Regular PCR Procedure

General PCR Protocols and Its Product Processes

Recommended Reagent Concentrations

Recommended Reaction Conditions

Initial Conditions

Temperature Cycling

"Hot Start" PCR

Asymmetric PCR for ssDNA Production

Detecting Products

Labelling PCR Products with Digoxigenin

Cleaning PCR Products

Sequencing PCR Products

Cloning PCR Products

AND ALWAYS REMEMBER:

Protocol for PCR using Taq DNA Polymerase
Protocol for PCR with Taq DNA Polymerase. Avoiding Contamination. PCR allows the production of more than 10 million copies of a target DNA sequence from ...www.fermentas.com/techinfo/pcr/dnaamplprotocol.htm -

General PCR Protocol
Detailed PCR protocol from the web site of the Department of Biology, University of Michigan, USA.www.mcdb.lsa.umich.edu/labs/maddock/protocols/PCR/general_pcr_protocol.html

Standard PCR
However, efficient sequencing of dsDNA generated by normal PCR is possible using the modification to the SequenaseTM protocol published by Bachmann et al. ...www.mcb.uct.ac.za/pcrcond.htm

PCR PROTOCOL
PCR PROTOCOL FOR cDNA ARRAYS ON MEMBRANES. Purpose: to amplify insert DNA from purified plasmid DNA derived from bacterial. plasmid libraries. ...www.daf.jhmi.edu/microarray/protocols/protocol6.pdf

Basic PCR Protocol
Basic PCR Protocol. CGLab, 7/2002. 1). Wipe down the bench area with bleach and a new paper towel. 2). Take the PCR components out of the freezer to thaw ...www.sfsu.edu/~biology/cgl/media/PCR%20Protocol-Basic.pdf

Long PCR Protocol
Protocol and guidelines for choice of conditions for PCR of long sequences (10 kb or larger). From Genetics Dept., Harvard Medical School,Boston, MA, USA.arep.med.harvard.edu/labgc/estep/longPCR_protocol.html

20-mer Polymerase Chain Reaction Procedure (for MJ Research ...
MJ Research thermal Cycler: 10-mer PCR for amplification of random genomic DNA fragments ... Edit (or choose a program if it has been set up) PCR Program. ...wheat.pw.usda.gov/~lazo/methods/lazo/pcrproto.html

Single tube confirmation PCR protocol
For characterization colonies of transformed clones of Saccharaomyces, from the web site of the Stanford Genome Technology Center, Palo Alto, CA, USA.www-sequence.stanford.edu/group/yeast_deletion_project/single_tube_protocol.html

Protocol for PCR with Hot Start Taq DNA Polymerase
Protocol for PCR with Hot Start Taq DNA Polymerase. How to Avoid Contamination. During PCR, usually more than 10 million copies of a template DNA can be ...www.fermentas.com/profiles/modifyingenzymes/pdf/protocols/protocolhotstart.pdf

A Basic Polymerase Chain Reaction Protocol
Here, a basic, straight-forward PCR protocol is. presented. Where appropriate, some of the choices for modifying this standard reaction ...www.idtdna.com/support/technical/TechnicalBulletinPDF/A_Basic_PCR_Protocol.pdf

PCR Reamplification Protocol
PCR Reamplification for Inadequate or Failed Amplifications. Change your standard PCR protocol for the locus as follows:. decrease the number of cycles by ...genome-lab.ucdavis.edu/Protocols/pcr_tips/pcr_reamplification.htm

Inverse PCR and Sequencing Protocol
Inverse PCR and Sequencing Protocol on 5 Fly Preps. For recovery of sequences flanking XP elements. This protocol is an adaptation of ...flystocks.bio.indiana.edu/pdfs/Exel_links/5__fly_iPCR_XP_pub.pdf

Videos and Animations for PCR

YouTube - BC on Autism 17: A Primer on PCR
YouTube - PCR
YouTube - The qPCR le film FR
Fast PCR Tutorial
PCR reaction
Direct download: PCR movie (1.1 MB)
Direct download: PCR movie (800 KB)
PCR Animation
LinkedIn-PCR Tutorial
PCR--Introduction of PCR

Genotyping by PCR

Methods for Mouse Genotyping by PCR (protocol 1)

1. Preparation of genomic DNA from the mouse tail.

1) Obtain about 5 mm of the mouse tail and cut it symmetrically into two pieces.
Note: Too long tail can result in the inhibition of PCR because of increased impurity.
Put the cut tail into 500 ul lysis buffer 9see below) in a 1.5 ml microfuge tube, which should be
with a rubber ring to prevent leakage of the content. Without DNA degration, tails can be stored at
-80 centigrade even after standing at room temperature for a couple of hours.
2) Incubate at 65 degree centigrade with gentle shaking overnight. When a part of tail tissue remains
because of inactivation of Proteinase K by the high temperature, addition of more Proteinase K is
recommended to lyse the tail completely.
3)--This step is optional--
Detect the quality of the genomic DNA by 1.0% agarose gel electrophoresis. 10 ul of the lysate is
enough for the detection. The sample may not be suitable for the following PCR unless >4kb DNA
is detected.
4) Heat the lysates at 95 degree centigrade for 10 minutes in a PCR machine or by boiling to inactivate
Proteinase K completely.
5) Spin the tail lysate briefly before transferring to a PCR tube to exclude the tissue debris. Proceed
directly to PCR using the tail DNA lysate as a template at a volume rate of 1/10 as follows.

2. PCR reactions.

Contents of PCR mixture for wildtype/knockout allele screening:
5 ul tail DNA solution: spin briefly before transferring to a PCR tube to avoid contamination of debris.
1 ul 10 uM primers (each upper and lower primer)
5 ul 10x KOD dash DNA polymerase (from TOYOBO Co. LTD.,Japan)
5 ul 2.0 mM dNTPs
32 ul dd H2O
Total volume of 50 ul

We recently found that the final volume can be reduced to 25 ul without mineral oil application.

Sequences of PCR primers: should be designed according to your target gene.
Primers for detecting wild-type allele
Primers for detecting knock-out allele

Methods for Mouse Genotyping by PCR (protocol 2)

Transgenic Genotyping from Tail Biopsies
Harvard University--MCB Department / HSCI
Remove .5-1 cm of the tail and place in 1.5 ml Eppendorf tube. (Store at -20oC until ready to digest).
Digest in Lysis Buffer* + Proteinase K (to 200 ug/ml final conc.).
Incubate in 55oC water bath overnight. (Vortex 1x after 1-2 h).
Add .5 ml Phenol:Chloroform:Isoamyl alcohol (25:24:1) to each tube and vortex for 30 sec.
Spin at top speed in a microcentrifuge for 5 minutes.
Transfer upper (aqueous) phase to new tube; make sure no debris from the interface is transferred.
Add 1 ml of 100% EtOH.
Vortex briefly or shake. Stringy white precipitate (the genomic DNA) should now be visible.
Spin briefly (<1 min) just enough to get the DNA to cling to the plastic, and decant supernatant.
Wash with 1 ml of 70% EtOH.
Let air dry until the pellet becomes partially translucent, but do NOT over-dry, or the DNA will not go into solution any longer.
Redissolve the pellet in 100 ml TE, pH 8.0.
Check concentration, and calculate the total yield, which should be around 10 to 50 mg.
Use 100 ng for subsequent PCR analysis.
*Lysis Buffer:
10 mM Tris-HCl, pH 8.0
25 mM EDTA, pH 8.0
100 mM NaCl
0.5% SDS

DNA from Tail Biopsies

Genotyping Transgenic Rodents by PCR

Isolation of DNA from Mouse Tail Biopsies

Lac-Z Detection in Tail Biopsies

Preparation of Mouse Tail DNA for Dot Blots or PCR

Universal Mouse Genotyping Protocol Using PCR

beta globin Primers

lacZ Primers

neo Primers

PCR Primer Design Tools

Primer3
PrimerQuest
Primer Premier
FastPCR
PrimerX
OligoMaster
PerlPrimer
Methprimer
NetPrimer
Oligo2002
CODEHOP
The Primer Generator
Primer Design Assistant
PROBEmer
GenomePride
Pride
TGGE-Star
Primer3 (UMass server)
Exon Locator and Extractor for Resequencing
AutoPrime -primer design software

PCR Troubleshooting

Ten Things That Can Kill Your PCR
Ten Things That Can Kill Your PCR. by Peter Frame. A blank PCR gel has got to be one of the most aggravating things about. molecular biology. ...www.mbi.ufl.edu/~rowland/protocols/pcr.htm

PCR trouble shooting, help, suggestions and advice
PCR trouble shooting, help, suggestions and advice. If your PCR amplification somehow performs unexpectedly, it is usually caused by one of the listed ...biologi.uio.no/bot/ascomycetes/PCR.troubleshooting.html

PCR Troubleshooting
Troubleshooting PCR. Polymerase Chain Reaction problems and solutions, PCR help.www.pcrstation.com/pcr-troubleshooting/

Troubleshooting Guide
MultiplexPCR Troubleshooting Guide. Poor amplification of some or all loci. Pipetting error /. reagents missing. Repeat experiment checking the ...
www.abgene.com/downloads/Guide_PCR-multiplex-v2-0208.pdf

PCR-Online.org - PCR Protocols, Troubleshooting and Information
Westernblotting.org: definitions, molecular biology links, protocols, troubleshooting and technical information for those interested in western blots and...
www.pcr-online.org/Troubleshooting.htm

PCR troubleshooting - MyBio
PCR troubleshooting - Web Resources. Optimizing DNA Amplification Protocols Optimizing DNA Amplification Protocols using the Eppendorf ?? Mastercycler ?? ...mybio.net/biowiki/PCR_troubleshooting

Troubleshooting PCR Why do I have non-specific bands when I run my ...
Appendix III:Troubleshooting. Successful PCR Guide. Takara Mirus Bio. 38. Causes.Trouble-shooting measures. Concentration of primers is too high ...www.takarabiousa.com/docs/PCR_TRBSHT.pdf -

Troubleshooting the PCR procedure Specific application of PCR ...
Troubleshooting the. PCR procedure. For a detailed discussion of the factors that. influence PCR and how to troubleshoot the ...www.roche-applied-science.com/PROD_INF/MANUALS/epitope/p18-19.pdf

Optimization and troubleshooting in PCR.
Optimization and troubleshooting in PCR. References. http://www.genome.org#References. This article cites 42 articles, 13 of which can be accessed free at: ...www.genome.org/cgi/reprint/4/5/S185.pdf -

EdgeBio ExcelaPure 96-Well UF PCR Purification Kit Troubleshooting ...
Troubleshooting Guide forExcelaPure 96-Well UF PCR Purification Kit>www.edgebio.com/tech/tsg/ExcelaPure96-wellUF_TSG.html

About PCR

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

Introduction to PCR

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

Polymerase chain reaction--PCR

From Wikipedia, the free encyclopedia

"PCR" redirects here. For other uses, see PCR (disambiguation).

A strip of eight PCR tubes, each containing a 100μl reaction.
The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
Developed in 1983 by Kary Mullis,[1] PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.[2][3] These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis won the Nobel Prize in Chemistry for his work on PCR.[4]

Contents

1 PCR principles and procedure
1.1 Procedure
2 PCR stages
2.1 PCR optimization
3 Application of PCR
3.1 Isolation of genomic DNA
3.2 Amplification and quantitation of DNA
3.3 PCR in diagnosis of diseases
4 Variations on the basic PCR technique
5 History
5.1 Patent wars
6 References
7 External links

PCR principles and procedure

PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.[5]
A basic PCR set up requires several components and reagents.[6] These components include:
DNA template that contains the DNA region (target) to be amplified.
Two primers, which are complementary to the DNA regions at the 5' (five prime) or 3' (three prime) ends of the DNA region.
A DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C.
Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis[7]
Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 20-150 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Procedure

Schematic drawing of the PCR cycle. (1) Denaturing at 94-96°C. (2) Annealing at ~65°C (3) Elongation at 72°C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses.
The PCR usually consists of a series of 20 to 40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps (Fig. 2). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.[8]
Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.[9]
Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C,[10][11] and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.

Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.

Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.
To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products.

PCR stages

The PCR process can be divided into three stages:
Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very specific and precise.[citation needed]
Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.
Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

PCR optimization

Main article: PCR optimization
In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.[12][13] Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.[6] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA.

Application of PCR

Isolation of genomic DNA
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.
Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constructs[14]. PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.

Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing. This technique may also be used to determine evolutionary relationships among organisms.


Amplification and quantitation of DNA

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.[15]
Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample – a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.
See also Use of DNA in forensic entomology

PCR in diagnosis of diseases

PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely.[citation needed] PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.[citation needed]
PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.[citation needed]
Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below).

Variations on the basic PCR technique

Main article: Variants of PCR
Allele-specific PCR: This diagnostic or cloning technique is used to identify or utilize single-nucleotide 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.[16] See SNP genotyping for more information.
Assembly PCR or Polymerase Cycling Assembly (PCA): Assembly PCR is the 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.[17]
Asymmetric PCR: Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.[18] A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Melting temperatureTm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.[19]
Helicase-dependent amplification: This technique is 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.[20]
Hot-start PCR: This is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase.[21] Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody[9] 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 instantly activated at elongation temperature.
Intersequence-specific (ISSR) PCR: a PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.[22]
Inverse PCR: a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.[23]
Ligation-mediated PCR: This method 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.[24]
Methylation-specific PCR (MSP): The MSP method was developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine,[25] 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.
Miniprimer PCR: Miniprimer PCR uses a novel thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides, instead of the approximately 20 nucleotides required by Taq. This method permits PCR targeting smaller primer binding regions, and is particularly useful to amplify unknown, but conserved, DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene. 16S rRNA miniprimer PCR was used to characterize a microbial mat community growing in an extreme environment, a hypersaline pond in Puerto Rico. In that study, deeply divergent sequences were discovered with high frequency and included representatives that defined two new division-level taxa, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity.[26] By enlarging the "sequence space" that may be queried by PCR primers, this technique may enable novel PCR strategies that are not possible within the limits of primer design imposed by Taq and other commonly used enzymes.
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: The use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes 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, i.e., 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 being 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: is a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.
Quantitative PCR (Q-PCR): is used to measure the quantity of a PCR product (preferably real-time). It is the method of choice to quantitatively measure 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. The method with currently the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR commonly refers to reverse transcription PCR (see below), which is often used in conjunction with Q-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 in real time.
RT-PCR: (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. 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 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.
Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (the only primers present are covalently linked to 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[27] (where conventional Solid Phase PCR can be improved by employing high Tm solid support primer with application of a thermal 'step' to favour solid support priming).
TAIL-PCR: Thermal asymmetric interlaced PCR is used to isolate 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.[28]
Touchdown 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 (3-5˚C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5˚C) 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.[29]
PAN-AC: This method uses isothermal conditions for amplification, and may be used in living cells.[30][31]
Universal Fast Walking: this method allows 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') [32] 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) [33], 5'RACE LaNe [34] and 3'RACE LaNe [35].

History

Main article: History of polymerase chain reaction
A 1971 paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro.[36] However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.[37]
At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90°C (>195°F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures.[2] So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.
A 1976 discovery of Taq polymerase a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F)) environments[10] paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation,[11] thus obviating the need to add new DNA polymerase after each cycle[3]. This allowed an automated thermocycler-based process for DNA amplification.
At the time he developed PCR in 1983, Mullis was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car.[38] He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase.
In Scientific American, Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."[39] He was awarded the Nobel Prize in Chemistry in 1993 for his invention,[4] seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis' work, and whether he had been the sole inventor of the PCR principle. (see main article: Kary Mullis)

Patent wars

The PCR technique was patented by Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.
A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega. The legal arguments have extended beyond the life of the original PCR and Taq polymerase patents, which expired on March 28, 2005[40]

References

^ Bartlett & Stirling (2003)—A Short History of the Polymerase Chain Reaction. In: Methods Mol Biol. 226:3-6
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External links

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Polymerase chain reaction
PCR at Home - Amateur Scientist article in the July 2000 issue of Scientific American on performing PCRs with low-cost household materials.
US Patent for PCR
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Step-through animation of PCR - From Cold Spring Harbor's Dolan DNA Learning Center. Adobe Flash required.