2013 :Polymerase Chain ReactionShakira Sulehri

Contents

Standard Polymerase Chain Reaction (PCR) PCR Reaction Components Thermal Cycling Profile for Standard PCR PCR Phases PCR Products Types of PCR Polymerase Chain Reaction: Uses

Polymerase chain reaction

Polymerase chain reaction was invented by Kary Mullis and his colleagues in the 1983. Nobel prize 1993. It has become the most widely used nucleic acid amplification technology and gold standard for amplification processes in diagnosis and research.

PCR is a technique for in vitro amplification of specific DNA sequences via the temperature mediated DNA polymerase enzyme by simultaneous 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.

PCR Reaction Components

Template: previously isolated and purified. Two primers: to flank the target sequence. Four deoxynucleosides triphosphate (dNTPs): to

provide energy and nucleosides for the synthesis of DNA.

Buffer system containing magnesium. DNA polymerase

PCR Reaction ComponentsTemplate: 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 M are generally optimal. Higher primer

concentrations may promote mispriming and accumulation of non specific product. Lower primer concentrations may be exhausted before the reaction is completed, resulting in lower yield of desired product.

Deoxynucleosides Triphosphate (dNTPs) Concentration: Balanced solution of all four dNTPs minimize polymerase error rate. Imbalanced dNTP mixture will reduce Taq DNA polymerase fidelity.The final concentration of dNTPs should be 50-500 M (each dNTP). They are usually included at conc. of 200 M for each.

Buffer system containing magnesium: Providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Generally, the Ph of the 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 l reaction. Too little will limit the amount of product, while too much can produce unwanted non specific products and decreased specificity.

DNA Polymerase:

The most widely characterized polymerase is that from Thermus aquaticus (Taq DNA Polymerase), which is a thermophilic bacterium lives in hot springs and capable of growing at 70 -75 C .

The purified protein (Taq enzyme) consist of a single polypeptide chain has a molecular weight of 95 Kd, and has an optimum polymerization temperature of 70 – 80 C .

Initial Denaturation: This step consists of heating the reaction to a temperature of 94-95°C which is held

for 1-9 minutes. Initial heating of the PCR mixture for 2 minutes at 94- 95C is enough to completely denature complex genomic DNA.

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

Denaturation: One to several minutes at 94-96 C, during which the DNA is denatured into single strands.

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

Extention: For fragments up to 3 kb primer extension is normally carried out at 72 C , during which the polymerase binds and extends a complementary DNA strand from each primer and add approximately 60 bases per second at 72C .

Post extension and holding: Cycling should conclude with a final extension at 72 C for 5 -15 minute to

promote completion of partial extension products and then holding at 4 c .

Thermal Cycling Profile for Standard PCR

DenaturationDenaturation

AnnealingAnnealing

ExtentionExtention

End of the 1st PCR Cycle results in two Copies of target sequence. The copies of both strands 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 be seen 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.

PCR Phases

Three 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.

Gel Electrophoresis

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

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

Electrophoretic "gels" are composed of either agarose or polyacrylamide. 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 small fragments of DNA. Polyacrylamide gels are also widely used for electrophoresis of proteins.

Agarose Gel Electrophoresis: It is a method used in biochemistry and molecular biology to separate DNA, or RNA

molecules based upon charge, size and shape.

A gel is made by dissolving agarose powder in boiling buffer solution, the solution is then cooled to approximately 50C 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 are prepared and added to the well by mixing them with solutions containing glycerol or sucrose, and then an electric current is run through the gel apparatus.

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

Factors, such as charge, size and shape, together with buffer conditions, gel concentration 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.

Advantages of PCR: Simplicity of the procedure. Sensitivity of the PCR.

Disadvantages of PCR: False positive results (cross contamination). False negative results (e.g. rare of circulating fetal

cells

Types 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. ….

Reverse Transcriptase-PCR

RT-PCR, one of the most sensitive methods for the detection and analysis of rare mRNA transcripts or other RNA present in low abundance.

RNA cannot serve as a template for PCR, so it must be first transcribed into cDNA with reverse transcriptase from Moloney murine leukemia virus or Avian myeloblastosis virus, and the cDNA copy is then amplified.

The technique is usually initiated by mixing short (12-18 base) polymers of thymidine (oligo dT) with messenger RNA such that they anneal to the RNA's polyadenylate tail. Reverse transcriptase is then added and uses the oligo dT as a primer to synthesize so-called first-strand cDNA.

Roche Molecular Biochemicals: PCR Application Manual. RT-PCRRoche Molecular Biochemicals: PCR Application Manual. RT-PCR

Nested PCR

Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (instead of one pair) of PCR primers are used to amplify a fragment.

The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a second pair of primers called nested primers bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one.

Nested PCR is a very specific PCR amplification.

Hot Start PCR Hot Start PCR significantly improve specificity, sensitivity and

yield of PCR

Some components essential for polymerase activity is separated from the reaction mixture until the temperature in the tubes has exceeded the optimal primer annealing temperature usually 55-65 C ˚.

The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step.

Real-Time PCR

QUANTITATION

Real-Time PCR

PCR was traditionally limited to end-point analysis using agarose gels

Limitations of end-point PCR:Poor precision

Low sensitivity

Short dynamic range

Low resolution

Size-based discrimination

Ethidium bromide for staining does not allow foraccurate quantitationRequires post-PCR processing

Real-time PCR instruments and chemistry allow for the detection and quantitation of

amplification throughout the reaction

End Point

Standard PCR

Real-Time PCRPlateau

phase

Linearphase

Exponentialphase

Area of detection forreal-time PCR

QUANTITATION

Theoretically there is a quantitative relationship between the amount of starting sample and the amount of PCR product at any given sample.

Real-time PCR detects the accumulation of amplicon during the reaction. The data is then measured at the exponential phase of the PCR reaction rather than end-point plateau. The exponential phase is the optimal point for analyzing data.

CT

Cycle threshold is related to the initial target copy number

Real-Time PCR

Applications:

Viral quantitation

Quantitation of gene expression

Microarray verification

Drug therapy efficacy

Pathogen detection

Genotyping

Advantages of real-time vs. end-point PCR:

Collects data in the exponential growth phase (vs end-point plateau)

Increase in fluorescent signal is proportional to number of amplicons generated

Increased dynamic range of detection

Does not require post-PCR processing

Increased sensitivity (detection down to 2-fold change)

Real-Time PCR

Detection Assays: SYBR Green Dye

SYBR Green I binds to double- stranded DNA. The resulting

DNA- dye-complex absorbs blue light) max = 498 nm (and emits green

light, (max = 522 nm) As DNA is amplified SYBR

fluorescence increases proportionally

Real-Time PCR

Non-specific dye used to detect the presence or absence of an

amplicon

Dual fluorophore-labeled oligonucleotide probe: e.g. TaqMan

Detection Assays: Sequence-specific probes

Real-Time PCR

Polymerase Chain Reaction: Uses

The polymerase chain reaction (PCR) is a technique widely used in: Molecular biology ,

Microbiology ,Genetics ,

Diagnostics clinical laboratories ,Forensic science ,

Environmental science ,Hereditary studies ,

Paternity testing, and Many other

applications ………………………………………

Diagnosis of a variety of human disorders:

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

detection of infectious agents, such as pathogenic bacteria, viruses or protozoa.

Cancer: Detection of malignant diseases by PCR. Recurrence of hematological cancers has also been evaluated. Detection of micro-metastasis in blood, lymph nodes and bone marrow.

Diagnosis of Genetic Diseases: Single point mutations can be detected by modified PCR techniques . 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 of pregnancy.

Prenatal Diagnosis of diseases e.g. Prenatal diagnosis of many of the inborn errors of metabolism is possible by DNA markers.

Mapping of entire genomes .

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

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

Amplification of forensic material.

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

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