POLYMERASE CHAIN REACTION (PCR) PCR stands for the Polymerase Chain Reaction and was developed in 1987

by Kary Mullis (which won him a Nobel Prize) and associates. With this technique it is possible to make virtually unlimited copies of a single DNA molecule even though it is initially present in a mixture containing many different DNA molecules. It is used to amplify a specific DNA (target) sequence lying between known positions (flanks) on a double-stranded (ds) DNA molecule. The polymerase chain reaction can be used to amplify both double and single stranded DNA. In order to perform PCR, one must know at least a portion of the sequence of the target DNA molecule that has to be copied. Generally, PCR amplifies small DNA targets 100-1000 base pairs (bp) long. It is technically difficult to amplify targets >5000 bp long. A pair of single stranded oligonucleotide primers, which have DNA sequences complementary to the flanking regions of the target sequence, must be synthesized. The primers are complementary to either end of the target sequence but lie on opposite strands. The primers are usually 20-30 nucleotides long and bind to complementary flanking region at 3' end.

The basic PCR principle is simple. As the name implies, it is a chain reaction: One DNA molecule is used to produce two copies, then four, then eight and so forth. This continuous doubling is accomplished by specific proteins known as polymerases, enzymes that are able to string together individual DNA building blocks to form long molecular strands. To do their job polymerases require a supply of DNA building blocks, i.e. the nucleotides consisting of the four bases adenine (A), thymine (T), cytosine (C) and guanine (G). They also need a small fragment of DNA, known as the primer, to which they attach the building blocks as well as a longer DNA molecule to serve as a template for constructing the new strand. If these three ingredients are supplied, the enzymes will construct exact copies of the templates (see box on page 67). This process is important, for
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example, when DNA polymerases double the genetic material during cell division. Besides DNA polymerases there are also RNA polymerases that string together RNA building blocks to form molecular strands. They are mainly involved in making mRNA, the working copies of genes.

Requirements: Thermal cycler (thermocycler) PCR amplification mix typically containing: Sample dsDNA with a target sequence Thermostable DNA polymerase Two oligonucleotide primers Deoxynucleotide triphosphates (dNTPs) Reaction buffer containing magnesium ions and other components

Components: Sterile deionised water 10X PCR buffer dNTP mix Primer Taq DNA polymerase MgCl2 Template DNA

Considerations: Template DNA Primers MgCl2 concentration Taq polymerase dNT

Procedure: 1. The DNA molecule carrying a target sequence is denatured by heat at 9095oC for 20 seconds. The two strands separate due to breakage of the hydrogen bonds holding them together. Oligonucleotide primers are added.

2. A reaction mixture containing all four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) and a thermostable DNA polymerase is added. A DNA polymerase (Taq) that is not denatured by the high temperature needed to separate the DNA strands is used. It is usually sourced from Thermus aquaticus, a bacterium isolated from hot springs. 3. The mixture is allowed to cool to a lower temperature (50-65oC). Each strand of DNA molecule becomes annealed with an oligonucleotide primer complementary to either end of the target sequence. Primer annealing takes 20 seconds.

4. The temperature is raised to 60-75oC and primers are extended by the action of DNA polymerase for 30 seconds. The polymerase synthesizes complementary sequence the 5' to 3' direction away from each of the primers. If the template contains an A nucleotide, the enzyme adds on a T nucleotide to the primer. If the template contains a G, it adds a C to the new chain. Polymerization continues until each newly synthesized strand has proceeded far enough to contain the site recognized by the other
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primer. At this point there would be exactly two copies of the target DNA sequence.

5. The mixture is heated again at 90-95oC to denature the molecules and separate the strands and the cycle repeated. Each new strand then acts as a template for the next cycle of synthesis. Thus amplification proceeds at an exponential (logarithmic) rate, i.e. amount of DNA produced doubles at each cycle. The amplified product at the end of PCR is called amplicon. PCR procedures: steps Denaturation: DNA fragments are heated at high temperatures, which reduce the DNA double helix to single strands. These strands become accessible to primers. Annealing: The reaction mixture is cooled down. Primers anneal to the complementary regions in the DNA template strands, and double strands are formed again between primers and complementary sequences. Extension: The DNA polymerase synthesises a complementary strand. The enzyme reads the opposing strand sequence and extends the primers by adding nucleotides in the order in which they can pair.

The whole process is repeated over and over The PCR steps are all carried out, one after the other, in bouts of cycling. Cycle 1 is as follows: During denaturation (about 1 min at 95C), the DNA strands separate to form single strands.

During annealing (about 1 min at temperatures ranging between 45C and 60C), one primer binds to one DNA strand and another binds to the complementary strand. The annealing sites of the primers are chosen so that they will prime DNA synthesis in the region of interest during extension.

During extension (about 1 min at 72C), the DNA synthesis proceeds through the target region and for variable distances into the flanking region, giving rise to 'long fragments' of variable lengths

When the second cycle starts, there are effectively two types of template: (1) the original DNA strands; and (2) the newly synthesised DNA strands, consisting of the target region and variable lengths of the flanking region at the 3' end. When the latter template is used in this cycle, only the target region is replicated.

In the third cycle, the newly synthesised target region DNA (i.e. without flanking regions) acts as template. The original DNA molecule is still present, and will be until the end of the reaction. However, after a few cycles, the newly synthesised DNA fragment quickly establishes itself as the predominant template. Cycles are typically repeated 25 to 45 times. Standardisation of the thermocycler's running conditions is essential for the reproducibility of results.

Applications of PCR: Amplification of small amounts of DNA for further analysis by DNA fingerprinting. The analysis of ancient DNA from fossils. Mapping the human (and other species) genome. The isolation of a particular gene of interest from a tissue sample. Generation of probes: large amount of probes can be synthesized by this technique. Production of DNA for sequencing: Target DNA in clone is amplified using appropriate primers and then its sequence determined. Helpful in conditions where amount of DNA is small. Analysis of mutations: Deletions and insertions in a gene can be detected by differences in size of amplified product.

Diagnosis of monogenic diseases (single gene disorders): For pre-natal diagnosis, PCR is used to amplify DNA from foetal cells obtained from amniotic fluid. PCR has also proved very important in carrier testing.

Detection of microorganisms: Especially of organisms and viruses that are difficult to culture or take longtime to culture or dangerous to culture. The PCR has even made it possible to analyze DNA from microscope slides of tissue preserved years before. Detection of microbial genes responsible for some aspect of pathogenesis or antibiotic resistance. Crucial forensic evidence may often be present in very small quantities, e.g. one human hair, body fluid stain (blood, saliva, semen). PCR can generate sufficient DNA from a single cell.

Limitations of PCR: PCR is an extremely sensitive technique but is prone to contamination from extraneous DNA, leading to false positive results. Another potential problem is due to cross-contamination between samples. It is for this reason that sample preparation, running PCR and post-amplification detection must be carried out in separate rooms. Concentration of Mg is very crucial as low Mg2+ leads to low yields (or no yield) and high Mg2+ leads to accumulation of nonspecific products. Non-specific binding of primers and primer-primer dimmer formation are other possible reasons for unexpected results. Reagents and equipments are costly, hence cant be afforded by small laboratories.

REAL-TIME PCR Traditionally, PCR is performed in a tube and when the reaction is complete the products of the reaction (the amplified DNA fragments) are analysed and visualised by gel electrophoresis. However, Real-Time PCR permits the analysis of the products while the reaction is actually in progress. This is achieved by using various fluorescent dyes which react with the amplified product and can be measured by an instrument. This also facilitates the quantitation of the DNA. Not only can one tell instantly "what" DNA is present in the sample but also "how much". Quantitative PCR (Q-PCR), as this technique is known, is used to measure

the quantity of a PCR product (usually in a real-time PCR procedure). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. PCR is therefore often used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Another advantage of Real-Time PCR is rapidity of the assay, since it is not necessary to perform electrophoresis or other procedure after the DNA amplification reaction. The development of fluorescent methods for a closed tube polymerase chain reaction has greatly simplified the process of quantification. Current

approaches use fluorescent probes that interact with the amplification products during the PCR to allow kinetic measurements of product accumulation. These probe methods include generic approaches to DNA quantification such as fluorescent DNA binding dyes. There are also a number of strand-specific probes that use the phenomenon of Fluorescent Resonance Energy Transfer (FRET). The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a very significant advance in PCR technology. The technology is very flexible and many alternative instruments and fluorescent probe systems are currently available. Real-time PCR assays can be completed very rapidly since no manipulations are required post-amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with size analysis on gels. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Further investigation of the realtime PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations. Real-time PCR has applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. Due to their capability in fluorimetry the real-time machines are also compatible with alternative amplification methods such as NASBA provided a fluorescence end-point is available.

Variations on the basic PCR technique

Allele-specific PCR: a diagnostic or cloning technique based on singlenucleotide polymorphisms (SNPs) (single-base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.[18] See SNP genotyping for more information.

Assembly PCR or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.

Asymmetric PCR: preferentially amplifies one DNA strand in a doublestranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.

Helicase-dependent amplification: similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.
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Hot start PCR: a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the denaturation temperature (e.g., 95C) 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 dissociate only after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.

Intersequence-specific

PCR

(ISSR):

PCR

method

for

DNA

fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.

Inverse PCR: is commonly used to identify the flanking sequences around genomic inserts. It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.

Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.

Methylation-specific PCR (MSP): developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine, and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
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Miniprimer PCR: uses a thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.

Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).

Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes. That is, their base pair length should be different enough to form distinct bands when visualized by gel electrophoresis.

Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.

Overlap-extension PCR or Splicing by overlap extension (SOE) : a genetic engineering technique that is used to splice together two or more DNA
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fragments that contain complementary sequences. It is used to join DNA pieces containing genes, regulatory sequences, or mutations; the technique enables creation of specific and long DNA constructs.

Quantitative PCR (Q-PCR): used to measure the quantity of a PCR product (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA, or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR (or QF-PCR) methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly refers to reverse transcription PCR (see below), often used in conjunction with Q-PCR.

Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends).

Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR

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(where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming).

Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.

Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.

PAN-AC: uses isothermal conditions for amplification, and may be used in living cells.

Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer which can lead to artefactual 'noise') by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends), 5'RACE LaNe and 3'RACE LaNe.

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