PCR--Polymerase Chain Reaction

PCR—from (Dr. Chen, Dept of Biochem. & Mol. Biology, Univ. College London)

Polymerase Chain Reaction
1) Add the following to a microfuge tube:10 ul reaction buffer1 ul 15 uM forward primer1 ul 15 uM reverse primer1 ul template DNA5 ul 2 mM dNTP8 ul 25 mM MgCl2 or MgSO4 (volume variable)water (to make up to 100 ul)
2) Place tube in a thermocycler. Heat sample to 95C, then add 0.5 -1 ul of enzyme (Taq, Tli, Pfu etc.). Add a few drops of mineral oil.
3) Start the PCR cycles according the following schemes:
a) denaturation - 94C, 30-90 sec.b) annealing - 55C (or -5C Tm), 0.5-2 min. c) extension - 72C, 1 min. (time depends on length of PCR product and enzyme used)repeat cycles 29 times
4) Add a final extension step of 5 min. to fill in any uncompleted polymerisation. Then cooled down to 4- 25C.
Note: Most of the parameters can be varied to optimise the PCR (more at Tavi's PCR guide):a) Mg++ - one of the main variables - change the amount added if the PCR result is poor. Mg++ affects the annealing of the oligo to the template DNA by stabilising the oligo-template interaction, it also stabilises the replication complex of polymerase with template-primer. It can therefore also increases non-specific annealing and produced undesirable PCR products (gives multiple bands in gel). EDTA which chelate Mg++ can change the Mg++ concentration.b) Template DNA concentration - PCR is very powerful tool for DNA amplification therefore very little DNA is needed. But to reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used, though too much template may increase the amount of contaminants and reduce efficiency.c) Enzymes used - Taq DNA polymerase has a higher error rate (no proof-reading 3' to 5' exonuclease activity) than Tli or Pfu. Use Tli, Pfu or other polymerases with good proof-reading capability if high fidelity is needed. Taq, however, is less fussy than other polymerases and less likely to fail. It can be used in combination with other enzymes to increase its fidelity. Taq also tends to add extra A's at the 3'end (extra A's are useful for TA cloning but needs to be removed if blunt end ligation is to be done). More enzymes can also be added to improve efficiency (since Taq may be damaged in repeated cycling) but may increase non-specific PCR products. Vent polymerase may degrade primer and therefore not ideal for mutagenesis-by-PCR work. d) dNTP - can use up to 1.5 mM dNTP. dNTP chelate Mg++, therefore amount of Mg++ used may need to be changed. However excessive dNTP can increase the error rate and possibly inhibits Taq. Lowering the dNTP (10-50 uM) may therefore also reduce error rate. Larger size PCR fragment need more dNTP. e) primers - up to 3 uM of primers may be used, but high primer to template ratio can results in non-specific amplification and primer-dimer formation (note: store primers in small aliquots). f) Primer design - check primer sequences to avoid primer-dimer formation. Add a GC-clamp at the 5' end if a restriction site is introduced there. One or two G or C at the 3' end is fine but try to avoid having too many (it can result in non-specific PCR products). Perfect complementarity of 18 bases or more is ideal. See Guide.g) Thermal cycling - denaturation time can be increased if template GC content is high. Higher annealing temperature may be needed for primers with high GC content or longer primers (calculate Tm). Using a gradient (if your PCR machine permits it) is a useful way of determining the annealing temperature. Extension time should be extended for larger PCR products; but reduced it whenever possible to limit damage to enzyme. Extension time is also affected by the enzymes used e.g for Taq - assume 1000 base/min (also check suppliers' recommendations, actual rate is much higher). The number of cycle can be increased if the number of template DNA is very low, and decreased if high amount of template DNA is used (higher template DNA is preferable for PCR cloning - lower error rate in the PCR).
h) Additives -
Glycerol (5-10%), formamide (1-5%) or DMSO (2-10%) can be added in PCR for template DNA with high GC content (they change the Tm of primer-template hybridisation reaction and the thermostability of polymerase enzyme). Glycerol can protects Taq against heat damage, while formamide may lower enzyme resistence.
0.5 -2M Betaine (stock solution - 5M) is also useful for PCR over high GC content and long stretches of DNA (Long PCR / LA PCR). Perform a titration to determine to optimum concentration (1.3 M recommended). Reduce melting temperature (92 -93 °C) and annealing temperature (1-2°C lower). It may be useful to use betaine in combination with other reagents like 5%DMSO. Betaine is often the secret (and unnecessarily expensive) ingredient of many commercial kits.
>50mM TMAC (tetramethylammonium chloride), TEAC (tetraethylammonium chloride), and TMANO (trimethlamine N-oxide) can also be used.
BSA (up to 0.8 µg/µl) can also improve efficiency of PCR reaction.
See also Dan Cruickshank's PCR additives and Alkami Enhancers for more.
i) PCR buffer
Higher concentration of PCR buffer may be used to improve efficiency.
This buffer may work better than the buffer supplied from commercial sources.16.6 mM ammonium sulfate67.7 mM TRIS-HCl, pH 8.8910 mM beta-mercaptoethanol170 micrograms/ml BSA1.5-3 mM MgCl2
j) The PCR product may be purified using a number of commercially available products or by gel-purification if the template needed to be removed. It can also be sequenced.
k) Trouble shooting see Tavi's page, MycoSite, Alkami Biosystems, Promega and Sigma.
l) PCR methods
Hot-start PCR - to reduce non-specific amplification. Can also be done by separating the DNA mixtures from enzyme by a layer of wax which melts when heated in cycling reaction. A number of companies also produce hot start PCR products, See Alkami Biosystem.
"Touch-down" PCR - start at high annealing temperature, then decrease annealing temperature in steps to reduce non-specific PCR product. Can also be used to determine DNA sequence of known protein sequence.
Nested PCR - use to synthesize more reliable product - PCR using a outer set of primers and the product of this PCR is used for further PCR reaction using an inner set of primers.
Inverse PCR - for amplification of regions flanking a known sequence. DNA is digested, the desired fragment is circularise by ligation, then PCR using primer complementary to the known sequence extending outwards.
AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA) - methods for creating genomic fingerprints from species with little-known target sequences by amplifying using arbitrary oligonucleotides. It is normally done at low and then high stringency to determine the relatedness of species or for analysis of Restriction Fragment Length Polymorphisms (RFLP).
RT-PCR (reverse transcriptase) - using RNA-directed DNA polymerase to synthesize cDNAs which is then used for PCR and is extremely sensitive for detecting the expression of a specific sequence in a tissue or cells. It may also be use to quantify mRNA transcripts. See also Quantiative RT-PCR, Competitive Quantitative RT-PCR, RT in situ PCR, Nested RT-PCR.
RACE (rapid amplificaton of cDNA ends) - used where information about DNA/protein sequence is limited. Amplify 3' or 5' ends of cDNAs generating fragments of cDNA with only one specific primer each (+ one adaptor primer). Overlapping RACE products can then be combined to produce full cDNA. See also Gibco manual.
DD-PCR (differential display) - used to identify differentially expressed genes in different tissues. First step involves RT-PCR, then amplification using short, intentionally nonspecific primers. Get series of band in a high-resolution gel and compare to that from other tissues, any bands unique to single samples are considered to be differentially expressed.
Multiplex-PCR - 2 or more unique targets of DNA sequences in the same specimen are amplified simultaneously. One can be use as control to verify the integrity of PCR. Can be used for mutational analysis and identification of pathogens.
Q/C-PCR (Quantitative comparative) - uses an internal control DNA sequence (but of different size) which compete with the target DNA (competitive PCR) for the same set of primers. Used to determint the amount of target template in the reaction.
Recusive PCR - Used to synthesise genes. Oligos used are complementary to stretches of a gene (>80 bases), alternately to the sense and to the antisense strands with ends overlapping (~20 bases). Design of the oligo avoiding homologous sequence (>8) is crucial to the success of this method.
Asymmetric PCR
In Situ PCR
Mutagenesis by PCR
Far too many to list properly.
For more information, protocols and links, go to PCR jump station, Alkami Biosystem, Fermentas, Promega, and Sigma, See also PCR primer, PCR notes and PCR manual at Roche and Qiagen.
Other PCR links - PCR lectures, radio-labelled probes, Thermocycler suppliers

PCR Protocol--Variants of PCR (2)

Special PCR Protocols (from biogate)

384-well PCR

Adjuvants in PCR reactions

Alpha-satellite DNA by PCR Preparation

Amplification of Genomic DNA using Alu PCR

Calculating Concentrations for PCR

Choice of Polymerases for PCR

Colony PCR

Core Sample PCR

Degenerate PCR

Degenerate PCR Primer Design

Degenerate PCR, a short guide

Designing PCR programs

Direct PCR from Whole Yeast Cells: Zymolyase Method

Disruption by Fusion PCR

Home-made Taq Polymerase Purification

Incorporation of Digoxigenin-dUTP into Plasmid Inserts Using PCR

Inverse PCR

Inverse PCR

Inverse PCR & Cycle Sequencing of P Element Insertions for STS Generation

Inverse PCR for PAC-end sequencing

Long PCR Reagents and Guidelines

Long-PCR Reagents and Guidelines

Methylated CpG Island Amplification

Methylation-Specific PCR

Multiplex PCR: Critical Parameters and Step-by-Step Protocol

PCR Additives

PCR Amplification of DNA

PCR Amplification of Inserts from Bacterial Cultures

PCR and multiplex PCR guide

PCR and multiplex PCR Troubleshooting

PCR of blood, hair or small tissue samples

PCR Primer Design

PCR Primer Design and Reaction Optimization

PCR protocol

PCR Technology

PCR to Amplify rRNA Gene Fragment

PEG Precipitation of PCR products

Polymerase Chain Reaction

Primary Amplification of Genomic DNA using DOP - PCR

Primer Design

primer design for PCR cloning

Purification of PCR products with Sephadex

Quantitative RT-PCR and Other PCR Procedures

RT In Situ PCR

Singel Nucleotide Primer Extension (SNuPE)

Single Primer ("Semi-Random") PCR

Single Tube Confirmation PCR Protocol

Site-directed Mutagenesis using PCR

SOEing PCR for mutagenesis

Standard PCR Protocol

Tail DNA for PCR (No Organic Solvents)

The In Situ PCR: Amplification and Detection in a Cellular Context

PCR Protocol--Variants of PCR

Variants of PCR

From Wikipedia, the free encyclopedia

This page assumes familiarity with the terms and components used in the Polymerase Chain Reaction (PCR) process.
The versatility of PCR has led to a large number of variants:

Contents
Basic modifications
Pretreatments and extensions
Buffer and temperature modifications
Primer modifications
Polymerase modifications
Mechanism modifications
Isothermal amplification methods
Additional reading
References
Basic modifications
Often only a small modification needs to be made to the 'standard' PCR protocol to achieve a desired goal:
One of the first adjustments made to PCR was the amplification of more than one target in a single tube. Multiplex-PCR can involve up to a dozen pairs of primers acting independently. This modification might be used simply to avoid having to prepare many individual reactions, or could allow the simultaneous analysis of multiple targets in a sample that has only a single copy of a genome. In testing for genetic disease mutations, six or more amplifications might be combined. In the standard protocol for DNA Fingerprinting, the 13 targets assayed are often amplified in groups of 3 or 4. Multiplex Ligation-dependent Probe Amplification (or MLPA) permits multiple targets to be amplified using only a single pair or primers, avoiding the resolution limitations of multiplex PCR.
VNTR PCR involves few modifications to the basic PCR process, but instead targets areas of the genome that exhibit length variation. The analysis of the genotypes of the sample usually involves simple sizing of the amplification products by gel electrophoresis. Analysis of smaller VNTR segments known as Short Tandem Repeats (or STRs) is the basis for DNA Fingerprinting databases such as CODIS.
Asymmetric PCR is used to preferentially amplify one strand of the target DNA. It finds use in some types of sequencing and hybridization probing, where having only one of the two complementary strands of the product is advantageous. PCR is carried out as usual, but with a limiting amount of one of the primers. When it becomes depleted, continued replication leads to an arithmetic increase in extension of the other primer[1]. A recent modification on this process, known as Linear-After-The-Exponential-PCR (or LATE-PCR), uses a limiting primer with a higher melting temperature Melting temperature (or Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction[2]. (Also see Overlap-extension PCR).
Some modifications are needed to perform long PCR. The original Klenow-based PCR process had trouble making a product larger than about 400 bp. However, early characterization of Taq polymerase showed that it could amplify targets up to several thousand bp long[3]. Since then, modified protocols have allowed targets of over 50,000 bp to be amplified[4].


Nested PCR
Nested PCR, another early modification, can be used to increase the specificity of DNA amplification. Two sets of primers are used in two successive reactions. In the first, one pair of primers is used to generate DNA products, which may also contain products amplified from non-target areas. The products from the first PCR are then used to start a second, using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set. The specificity of all of the primers is combined, usually leading to a single product. Nested PCR is often more successful in specifically amplifying long DNA products than conventional PCR, but it requires more detailed knowledge of the sequence of the target.
Quantitative PCR (or Q-PCR) is used to measure the specific amount of target DNA (or RNA) in a sample. The normal PCR process is performed in a way that is largely qualitative - the amount of final product is only slightly proportional to the initial amount of target. By carefully running the amplification only within the phase of true exponential increase (avoiding the later 'plateau' phase), the amount of product is more proportional to the initial amount of target. Thermal cyclers have been developed which can monitor the amount of product during the amplification, allowing quantitation of samples containing a wide range of target copies. A method currently used is Quantitative Real-Time PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product as the amplification progresses. It is often confusingly referred to as RT-PCR, the same acronym used for PCR combined with Reverse Transcriptase (see below), which itself might be used in conjunction with Q-PCR. More appropriate acronyms are QRT-PCR or RTQ-PCR.
Hot-start PCR is a technique that modifies the way that a PCR mixture is initially heated. During this step the polymerase is active, but the target has not yet been denatured and the primers may be able to bind to non-specific locations (or even to each other). The technique can be performed manually by heating the reaction components to the melting temperature (e.g. 95°C) before adding the polymerase[5]. Alternatively, specialized 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. 'Hot-start/cold-finish PCR' is achieved with new hybrid polymerases that are inactive at ambient temperature and are only activated at elevated temperatures.
Another simple modification can also decrease non-specific amplification. In Touchdown PCR, the temperature used to anneal the primers is gradually decreased in later cycles. The annealing temperature in the early cycles is usually 3-5°C above the standard Tm of the primers used, while in the later cycles it is a similar amount below the Tm. The initial higher annealing temperature leads to greater specificity for primer binding, while the lower temperatures permit more efficient amplification to the end of the reaction[6].
Other common modifications to PCR allow it to amplify low copy targets. The original report on Taq polymerase[3] showed how the use of up to 60 cycles could amplify targets diluted to just one copy per reaction tube. A later report[7] showed how multiple genetic loci could be amplified and analyzed from a single sperm. Modified protocols[8] have allowed the identification of just one copy of the HIV genome within the DNA of up to 70,000 host cells.
Assembly PCR (also known as Polymerase Cycling Assembly or PCA) is the artificial synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotide building blocks alternate between sense and antisense directions, and the overlaps determine the order of oligonucleotides, thereby selectively producing the final long DNA product[9].
In Colony PCR, bacterial colonies are rapidly screened by PCR for correct DNA vector constructs. Colonies are sampled with a sterile toothpick and dabbed into a master mix. To free the DNA for amplification, PCR is either started with an extended time at 95°C (when standard polymerase is used), or with a shortened denaturation step at 100°C and special chimeric DNA polymerase[10]. Colonies from the master mix that shows the desired product are then tested individually.
The Digital polymerase chain reaction simultaneously amplifies thousands of samples, each in a separate droplet within an emulsion.
Pretreatments and extensions
The basic PCR process can sometimes precede or follow another technique:
RT-PCR (or Reverse Transcription PCR) is a common method used to amplify, isolate, or identify a known sequence from a cell's or tissue's RNA. PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. The two reactions are compatible enough that they can be run in the same mixture tube, with the initial heating step of PCR being used to inactivate the transcriptase[3]. Also, the Tth polymerase described below exhibits RT activity, and can carry out the entire combined reaction. RT-PCR is widely used in expression profiling, which determines the expression of a gene or identifies the sequence of an RNA transcript (including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, 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 an RT-PCR method named RACE-PCR, short for Rapid Amplification of cDNA Ends. (Note that the acronym RT-PCR has more recently been applied to Real-Time PCR, a version of Quantitative PCR described above.)
Since PCR is based on components of DNA replication, it is not surprising that it can easily be combined[1] with DNA sequencing. In its simplest form, the products of an 'asymmetric PCR' (above) are diluted into a new reaction containing sequencing components, which are then extended by Taq polymerase.
Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' that are first ligated to fragments of the target DNA. PCR primers are then chosen from the linker sequences, and used to amplify the unknown target fragments. It has been used for DNA sequencing, genome walking, and DNA footprinting[11]. A related technique, Amplified fragment length polymorphism, looks at fragments of a genome that differ in length.
Methylation-specific PCR (or MSP) was developed to study patterns of methylation at CpG islands in genomic DNA[12]. Target DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two amplifications are then carried out on the modified DNA, using primer sets that distinguish between the modified and unmodified templates. One primer set recognizes DNA with cytosines to amplify the previously methylated DNA, and the other set recognizes DNA with uracil or thymine to amplify unmethylated target. MSP using Q-PCR can also be performed to obtain quantitative information about methylation.
Buffer and temperature modifications
Adjustments to the 'small' components in PCR can sometimes be useful:
The divalent magnesium ion (Mg++) is crucial to the activity of the polymerase used in PCR. Since many of the other components used in an amplification will also bind Mg++, it's exact concentration available to the enzyme is difficult to control. In general, lower concentrations will increase replication fidelity, while higher concentrations will introduce more mutations (either of which may be desired).
The use in PCR of modified dNTPs can help to control 'carryover' contamination. The PCR process can be carried out using dUTP, an analog of the normal dTTP. Later amplifications are then treated with an enzyme that destroys DNA containing the analog, but leaving the normal target DNA unmodified[13]. Thus, targets that represent contamination from earlier amplifications are selectively destroyed.
A wide variety of other chemicals can be added to PCR, for a variety of effects. Mild denaturants (such as DMSO) can increase amplification specificity by destabilizing non-specific primer binding. Certain chemicals (such as glycerol) can act as stabilizers for the activity of the polymerase during amplification. Detergents (such as Triton X-100) can prevent having the polymerase stick to itself, or to the walls of the reaction tube.
The temperature changes carried out by the thermal cycler will also affect amplification. A particular set of primers are usually tested using different annealing temperatures to determine their optimum. The time given to the polymerase to fully copy the templates may need to be adjusted, depending on their lengths. Longer extension times can also lead to higher yields after the reaction has entered the 'plateau' phase. When amplifying low-copy targets, the total number of cycles performed must be increased.
The polymerases that perform replication during PCR sometime incorporate incorrect bases. This is of no consequence to most assays that test the bulk of the amplified product - the errors are scattered within the product at random, and aren't seen by the assay. However, it is best to perform high-fidelity PCR when the products are individually cloned (for sequencing or expression). A different DNA polymerase (such as Pfu, with a proofreading activity missing in Taq) might be used, and the Mg++ and dNTP concentrations might be adjusted to maximize the number of products that exactly match the original target DNA. Some researchers choose to do the opposite, purposefully running PCR under low-fidelity conditions to produce a spectrum of mutations in the amplified product.
(For additional details, see the auxiliary article PCR optimization.)
Primer modifications
Adjustments to the synthetic oligonucleotides used as primers in PCR are a rich source of modification:
Normally PCR primers are chosen from an invariant part of the genome, and might be used to amplify a polymorphic area between them. In Allele-specific PCR the opposite is done. At least one of the primers is chosen from a polymorphic area, with the mutations located at (or near) its 3'-end. Under stringent conditions, a mismatched primer will not initiate replication, whereas a matched primer will. The appearance of an amplification product therefore indicates the genotype. (For more information, see SNP genotyping.)
InterSequence-Specific PCR (or ISSR-PCR) is method for DNA fingerprinting that uses primers selected from segments repeated throughout a genome to produce a unique fingerprint of amplified product lengths[14]. The use of primers from a commonly repeated segment is called Alu-PCR, and can help amplify sequences adjacent (or between) these repeats.
Primers can also be designed to be 'degenerate' - able to initiate replication from a large number of target locations. Whole genome amplification (or WGA) is a group of procedures that allow amplification to occur at many locations in an unknown genome, and which may only be available in small quantities. Other techniques use degenerate primers that are synthesized using multiple nucleotides at particular positions (the polymerase 'chooses' the correctly matched primers). Also, the primers can be synthesized with the nucleoside analog inosine, which hybridizes to three of the four normal bases. A similar technique can force PCR to perform Site-directed mutagenesis. (also see Overlap extension polymerase chain reaction)
Normally the primers used in PCR are designed to be fully complementary to the target. However, the polymerase is tolerant to mis-matches away from the 3' end. Tailed-primers include non-complementary sequences at their 5' ends. A common procedure is the use of linker-primers, which ultimately place restriction sites at the ends of the PCR products, facilitating their later insertion into cloning vectors.
An extension of the 'colony-PCR' method (above), is the use of vector primers. Target DNA fragments (or cDNA) are first inserted into a cloning vector, and a single set of primers are designed for the areas of the vector flanking the insertion site. Amplification occurs for whatever DNA has been inserted[3].
PCR can easily be modified to produce a labeled product for subsequent use as a hybridization probe. One or both primers might be used in PCR with a radioactive or fluorescent label already attached, or labels might be added after amplification. These labeling methods can be combined with 'asymmetric-PCR' (above) to produce effective hybridization probes.
Polymerase modifications
There are many choices for the all-important DNA polymerase used in PCR:
The Klenow fragment, derived from the original DNA Polymerase I from E. coli, was the first enzyme used to demonstrate PCR. It is inactivated in the denaturation step of PCR, and had to be replenished during each cycle.
The bacteriophage T4 DNA polymerase was also tested shortly after the first reports of PCR. It has a higher fidelity of replication than the Klenow fragment. Since it is also destroyed by heat, it has seen little use since the development of thermostable polymerases.
The DNA polymerase from Thermus aquaticus (or Taq), was the first thermostable polymerase used in PCR[3], and is still the one most commonly used. The enzyme can be isolated from its 'native' bacterial source, or from a cloned gene expressed in E. coli.
The Stoffel fragment is produced from a truncated gene for Taq polymerase, expressed in E. coli. It is missing the 'forward' nuclease activity, and may be able to amplify longer targets than the native enzyme.
The Faststart polymerase is a variant of Taq polymerase that only becomes active after the first denaturation step of PCR, thereby avoiding problems during the first cycle. (see Hot-start PCR above)
A thermostable polymerase has also been isolated from the archeozoic organism Pyrococcus furiosus. Unlike Taq polymerase, Pfu DNA polymerase includes a 'proofreading' activity, leading to about a 5-fold decrease in the error rate of replication[15]. Since these errors accumulate during every cycle of PCR, Pfu is the preferred polymerase when products are to be individually cloned for sequencing or expression.
An extremely thermostable DNA polymerase has been isolated from Thermococcus litoralis, and is marketed as Vent polymerase.
Another thermostable polymerase has been isolated from Thermus thermophilus, and is known as Tth polymerase. In the presence of Mn++ ions, it exhibits a reverse transcriptase activity, allowing PCR amplification to be initiated by RNA targets.
But not Bst polymerase, isolated from the thermophilic bacterium Bacillus stearothermophilus. This was an early candidate to be tested for PCR. It was later found to be unsuitable for continued amplification - it is irreversibly inactivated during the denaturation step. This highlights the point that a good polymerase for PCR should both be active at a higher temperature (for specificity), and should also be able to survive the near-boiling temperatures of the PCR process.
Mechanism modifications
Sometimes even the basic mechanism of PCR can be modified:
Unlike normal PCR, Inverse PCR allows amplification and sequencing of DNA that surrounds a known sequence. It involves initially subjecting the target DNA to a series of restriction enzyme digestions, and then circularizing the resulting fragments by self ligation. Primers are designed to be extended outward from the known segment, resulting in amplification of the rest of the circle. This is especially useful in identifying sequences to either side of various genomic inserts[16].
Similarly, Thermal Asymmetric InterLaced PCR (or TAIL-PCR) is used to isolate unknown sequences flanking a known area of the genome. 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[17].
Isothermal amplification methods
Some amplification protocols have been developed that only remotely resemble PCR:
Helicase-dependent amplification is a technique that is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension steps. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation[18].
PAN-AC also uses isothermal conditions for amplification, and may be used to analyze living cells[19][20].
Additional reading
PCR Applications Manual (from Roche Diagnostics).]

[edit] References
1. ^ a b Innis MA, Myambo KB, Gelfand DH, Brow MA. (1988). "DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA". Proc Natl Acad Sci USA 85: 9436-4940. PMID 3200828.
2. ^ Pierce KE and Wangh LJ (2007). "Linear-after-the-exponential polymerase chain reaction and allied technologies Real-time detection strategies for rapid, reliable diagnosis from single cells". Methods Mol Med. 132: 65-85. PMID 17876077.
3. ^ a b c d e Saiki et al. "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science vol. 239 pp. 487-91 (1988).
4. ^ Cheng S, Fockler C, Barnes WM, Higuchi R. "Effective amplification of long targets from cloned inserts and human genomic DNA." Proc Natl Acad Sci vol. 91(12) pp. 5695-9 (1994).
5. ^ Q. Chou, M. Russell, D.E. Birch, J. Raymond and W. Bloch (1992). "Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications". Nucleic Acids Research 20: 1717-1723.
6. ^ Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991). "'Touchdown' PCR to circumvent spurious priming during gene amplification.". Nucl Acids Res 19: 4008.
7. ^ Boehnke M et al. "Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm." Am J Hum Genet vol. 45(1) pp. 21-32 (1989).
8. ^ Kwok S et al. "Identification of HIV sequences by using in vitro enzymatic amplification and oligomer cleavage detection." J. Virol. vol. 61(5) pp. 1690-4 (1987).
9. ^ Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides". Gene 164: 49-53. PMID 7590320.
10. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes", in Kieleczawa J: DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett, pp. 241-257. ISBN 0-7637338-3-0.
11. ^ Mueller PR, Wold B (1988). "In vivo footprinting of a muscle specific enhancer by ligation mediated PCR". Science 246: 780-786. PMID 2814500.
12. ^ Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB (1996). "Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands". Proc Natl Acad Sci U S A 93 (13): 9821-9826. PMID 8790415.
13. ^ Longo MC, Berninger MS, Hartley JL "Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions." Gene vol. 93(1) pp. 125-8 (1990).
14. ^ E. Zietkiewicz, A. Rafalski, and D. Labuda (1994). "Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification". Genomics 20 (2): 176-83.
15. ^ Cline J,Braman JC, Hogrefe HH "PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases." Nucleic Acids Research vol. 24(18) pp. 3546-51 (1996).
16. ^ Ochman H, Gerber AS, Hartl DL (1988). "Genetic applications of an inverse polymerase chain reaction". Genetics 120: 621-623. PMID 2852134.
17. ^ Y.G. Liu and R. F. Whittier (1995). "Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking". Genomics 25 (3): 674-81.
18. ^ Myriam Vincent, Yan Xu and Huimin Kong (2004). "Helicase-dependent isothermal DNA amplification". EMBO reports 5 (8): 795–800.
19. ^ David, F.Turlotte, E., (1998). "An Isothermal Amplification Method". C.R.Acad. Sci Paris, Life Science 321 (1): 909-914.
20. ^ Fabrice David (September-October 2002). Utiliser les propriétés topologiques de l’ADN: une nouvelle arme contre les agents pathogènes. Fusion.(in French)
Retrieved from "http://en.wikipedia.org/wiki/Variants_of_PCR"

Introduction o PCR

General PCR introduction

1. IntroductionIn 1983 Kary B. Mullis was driving through California on a moonlight night (Mullis, 1990). He was pondering how to use DNA polymerase with oligonucleotide primers in order to identify a given nucleotide at a given position in a complex DNA molecule, such as the human genome. During this drive he invented or discovered the elegant method of making unlimited DNA copies from a single copy of DNA, and called the method: "Polymerase Chain Reaction" (PCR). A couple of months later he conducted the first successful experiment. Ten years after his drive in California, he was awarded the Nobel Prize in Stockholm for his brilliant discovery (Carr, 1993).
PCR was first published in 1985 (Saiki et al., 1985) with Klenow polymerase used as the elongation enzyme. Due to the heat instability of the Klenow polymerase, new enzyme had to be added for every new cycle, and the maximum limit of the product length was 400 bp. In 1988 the first report using DNA polymerase from Thermophilus aquaticus (Taq-polymerase) was published (Saiki et al., 1988). This polymerase greatly enhanced the value of PCR, and the introduction of the automatic programmable heating block in the same report also took the tedious need for three different water baths out of the procedure. Currently the PCR technique is utilized in most molecular biology laboratories as a routine tool which is suitable for performing a great number of different experiments. The method is frequently chosen for conducting experiments, such as cloning, making mutations, sequencing, detecting, typing, etc. (Erlich et al., 1991).

2. AnimationThe basic molecular events of PCR are illustrated in an animation of the liquid phase DNA amplification, which is a prerequisite of the solid phase DNA amplification. The whole animation can be seen in the DIAPOPS animation.

3. The basic reactionPCR is based on the recognition by a short piece of DNA (the primer) of a sequence on a larger, single stranded fragment of DNA (template strand). When the primer recognizes the template and binds (anneals) to the recognition sequence, the 3'-end of the primer is used by DNA polymerase to synthesize a new DNA strand (elongation). When the temperature is raised, the new DNA strand will melt away (denature) from the template, and the template is once again open for annealing of a new primer when the temperature is decreased. By adding a second primer which recognizes the template strand complementary to the first template, the elongation can proceed in the direction of the first primer. In the first round of elongation, this will ideally double the amount of template strands. In the second temperature cycling, half of the templates for the first primer will be new-synthesized fragments, all terminated where the second primer annealed. When these new fragments are recognized by the first primer, the elongation cannot proceed beyond the second primer, and the synthesized fragments will have a fixed length determined by the distance of the annealing sites of the two primers. New production of template strands take place in every temperature cycle. In this way the DNA sequence between the two primer sequences is amplified exponentially, yielding high concentrations of double-stranded DNA of the same length. The newly-formed double stranded DNA is denatured at 94-97ºC. Primers anneal at 35-72ºC (the exact temperature is primer- and assay dependent), and the new product is synthesized at 72ºC, which is the optimal temperature for the Taq-polymerase.

4. ConclusionPCR is capable of producing large amounts of DNA fragments from a single piece of template DNA as the amplification increases the amount of fragments produced exponentially. In theory, it is possible to detect a single copy of template DNA by PCR using simple methods. For this reason PCR is used to identify nucleic acid sequences that are only present in very small numbers in the sample to be analyzed.

Lecture of PCR-2
Introduction to PCR. Molecular biology relies on techniques that enable the detection or ... With the introduction of the Polymerase Chain Reaction (PCR), ...www.modares.ac.ir/elearning/mnaderi/Genetic%20Engineering%20course%20II/Pages/Lecture2.htm
PCR Technology
Introduction. Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in molecular biology and for good reason: it is a ...www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.html
Introduction to PCR
Either way, the DNA is extracted from the source and is amplified via PCR (the Polymerase Chain Reaction). This allows very minute amounts of DNA to be ...nature.umesci.maine.edu/forensics/p_intro.htm
6.1 Polymerase Chain Reaction (PCR) Introduction6.1 Polymerase Chain Reaction (PCR). Introduction. T. he polymerase chain reaction technique employs oligonucleotide primers to amplify segments of ...www.fws.gov/policy/library/fh_handbook/Volume_1/Chapter_6.pdf
Real-Time PCR Introduction [M.Tevfik DORAK]
Overview by MT Dorak, University of Alabama at Birmingham, USA.dorakmt.tripod.com/genetics/realtime.html
YouTube - EDIROL PCR Introduction
This is a video introduction to our new PCR MIDI controllers.www.youtube.com/watch?v=vfiK7Fl75ZQ

PCR Protocol--PCR SSCP

PCR SSCP

Protocol: Mutation Detection by SSCP PCR.
A protocol for mutation detection by single-strand conformational polymorphism (SSCP) by PCR from the neurogenetics laboratory in the Neurological Sciences ...www.ohsu.edu/nsi/faculty/reddyh/lab/protsscp.html
step by step sscp
Step by Step SSCP. Travis Glenn. Laboratory of Molecular Systematics. Smithsonian Institution. Washington, DC 20560. phone: 301-238-3444. fax: 301-238-3059 ...www.uga.edu/srel/DNA_Lab/SSCP'96V2.rtf
Springer Protocols: Abstract: Multiple Fluorescence-Based PCR-SSCP analysis with primer, post- and internal labeling.
Springer Protocols is the largest subscription-based electronic database of reproducible laboratory protocols in the Life and Biomedical Sciences.www.springerprotocols.com/Abstract/doi/10.1385/0-89603-499-2:51
PCR-SSCP: a practical approach (detailed SSCP protocols)
The multiplexed PCR-SSCP analysis described here (Protocol 5) is essentially a two step procedure, each strand of the target DNA sequence is labelled as it ...europium.csc.mrc.ac.uk/WebPages/Database/Methods/pcrpract.htm
Optimization of Nonisotopic PCR–Single-Strand Conformation Polymorphism.
The protocol used for the GCK gene allowed us to establish a successful strategy for the development of PCR-SSCP on other genes such as BRCA1 (breast cancer ...www.clinchem.org/cgi/content/full/43/11/2190
Genomic Variation Laboratory – SSCP Protocol
Feb 27, 2003 ... SSCP Protocol. MDE gel (BioWhittaker Molecular Applications) final ... 3) For single strands, mix 2 ul PCR product for each sample with 10 ...http://genome-lab.ucdavis.edu/Protocols/SSCP%20Protocols.htm
Evaluating Duplicate Gene Expression using RT-PCR/SSCP Analysis (Wendel Lab.)
The subsequent PCR reaction follows the protocol you have predetermined to work best .... 1995, Identification of DNA polymorphism by asymmetric-PCR SSCP. ...
http://www.eeob.iastate.edu/faculty/WendelJ/rt-pcr_sscp.htm
Sensitive detection of p53 gene mutations by a 'mutant enriched PCR SSCP technique.
In the past, the existing PCR-SSCP technique as established by Orita et al. ... Figure 1 gives a schematic view of the protocol applied whereby the ...nar.oxfordjournals.org/cgi/content/full/26/5/1356
Single-strand conformational polymorphism.
cantly to its utility. SSCP PROTOCOL. The following is an example of an SSCP protocol that we used for detection. $138 PCR Methods and Applications ...www.genome.org/cgi/reprint/4/3/S137.pdf?ck=nck

PCR Protocol--PCR RFLP

PCR RFLP

Restriction fragment length polymorphism (From Wikipedia, the free encyclopedia):
A Restriction Fragment Length Polymorphism (or RFLP, often pronounced as "rif-lip") is a variation in the DNA sequence of a genome which can be detected by a laboratory technique known as gel electrophoresis. Analysis of RFLP variation was an important tool in genome mapping, localization of genetic disease genes, determination of risk for a disease, genetic fingerprinting, and paternity testing.
Contents
1 Analysis technique
2 Examples
3 Applications
4 Alternatives
5 References
5.1 External links

PCR-RFLP Method
18-Oct-02.www.ihwg.org/components/cytokine/mon8Lin.htm
Examples of PCR-RFLP for Nematode Diagnostics
Examples of Restriction Fragment Length Polymorphism (RFLP)electrophoresis slabs for different nematodes, from University of Nebrasca.nematode.unl.edu/its_id/EXAMPLES/index.htm
Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences
Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences.www.kfunigraz.ac.at/~binder/thesis/node64.html
RFLP/PCR Polymorphism Query Form
Search for RFLP and PCR based polymorphisms by strain, locus symbol, or map position. To search for SNPS, use the SNP Query Form. ...www.informatics.jax.org/searches/polymorphism_form.shtml
PCR, RFLP and Gene Therapy
Lecture 24:Genetic Engineering: PCR, RFLP Analysis & Gene Therapy. The Polymerase Chain Reaction (PCR) Can Make Millions of Copies of DNA in a Short Time ...http://members.aol.com/BearFlag45/Biology1A/LectureNotes/lec24.html
Handbook for DNA isolation, RAPD-PCR and PCR-RFLP
General protocol. We use agarose gels for checking the quality of DNA isolates, PCR products, and PCR-RFLP products, and for scoring RAPD products. ...www.toyen.uio.no/botanisk/brochmann/handbook.htm
PCR-RFLP PROTOCOL FOR ALLELES A AND B PCR ...BOVINE KAPPA-CASEIN PCR-RFLP PROTOCOL. FOR ALLELES A AND B. Laboratory of J.F. Medrano. Department of Animal Science. University of California. ...animalscience.ucdavis.edu/laboratory/

PCR Protocol--Competitive Quantitative RT-PCR

Competitive and/or Quantitative RT-PCR

Competitive RT-PCR (Dieter Kaufmann Lab.)

Quantitative RT-PCR (Morimoto Lab.)

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures (Jack Vanden Heuvel Lab.)
http://www.cas.psu.edu/docs/CASDEPT/VET/jackvh/jvhpcr.html

Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample (Biological Procedures Online)
http://www.biologicalprocedures.com/bpo/arts/1/20/m20.htm

Quantitative Measurement of mRNA by Competitive RT-PCR. (Springer Protocols)

Improved quantitative real-time RT–PCR for expression profiling of Individual Cells. (Nucleic Acid Research)

Competitive Quantitative RT-PCR (Ambion)

Comparative Study of Different Standardization Concepts in Quantitative Competitive Reverse Transcription-PCR Assays. (JCM)

Procedure for Competitive Quantitative Reverse Transcription PCR. (Transgenomic)