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Polymerase Chain Reaction

Polymerase Chain Reaction



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Polymerase chain reaction - Wikipedia, the free encyclopedia
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Polymerase chain reaction
From Wikipedia, the free encyclopedia

The polymerase chain reaction (PCR) is a biochemistry and molecular biology technique[1] for exponentially amplifying DNA, via enzymatic replication, without using a living organism (such as E. coli or yeast). As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations.

PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the
identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing.
PCR was invented by Kary Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, California for Cetus
Corporation, one of the first biotechnology companies. There, he was charged with making short chains of DNA for other scientists.

Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car[2]. 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. Mullis has said that before his trip was over, he was already savoring the prospects of a Nobel Prize. He
shared the Nobel Prize in Chemistry with Michael Smith in 1993.

As Mullis has written in Scientific American: "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."[3]

1 PCR in practice
1.1 Primer (molecular biology)
1.2 Procedure
1.3 Example
1.4 PCR optimization

1.4.1 Hairpins
1.4.2 Polymerase errors
1.4.3 Size and other limitations
1.4.4 Non-specific priming

1.5 Practical modifications to the PCR technique
1.6 Recent developments in PCR techniques

2 Uses of PCR
2.1 Genetic fingerprinting
2.2 Paternity testing
2.3 Detection of hereditary diseases
2.4 Cloning genes
2.5 Mutagenesis
2.6 Analysis of ancient DNA
2.7 Genotyping of specific mutations
2.8 Comparison of gene expression

3 History
4 Patent wars
5 References
6 External links

PCR in practice
PCR is used to amplify specific regions of a DNA strand. This can be a single gene, just 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). Some PCR methods can copy DNA fragments of up to 40 kb in size[4], which is still much less than the total nuclear DNA content of a eukaryotic cell - for comparison, the haploid genome of a human cell consists of about three billion DNA base pairs (3 Gb).

PCR, as currently practiced, requires several basic components[5]. These components are:
DNA template that contains the region of the DNA fragment to be amplified

One or moreprimers, which are complementary to the DNA regions at the 5' and 3' ends of the DNA region that is to be amplified (see
following section on primers)
a DNA polymerase (e.g. Taq polymerase or another DNA polymerase with a temperature optimum at around 70\u00b0C), used to synthesize a DNA
copy of the region to be amplified

Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA
Buffer solution, which provides a suitable chemical environment for optimum activity and stability of the DNA polymerase
Divalent cation, 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 [6]
Monovalent cation potassium ions
The PCR is carried out in small reaction tubes (0.2-0.5 ml volumes), containing a reaction volume typically of 15-100 \u00b5l, that are inserted into a thermal cycler. This is a machine that
eats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. Most thermal cyclers have heated lids to prevent condensation on the inside of
the reaction tube caps. Alternatively, a layer of oil may be placed on the reaction mixture to prevent evaporation. These machines cost more than $2,500 USD, as of 2004.
Primer (molecular biology)

To selectively PCR-amplify a DNA fragment, suitable primers need to be designed and synthesized. Primers are short oligonucleotides, i.e., chemically synthesized, single-stranded DNA fragments \u2014 often not more than 50 and usually only 18 to 25 base pairs long \u2014 containing nucleotides that are complementary to the nucleotides at both ends of the DNA fragment to be amplified. These complementary bases in primer and DNA template facilitate annealing of the primer to the DNA template to which the DNA polymerase can bind and begin with the

PCR tubes in a stand after a colony PCR
Figure 1: A thermal cycler for
Polymerase chain reaction - Wikipedia, the free encyclopedia
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synthesis of a new DNA strand that is complementary to the DNA template (as described below).

The length of the primers and their desired melting temperature (Tm) depend on a number of considerations. The T m of a primer -- not to be confused with the T m of the template DNA --
is defined as the temperature at which half of the primer binding sites at the DNA template are occupied. As the Tm increases with the length of the primer (provided the content of
guanine (G) and cytosine (C) relative to adenine (A) and thymine (T) base residues remains constant), primers that are short (<15 base pairs) require a lower annealing temperature
(<50\u00b0C) for efficient amplification. Owing to the inherently less complex base composition of short primers, these can potentially anneal at several positions on a DNA template, which
would result in undesirable non-specific amplification. On the other hand, using a primer that is very long (>40 base pairs) requires annealing temperatures that are above 80 \u00b0C, i.e., at
temperatures that impinge on the activity and stability of the DNA polymerase. The optimum length of a primer (with a G+C content of 40-60%) is generally from 15 to 40 nucleotides
with an annealing temperature between 50\u00b0C and 74\u00b0C.

Some PCR applications require the use ofdegenerate primers, which are mixtures of primers having one or more differences in bases at specific positions. The use of degenerate primers is called for in instances where the exact sequence of a DNA template is unknown, or amplification of DNA fragments with slightly different sequence is desired. For example, they may be required if a homologous gene (i.e., a gene with similar function, but dissimilar DNA sequence) is to be amplified from different organisms. Another common use for degenerate primers is required when primer design can only be performed on protein sequence. As several different nucleotide codons can code for one amino acid (see Degeneracy of the genetic

code at Genetic code), most protein sequences can be encoded by several different DNA sequences. A primer sequence corresponding to the codon for the amino acid isoleucine may be
"ATH", where A stands for adenine, T for thymine, and H for adenine, thymine, or cytosine. Use of degenerate primers can greatly reduce the specificity of the PCR amplification. This
problem can be partly overcome by using touchdown PCR.
The above-mentioned considerations make primer design a multi-step process to ensure good yield and quality of the PCR product:

The GC-content should be between 40-60%; ideally there should be an even distribution of G+C and A+T along the primer.
The calculated Tm for both primers used in reaction should not differ >5\u00b0C.
The ideal annealing temperature usually is 5\u00b0C below the calculated primer Tm. However, it should be chosen empirically for individual conditions.
Inner self-complementary hairpins of >4 and of dimers >8 should be avoided along with long runs (>4) of G or C residues.
Primer 3' terminus design is critical to PCR success since the nascent DNA strand extends from the 3' end of the primer. The primer 3' end should not contain more than 3-4 bases
that are complementary to any region within itself or the other primer used in the reaction and must correctly match the bases in the template.
It is often preferable to have a G or C nucleotide at the final 3' terminus of the primer ("G-C clamp"), as this enhances efficient strand elongation in the PCR; however having more
than three G or C residues within the five terminal bases at the 3' end should be avoided.

While it is very useful to follow these guidelines, in practice, it is often impossible to design primers that fulfil all of the above criteria. Because of the high number of variables that can affect PCR, even suboptimally designed primers may work well, so it is generally advisable to determine optimum conditions empirically, e.g., by using different primer combinations or changing selected PCR conditions. There are computer programs that can assist in designing primers (see External links), but the final call can often only be made after experimental trials.

The PCR usually consists of a series of 20 to 35 cycles. Most commonly, PCR is carried out in three steps (Fig. 2), often preceded by
one temperature hold at the start and followed by one hold at the end.

Prior to the first cycle, during an initialization step, the PCR reaction is often heated to a temperature of 94-96\u00b0C (or 98\u00b0C if
extremely thermostable polymerases are used), and this temperature is then held for 1-9 minutes. This first hold is employed to
ensure that most of the DNA template and primers are denatured, i.e., that the DNA ismelted by disrupting the hydrogen bonds
between complementary bases in two DNA strands. Also, some PCR polymerases require this step for activation (see hot-start
PCR). Following this hold, cycling begins, with one step at 94-98\u00b0C for 20-30 seconds (denaturation step).


The denaturation is followed by theannealing step. In this step the reaction temperature is lowered so that the primers can attach to the single-stranded DNA template. The temperature at this step depends on the Tm of the primers (see above), and is usually between 50-64\u00b0C for 20-40 seconds.


The annealing step is followed by an extension/elongation step during which the DNA polymerase copies the DNA template,
starting at the primers annealed to both of its strands. The temperature at this step depends on the DNA polymerase used. Taq
polymerase has a temperature optimum of 70-74\u00b0C; thus, in most cases, during the extension a temperature of 72\u00b0C is used. The
extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a
practical rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute. A

final elongation step is frequently used after the last cycle to ensure that any remaining single-stranded DNA is completely

copied. This differs from the other elongation steps only in that it is longer--typically 10-15 minutes. A final hold of 4-15\u00b0C for
an indefinite time is often employed to allow short-term storage of the reaction, especially if reactions are run overnight, and
cannot be removed immediately after the cycling.

The times and temperatures given in this example are taken from a PCR program that was successfully used on a 250 bp fragment of
the C-terminus of the insulin-like growth factor (IGF).
Figure 2: Schematic drawing of the PCR cycle. (1)
Denaturing at 94-96\u00b0C. (2) Annealing at ~65\u00b0C (3)
Elongation at 72\u00b0C. Four cycles are shown here.
Polymerase chain reaction - Wikipedia, the free encyclopedia
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The reaction mixture consists of

1.0 \ue000l DNA template (100 ng/\ue000l)
2.5 \ue000l of primer, 1.25 \ue000l per primer (100 ng/\ue000l)
1.0 \ue000l Pfu-Polymerase
1.0 \ue000l nucleotides
5.0 \ue000l buffer solution
89.5 \ue000l water

A 200 \ue000l reaction tube containing the 100 \ue000l mixture is inserted into the thermocycler.
The PCR process consists of the following steps:
Initialization. The mixture is heated at 96\u00b0C for 5 minutes to ensure that the DNA strands as well as the primers have melted.
The DNA Polymerase can be present at initialization, or it can be added after this step.
Melting, where it is heated at 96\u00b0C for 30 seconds. For each cycle, this is usually enough time for the DNA to denature.

Annealing by heating at 68\u00b0C for 30 seconds: The primers are moving around, caused by Brownian motion. Hydrogen bonds
along short stretches of DNA are constantly formed and broken between the single-stranded primer and the single-stranded
template. Stable bonds are only formed when the primer sequence exactly fits the template sequence, and on that short piece of
double-stranded DNA (template and primer), the polymerase can attach and start copying the template. Once this extension has
created a longer double-stranded DNA segment, the Tm of this double-stranded region is now greater than the annealing or
extension temperature.


Elongation by heating 72\u00b0C for 45 seconds: This is the ideal working temperature for the polymerase. The combined hydrogen bonds between the extended primer and the DNA template are now strong enough to withstand forces breaking these attractions at the higher temperature. Primers that are on positions with no exact match, melt away from the template (because of the higher temperature) and are not extended.

The DNA polymerase condenses the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand, i.e., the polymerase adds dNTP's that
are complementary to the template in 5' to 3' direction, thus reading the template in 3' to 5' direction.
Steps 2-4 are repeated 25-35 times, but with good primers and fresh polymerase, 15 to 20 cycles may be sufficient.

Mixture is held at 7\u00b0C. This is useful if one starts the PCR in the evening just before leaving the lab, so it can run overnight. The DNA will not be damaged at 7\u00b0C after just one


The correct PCR product can be identified by its size, using agarose gel electrophoresis. Agarose gel electrophoresis is a procedure that consists of loading DNA into small wells of an agarose gel and then applying an electric current to the gel. As a result, the smaller DNA strands move faster than the larger strands through the gel toward the positive current. The size of the PCR product can be determined by comparing it with aDNA ladder, which contains DNA fragments of known size, also loaded onto the gel (Fig. 3).

PCR optimization

Since PCR is very sensitive, i.e., requiring only a few DNA molecules for amplification across several orders of magnitude, adequate measures to avoid contamination from any DNA
present in the lab environment ( bacteria, viruses, lab staff's skin etc.) should be taken. Thus DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the
subsequent reaction product analysis, should all be performed in separate areas. In practice, one area should be dedicated to reaction assembly before the PCR and another area to
post-PCR processing, such as electrophoresis or purification of PCR products. For the preparation of reaction mixtures, a laminar flow cabinet with UV lamp is recommended, and
pipettes with filter tips should be used. Fresh gloves should be used for each PCR step as well as displacement pipettes with aerosol filters. The reagents for PCR should be prepared
separately and used solely for this purpose. Aliquots should be stored separately from other DNA samples. A control reaction, omitting template DNA (also called negative control),
should always be performed alongside experimental PCRs, to check for possible contamination of reagents with extraneous DNA or for primer multimer formation.


Secondary structures in the DNA, caused by base-pairing between nucleotides on the same strand of the molecule, can cause folding or even knotting of the DNA template or the primers, leading to decreased yield or total failure of the reaction. Hairpins, direct folding of the DNA caused by a run of complementary bases or an inversion, are the most common problems of this sort.

Typically, this calls for choosing different primers; secondary structures in the template DNA are not as serious as those in the primers, as the DNA polymerase will "flatten out" most
secondary structures unless they are particularly robust.
However, if use of hairpin-forming primers is necessary, as may be the case in PCR splicing and cloning, the problem can be ameliorated somewhat by use of DMSO or glycerol; these
chemicals can be added to the PCR mastermix to interrupt secondary structures.
Polymerase errors

Taq polymerase lacks a 3' to 5' exonuclease activity. This makes it impossible for it to do error proofreading, i.e., check the last base it has inserted and excise it if the base does not
match with the base in the complementary strand. This lack in 3' to 5' proofreading results in a high error rate of approximately 1 in 10,000 bases, which, if an error occurs early in the
PCR, can cause accumulation of a large proportion of amplified DNA with incorrect sequence in the final product. Several "high-fidelity" DNA polymerases, having engineered 3' to 5'
exonuclease activity have become available that permit more accurate amplification for use in amplification for sequencing or cloning. Examples of polymerases with 3' to 5' exonuclease
activity include: KOD DNA polymerase, a recombinant form of Thermococcus kodakaraensis KOD1; Vent, which is extracted from Thermococcus litoralis; Pfu DNA polymerase, which
is extracted from Pyrococcus furiosus; and Pwo, which is extracted fromPyrococcus woesii.

Size and other limitations

PCR works readily with DNA of up two to three thousand base pairs in length. However, above this size, product yields often decrease, as with increasing length stochastic effects such as premature termination by the polymerase begin to affect the efficiency of the PCR. It is possible to amplify larger pieces of up to 50,000 base pairs with a slower heating cycle and special polymerases. These are polymerases fused to a processivity-enhancing DNA-binding protein, making them literally "stick" to the DNA longer[7][8].

Other valuable properties of the prototype chimeric polymerases TopoTaq (http://www.fidelitysystems.com/TopoTaq.html) and PfuC2 include enhanced thermostability, specificity and resistance to contaminants and inhibitors[9][10]. They were engineered using unique Helix-hairpin-Helix (HhH) DNA binding domains of Topoisomerase V[11] from hyperthermophile Methanopyrus kandleri. Chimeric polymerases overcome many limitations of native enzymes and are used in direct PCR amplification from cell cultures and even food samples, thus by-passing laborious DNA isolation steps altogether. A robust strand displacement activity of the hybrid TopoTaq polymerase helps solving PCR problems with #Hairpins and G-loaded double helices, because helices with a high G-C context possess a higher melting temperature[12].

Non-specific priming

Negative Gel electrophoresis image of a standard
PCR. Two sets of specific primers were used to
amplify one gene from three separate tissues. As the
gel shows, Tissue #1 lacks that gene, whereas Tissue
#2 and #3 possess that gene.

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