at conc. of 200 Q M foreach.

Buffer system containing magnesium: Providing a suitable chemical environment

for optimum activity and stability of the DNA polymerase. Generally, the Ph ofthe reaction buffer is (Ph 8.3 9.0) will give the optimal results.Mg+ Concentration,The optimal MgCl2 concentration may vary from approximately

1mM-5mM, 1.5 mM is optimal in most cases.

DNA Polymerase: The recommended amount is 0.5 2.5 units/50 Q l reaction. Toolittle will limit the amount of product, while too much can produce unwantednon specific products and decreased specificity.


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DNAPolymerase:

The most widely characterized polymerase is thatfrom Thermus aquaticus (Taq DNA Polymerase),which is a thermophilic bacterium lives in hot

springs and capable of growing at 70 -75 C r.

The purified protein (Taq enzyme) consist of a singlepolypeptide chain has a molecular weight of 95 Kd,and has an optimum polymerization temperature of

70 80 C r.


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Initial Denaturation:This step consists of heating the reaction to a temperature of 94-95Cwhich is held for 1-9 minutes. Initial heating of the PCR mixture for 2minutes at 94- 95C ris enough to completely denature complexgenomic DNA.

Each cycle includes three successive steps: Each cycletakes as little as few minutes and it usually takes fewer than 20 cyclesto produce as much amplified DNA as one needs.

Denaturation: One to several minutes at 94-96 Cr, during which the DNAis denatured into single strands.

Annealing: One to several minutes at 50-65 C r, during which the primershybridize or "anneal" (by way of hydrogen bonds) to theircomplementary sequences on either side of the target sequence; and

Extention: For fragments up to 3 kb primer extension is normally carriedout at 72 C r, during which the polymerase binds and extends acomplementary DNA strand from each primer and add approximately60 bases per second at 72C r.

Post extension and holding:Cycling should conclude with a final extension at 72 C r for 5 -15minute to promote completion of partial extension products and then

holding at 4 c r.

Thermal Cycling Profile for Standard PCR


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DenaturationDenaturation

AnnealingAnnealing

ExtentionExtention


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End of the 1st PCR Cycle results in two Copies of target sequence. The copies of bothstrands then serve as templates for the next round of synthesis.

As amplification proceeds , the DNA sequence between primers doubles after each cycles(The amplification of the target sequence proceeding in an exponential fashion( 1 2 4

8 16.) up to million of times the starting amount until enough is present to beseen by gel electrophoresis.

Number of Cycles: The number of cycles required for optimum amplification varies depending on the amount

of the starting material. In optimal reaction, less than 10 template molecules can be amplified in less than 40 cycles

to a product that is easily detectable on a gel stained with ethidium bromide.

Most PCR should , therefore, include only 25 35 cycles. As cycle increases, nonspecific products can accumulate. After 20- 40 cycles of heating and cooling build up over a million copies of original DNA

molecules.


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PCR PhasesThree phases: Exponential: Exact doubling of product is accumulating at every cycle

(assuming 100% reaction efficiency). The reaction is very specific and precise.

Linear: The reaction components are being consumed, the reaction is slowing,

and products are starting to degrade.

Plateau: The reaction has stopped, no more products are being made and if left

long enough, the PCR products will begin to degrade.


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Gel Electrophoresis

Following amplification, the PCR products are usually loadedinto wells of an agarose gel and electrophoresed.

Gel electrophoresis is a method used to separate or purifysamples of DNA , RNA , or protein using an electric currentapplied to a gel matrix (porous sponge like matrix).

Electrophoretic "gels" are composed of eitheragarose orpolyacrylamide. These two substrates differ in resolving power,and also in the difficulty of setting them up .

Agarose gels are used much more commonly except for smallfragments of DNA. Polyacrylamide gels are also widely used forelectrophoresis of proteins.


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Agarose Gel Electrophoresis:

It is a method used in biochemistry and molecular biology to separate DNA, orRNA molecules based upon charge, size and shape.

A gel is made by dissolving agarose powder in boiling buffer solution, thesolution is then cooled to approximately 50C r and poured where it solidifies.The gel is submerged in a buffer filled chamber which contain electrodes.

The gel tray has combs attached to create wells in the gel, the samples areprepared and added to the well by mixing them with solutions containingglycerol or sucrose, and then an electric current is run through the gelapparatus.

The DNA fragments are separated by charge and the relative sizes of fragmentsare determined by comparing to a standard DNA ladder.

Factors, such as charge, size and shape, together with buffer conditions, gelconcentration and voltage, affect the mobility of molecules in gels.

Since PCR amplifications can generate microgram quantities of product,amplified fragments can be visualized easily following staining with a chemical

stain such as ethidium bromide.


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Advantages of PCR:

Useful non- invasive procedure.

Simplicity of the procedure. Sensitivity of the PCR.

Disadvantages of PCR:

False positive results (cross contamination).

False negative results (e.g. rare of circulating fetalcells


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Variants of PCR

Reverse transcriptase-PCR. Nested-PCR. Hot-start PCR.

Quantitative PCR. Multiplex-PCR. Mutagenesis by PCR. Allele specific PCR. Inverse PCR. Asymmetric PCR. In Situ PCR. .


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PCR-Reverse Transcriptase

RT-PCR, one of the most sensitivemethods for the detection andanalysis of rare mRNA transcriptsor other RNA present in lowabundance.

RNA cannot serve as a template forPCR, so it must be first transcribedinto cDNA with reversetranscriptase from Moloney murineleukemia virus or Avianmyeloblastosis virus, and the cDNAcopy is then amplified.

The technique is usually initiated bymixing short (12-18 base) polymersof thymidine (oligo dT) withmessenger RNA such that theyanneal to the RNA's polyadenylatetail. Reverse transcriptase is thenadded and uses the oligo dT as aprimer to synthesize so-called first-

strand cDNA.

Roche Molecular Biochemicals: PCR Application Manual. RTRoche Molecular Biochemicals: PCR Application Manual. RT--PCRPCR


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Nested PCR

Nested PCR is a variation of thepolymerase chain reaction(PCR), in that two pairs (insteadof one pair) of PCR primers areused to amplify a fragment.

The first pair of PCR primersamplify a fragment similar to astandard PCR. However, asecond pair of primers callednested primers bind inside thefirst PCR product fragment to

allow amplification of a secondPCR product which is shorterthan the first one.

Nested PCR is a very specificPCR amplification.

http://www.pcrstation.com/images/nested -pcr.gif


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Hot Start PCR

Hot Start PCR significantly improvespecificity, sensitivity and yield ofPCR.

Some components essential forpolymerase activity is separated

from the reaction mixture until thetemperature in the tubes hasexceeded the optimal primerannealing temperature usually 55-65 C .

The technique may be performedmanually by heating the reactioncomponents to the melting

temperature (e.g., 95C) beforeadding the polymerase. Specializedenzyme systems have beendeveloped that inhibit thepolymerase's activity at ambienttemperature, either by the bindingof an antibody or by the presence ofcovalently bound inhibitors thatonly dissociate after a high-

temperature activation step.


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Quantitative PCR

The determination or quantitation of the number of copies of a givengene achieves accurate estimation of DNA and RNA targets.

Real Time PCR Traditional PCR has advanced from detection at the end-point of the

reaction to detection while the reaction is occurring (Real-Time).

Real-time PCR uses a fluorescent reporter signal to measure theamount ofamplicon as it is generated. This kinetic PCR allows for datacollection after each cycle of PCR instead of only at the end of the 20to 40 cycles.

Time PCRwww. AppliedBiosystem.com Real


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The real time system reduces the time required for PCR amplification and analysis fromhours to minutes.

Monitor amplification online and in real-time.

Quickly and accurately quantify results.

Analyze melting characteristics of PCR product.

Combining online detection and continuous fluorescence monitoring allowed more rapidquantification of PCR product.

The recent development of real time PCR clearly demonstrates

many advantages over other existing method with: high accuracy,wide dynamic range , specificity , sensitivity , reduced carry over contamination and rapidaccurate and simultaneous quantification of multiple samples.


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Polymerase Chain Reaction: Uses

is a technique widely(PCR)The polymerase chain reactionused in:

Molecular biology,

Microbiology,Genetics,

Diagnostics clinical laboratories,Forensic science,

Environmental science,Hereditary studies,

Paternity testing, andMany other

applications


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Diagnosis of a variety of human disorders :

:Infectious agents One area where the PCR technique will undoubtedly become a routine method, is the

detection ofinfectious agents, such as pathogenic bacteria, viruses or protozoa.

Cancer: Detection ofmalignant diseases by PCR. Recurrence of hematological cancers has also been evaluated.

Detection ofmicro-metastasis in blood, lymph nodes and bone marrow.

Diagnosis ofGenetic Diseases: Single point mutations can be detected by modified PCR techniques such as the ligase

chain reaction (LCR) and PCR-single-strand conformational polymorphisms (PCR-SSCP)analysis.

Detection of variation and mutation in genes using primers containing sequences that were

not completely complementary to the template.

Prenatal Sexing and prenatal diagnosis of diseases: Prenatal sexing is often required in families with inherited sex linked diseases. In these

cases chorionic villus samples are ideal material for fetal sexing in the first trimester ofpregnancy.

Prenatal Diagnosis of diseasese.g. Prenatal diagnosis of many of the inborn errors of metabolism is possible by DNA

markers.


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Major role in the human genome project, replacing a singlepolymerase with a blend of a thermostable polymerase and aproofreading (Pwo DNA polymerase) made PCR an indispensable tool

in the analysis and mapping of entire genomes by greatly extendingthe length of the sequence that could be amplified, increasing theamount of PCR product and providing higher fidelity during PCR.

Identify the level of expression of genes in extremely small samples ofmaterial, e.g. tissues or cells from the body by reverse transcription-PCR (RT-PCR).

Multiplex PCR , made it possible to compare two or more complexgenomes, for instance to detect chromosomal imbalances.

Combining in situ hybridization with PCR made possible thelocalization of single nucleic acid sequences on one chromosomewithin an eukaryotic organism.

Amplification ofarchival and forensic material.

Identify testing for transplantation, HLA Typing.

PCR is used in research laboratories in DNA cloning procedures,Southern blotting, DNA sequencing, recombinant DNA technology.


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Real Time PCR : Uses

Viral load (HIV,HCV,HBV,),

Bacterial load (Salmonella, Mycobacterium, Listeria, Salmonella,Campylobacter..),

Fungal load ( Candida, Cryptococcus, Aspergillus,.)

Minimal residual disease, Chromosomal translocations, Singlenucleotide polymorphism (SNPs)

Gene transfer estimation, Biodistribution of vector


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References & Online Further Reading

www.Roche Molecular Biochemicals: PCR Applications Manual www.Roche Molecular Biochemicals: PCR Techniques www. AppliedBiosystem.COM Real Time PCR Trends in molecular medicine Vol. 8 No.6 june 2002. Quantification using real time PCR

technology, application and limitation. Robert F. Weaver. Molecular Biology. Fourth Edition. Page 600. McGraw-Hill International Edition.

ISBN 978-0-07-110216-2 Robert F. Mueller,Ian D. Young. Emery's Elements ofMedical Genetics: ISBN. 044307125X

Velasco J .A new view of malignancy New York Times. April 9, 2002.. Watson JD, Crick FHC. Molecular structure of nucleic acids .Nature .1953;171:737738 PubMed Stites DP . Medical immunology. Section II. Page 270 WWW.medscape.com www. pubmedcentral.nih http://en.wikipedia.org/wiki/RT-PCR

https://reader009.{domain}/reader009/html5/0518/5afdf033e8252/5afdf04f8fb96.jpg http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/mbglossary/mbgloss.htm http://www.dnalc.org/ddnalc/resources/electrophoresis.html

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