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Polymerase chain reaction

2007 Schools Wikipedia Selection. Related subjects: General Biology

   Polymerase chain reaction (PCR) is a molecular biology technique for
   enzymatically replicating DNA without using a living organism, such as
   E. coli or yeast. Like amplification using living organisms, the
   technique allows a small amount of DNA to be amplified exponentially.
   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, 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 1 one
   night in his car. 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 the 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."

PCR in practice

   Image:Pcr mjhgjgjhgjhgjuachine.jpg
   Figure 1: A thermal cycler for PCR

   PCR is used to amplify specific regions of a DNA strand. This can be a
   single gene, just a part of a gene, or non-coding sequence. PCR
   typically amplifies only short DNA fragments, usually up to 10 kilo
   base pairs (kb). Certain methods can copy fragments up to 47 kb in
   size, which is still much less than the chromosomal DNA of a eukaryotic
   cell - for example, a human cell contains about three billion base
   pairs.

   PCR, as currently practiced, requires several basic components. These
   components are:
     * DNA template, which contains the region of the DNA fragment to be
       amplified
     * Two primers, which determine the beginning and end of the region to
       be amplified (see following section on primers)
     * Taq polymerase (or another durable polymerase), a DNA polymerase,
       which copies the region to be amplified
     * Deoxynucleotide triphosphates, (dNTPs) from which the DNA
       polymerase builds the new DNA
     * Buffer, which provides a suitable chemical environment for the DNA
       Polymerase

   The PCR process is carried out in a thermal cycler. This is a machine
   that heats and cools the reaction tubes within it to the precise
   temperature required for each step of the reaction. To prevent
   evaporation of the reaction mixture (typically volumes between 15-100µl
   per tube), a heated lid is placed on top of the reaction tubes, or a
   layer of oil is put on the surface of the reaction mixture. These
   machines cost more than $2,500 USD, as of 2004.

Primers

   The DNA fragment to be amplified is determined by selecting primers.
   Primers are short, artificial DNA strands — often not more than 50 and
   usually only 18 to 25 base pairs long — that are complementary to the
   beginning or the end of the DNA fragment to be amplified. They anneal
   by adhering to the DNA template at these starting and ending points,
   where the DNA polymerase binds and begins the synthesis of the new DNA
   strand.

   The choice of the length of the primers and their melting temperature
   (T[m]) depends on a number of considerations. The melting temperature
   of a primer -- not to be confused with the melting temperature of the
   template DNA -- is defined as the temperature at which half of the
   primer binding sites are occupied. Primers that are too short would
   anneal at several positions on a long DNA template, which would result
   in non-specific copies. On the other hand, the length of a primer is
   limited by the maximum temperature allowed to be applied in order to
   melt it, as melting temperature increases with the length of the
   primer. Melting temperatures that are too high, i.e., above 80 °C, can
   cause problems since the DNA polymerase is less active at such
   temperatures. The optimum length of a primer is generally from 15 to 40
   nucleotides with a melting temperature between 55°C and 65°C.

   Sometimes degenerate primers are used. These are actually mixtures of
   similar, but not identical, primers. They may be convenient if the same
   gene is to be amplified from different organisms, as the genes
   themselves are probably similar but not identical. The other use for
   degenerate primers is when primer design is based on protein sequence.
   As several different codons can code for one amino acid, it is often
   difficult to deduce which codon is used in a particular case. Therefore
   primer sequence corresponding to the amino acid isoleucine might be
   "ATH", where A stands for adenine, T for thymine, and H for adenine,
   thymine, or cytosine. (See genetic code for further details about
   codons.) Use of degenerate primers can greatly reduce the specificity
   of the PCR amplification. This problem can be partly solved by using
   touchdown PCR.

   The above mentioned considerations make primer design a very exacting
   process, upon which product yield depends:
     * GC-content should be between 40-60%.
     * Calculated T[m] for both primers used in reaction should not differ
       >5°C, and T[m] of the amplification product should not differ from
       primers by >10°C.
     * Annealing temperature usually is 5°C below the calculated lower
       T[m]. However, it should be chosen empirically for individual
       conditions.
     * Inner self-complementary hairpins of >4 and of dimers >8 should be
       avoided.
     * Primer 3' terminus design is critical to PCR success since the
       primer extends from the 3' end. The 3' end should not be
       complementary over greater than 3-4 bases to any region of the
       other primer (or even the same primer) used in the reaction and
       must provide correct base matching to the template.

   There are computer programs to help design primers (see External
   links).

Procedure

   The PCR process usually consists of a series of twenty to thirty-five
   cycles. Each cycle consists of three steps (Fig. 2).
    1. The double-stranded DNA has to be heated to 94-96°C (or 98°C if
       extremely thermostable polymerases are used) in order to separate
       the strands. This step is called denaturing; it breaks apart the
       hydrogen bonds that connect the two DNA strands. Prior to the first
       cycle, the DNA is often denatured for an extended time to ensure
       that both the template DNA and the primers have completely
       separated and are now single-strand only. Time: usually 1-2
       minutes, but up to 5 minutes. Also certain polymerases are
       activated at this step (see hot-start PCR).
    2. After separating the DNA strands, the temperature is lowered so the
       primers can attach themselves to the single DNA strands. This step
       is called annealing. The temperature of this stage depends on the
       primers and is usually 5°C below their melting temperature
       (45-60°C). A wrong temperature during the annealing step can result
       in primers not binding to the template DNA at all, or binding at
       random. Time: 1-2 minutes.
    3. Finally, the DNA polymerase has to copy the DNA strands. It starts
       at the annealed primer and works its way along the DNA strand. This
       step is called elongation. The elongation temperature depends on
       the DNA polymerase. Taq polymerase elongates optimally at a
       temperature of 72 Celsius. The time for this step depends both on
       the DNA polymerase itself and on the length of the DNA fragment to
       be amplified. As a rule-of-thumb, this step takes 1 minute per
       thousand base pairs. 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 all other elongation
       steps, only in that it is longer, typically 10-15 minutes. This
       last step is highly recommendable if the PCR product is to be
       ligated into a T vector using TA-cloning.

   Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at
   94-96°C. (2) Annealing at (eg) 68°C. (3) Elongation at 72°C
   (P=Polymerase). (4) The first cycle is complete. The two resulting DNA
   strands make up the template DNA for the next cycle, thus doubling the
   amount of DNA duplicated for each new cycle (a total of three cycles is
   shown above).
   Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at
   94-96°C. (2) Annealing at (eg) 68°C. (3) Elongation at 72°C
   (P=Polymerase). (4) The first cycle is complete. The two resulting DNA
   strands make up the template DNA for the next cycle, thus doubling the
   amount of DNA duplicated for each new cycle (a total of three cycles is
   shown above).

Example

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

   The reaction mixture consists of
     * 1.0 µl DNA template (100 ng/µl)
     * 2.5 µl of primer, 1.25 µl per primer (100 ng/µl)
     * 1.0 µl Pfu-Polymerase
     * 1.0 µl nucleotides
     * 5.0 µl buffer solution
     * 89.5 µl water

   A 200 µl reaction tube containing the 100 µl mixture is inserted into
   the thermocycler.

   The PCR process consists of the following steps:
    1. Initialization. The mixture is heated at 96°C 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.
    2. Melting, where it is heated at 96°C for 30 seconds. For each cycle,
       this is usually enough time for the DNA to denature.
    3. Annealing by heating at 68°C for 30 seconds:The primers are
       jiggling around, caused by the Brownian motion. Short bondings are
       constantly formed and broken between the single stranded primer and
       the single stranded template. The more stable bonds last a little
       bit longer (primers that fit exactly) and on that little piece of
       double stranded DNA (template and primer), the polymerase can
       attach and starts copying the template. Once there are a few bases
       built in, the Tm of the double-stranded region between the template
       and the primer is greater than the annealing or extension
       temperature.
    4. Elongation by heating 72°C for 45 seconds:This is the ideal working
       temperature for the polymerase. The primers, having been extended
       for a few bases, already have a stronger hydrogen bond to the
       template than the forces breaking these attractions. Primers that
       are on positions with no exact match, melt away from the template
       (because of the higher temperature) and are not extended.

   The bases (complementary to the template) are coupled to the primer on
   the 3' side (the polymerase adds dNTP's from 5' to 3', reading the
   template from 3' to 5' side, bases are added complementary to the
   template)
    1. Steps 2-4 are repeated 25 times, but with good primers and fresh
       polymerase, 15 to 20 cycles is sufficient.
    2. Mixture is held at 7°C. 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°C after just one night.

   The PCR product can be identified by its size using agarose gel
   electrophoresis. Agarose gel electrophoresis is a procedure that
   consists of injecting DNA into 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 a DNA ladder, which contains DNA fragments of known size, also
   within the gel (Fig. 3).

PCR optimization

   Since PCR is very sensitive, adequate measures to avoid contamination
   from other 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 be performed in separate
   areas. For the preparation of reaction mixture, a laminar flow cabinet
   with UV lamp is recommended. 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 (inner control), omitting template DNA, should always
   be performed, to confirm the absence of contamination or primer
   multimer formation.

Difficulties with polymerase chain reaction

   Polymerase chain reaction is not perfect, and errors and mistakes can
   occur. These are some common errors and problems that may occur.

Polymerase errors

   Taq polymerase lacks a 3' to 5' exonuclease activity. This makes it
   impossible for it to check the base it has inserted and remove it if it
   is incorrect, a process common in higher organisms. This in turn
   results in a high error rate of approximately 1 in 10,000 bases, which,
   if an error occurs early, can alter large proportions of the final
   product.

   Other polymerases are available for accuracy in vital uses such as
   amplification for sequencing. 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 from Pyrococcus
   woesii.

Size limitations

   PCR works readily with DNA of lengths two to three thousand basepairs,
   but above this length the polymerase tends to fall off, and the typical
   heating cycle does not leave enough time for polymerisation to
   complete. It is possible to amplify larger pieces of up to 50,000 base
   pairs with a slower heating cycle and special polymerases. These
   special polymerases are often polymerases fused to a DNA-binding
   protein, making them literally "stick" to the DNA longer.

Non specific priming

   The non specific binding of primers is always a possibility due to
   sequence duplications, non-specific binding and partial primer binding,
   leaving the 5' end unattached. This is increased by the use of
   degenerate sequences or bases in the primer. Manipulation of annealing
   temperature and magnesium ion (which stabilise DNA and RNA
   interactions) concentrations can increase specificity. Non-specific
   priming can be prevented during the low temperatures of reaction
   preparation by use of "hot-start" polymerase enzymes where the active
   site is blocked by an antibody or chemical that only dislodges once the
   reaction is heated to 95˚C during the denaturation step of the first
   cycle.

   Other methods to increase specificity include Nested PCR and Touchdown
   PCR.

Practical modifications to the PCR technique

     * Nested PCR - Nested PCR is intended to reduce the contaminations in
       products due to the amplification of unexpected primer binding
       sites. Two sets of primers are used in two successive PCR runs, the
       second set intended to amplify a secondary target within the first
       run product. This is very successful, but requires more detailed
       knowledge of the sequences involved.
     * Intersequence specific (ISSR) PCR
     * Ligation-mediated PCR

     * Inverse PCR - Inverse PCR is 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 digestions and self ligation before cutting by
       an endonuclease, resulting in known sequences at either end of the
       unknown sequence.

     * RT-PCR - RT-PCR (Reverse Transcription PCR) is the method used to
       amplify, isolate or identify a known sequence from a cell or
       tissues RNA library. Essentially normal PCR preceded by
       transcription by Reverse transcriptase (to convert the RNA to cDNA)
       this is widely used in expression mapping, determining when and
       where certain genes are expressed.

     * Assembly PCR - Assembly PCR is the completely artificial synthesis
       of long gene products 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 serve to order the PCR fragments so
       that they selectively produce their final product.

     * 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 ideal. 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. A recent modification on this process, known
       as Linear-After-The-Exponential-PCR ( LATE-PCR), uses a limiting
       primer with a higher melting temperature ( Tm) than the excess
       primer to maintain reaction efficiency as the limiting primer
       concentration decreases mid-reaction.

     * Quantitative PCR - Q-PCR (Quantitative PCR) is used to rapidly
       measure the quantity of PCR product (preferably real-time), thus is
       an indirect method for quantitatively measuring starting amounts of
       DNA, cDNA or RNA. This is commonly used for the purpose of
       determining whether a sequence is present or not, and if it is
       present the number of copies in the sample. There are 3 main
       methods which vary in difficulty and detail.

     * Quantitative real-time PCR is often confusingly known as RT-PCR
       (Real Time PCR) and RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate
       contractions. RT-PCR can also refer to reverse transcription PCR,
       which even more confusingly, is often used in conjunction with
       Q-PCR. This method uses fluorescent dyes and probes to measure the
       amount of amplified product in real time.

     * Touchdown PCR - Touchdown PCR is a variant of PCR that reduces
       nonspecific primer annealing by more gradually lowering the
       annealing temperature between cycles. As higher temperatures give
       greater specificity for primer binding, primers anneal first as the
       temperature passes through the zone of greatest specificity.

     * Hot-start PCR is a technique that reduces non-specific priming that
       occurs during the preparation of the reaction components. The
       technique may be performed manually by simply heating the reaction
       components briefly at the melting temperature before adding the
       polymerase. Specialized enzyme systems have been developed that
       inhibit the polymerase's activity at ambient temperature, either by
       the binding of an antibody or by the presence of covalently bound
       inhibitors that only dissociate after a high-temperature activation
       step.

     * Colony PCR - Bacterial clones ( E.coli) can be screened for the
       correct ligation products. Selected colonies are picked with a
       sterile toothpick from an agarose plate and dabbed into the master
       mix or sterile water. Primers (and the master mix) are added - the
       PCR protocol has to be started with an extended time at 95^^C.

     * RACE-PCR - Rapid amplification of cDNA ends.

     * Multiplex-PCR - The use of multiple, unique primer sets within a
       single PCR reaction to produce amplicons of varying sizes specific
       to different DNA sequences. By targeting multiple genes at once,
       additional information may be elicited from a single test run that
       otherwise would require several times the reagents and technician
       time to perform. Annealing temperatures for each of the primer sets
       must be optimized to work correctly within a single reaction and
       amplicon sizes should be separated by enough difference in final
       base pair length to form distinct bands via gel electrophoresis.

     * Methylation Specific PCR - Methylation Specific PCR (MSP) 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
       PCR reactions 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.

Recent developments in PCR techniques

     * A more recent method which excludes a temperature cycle, but uses
       enzymes, is helicase-dependent amplification.
     * TAIL-PCR, developed by Liu et al. in 1995, is the thermal
       asymmetric interlaced PCR.
     * Meta-PCR, developed by Andrew Wallace, allows to optimize
       amplification and direct sequence analysis of complex genes.
       Details at National Genetic Reference Laboratory, Manchester, UK

Uses of PCR

   PCR can be used for a broad variety of experiments and analyses. Some
   examples are discussed below.

Genetic fingerprinting

   Genetic fingerprinting is a forensic technique used to identify a
   person by comparing his or her DNA with a given sample. An example is
   blood from a crime scene being genetically compared to blood from a
   suspect. The sample may contain only a tiny amount of DNA (obtained
   from a source such as blood, semen, saliva, hair, or other organic
   material)). Theoretically, just a single strand is needed. First, one
   breaks the DNA sample into fragments; then amplifies them using PCR.
   The amplified fragments are then separated using gel electrophoresis.
   The overall layout of the DNA fragments is called a DNA fingerprint.
   Since there is a very tiny possibility that two individuals may have
   the same sequences (one in several million), the technique is more
   effective at acquitting a suspect than proving the suspect guilty. This
   small possibility was exploited by defense lawyers in the controversial
   O.J. Simpson case. A match however usually remains an extremely strong
   indicator also in the question of guilt.

Paternity testing

   Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father.
   (2) Child. (3) Mother. The child has inherited some, but not all of the
   fingerprint of each of its parents, giving it a new, unique
   fingerprint.
   Enlarge
   Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father.
   (2) Child. (3) Mother. The child has inherited some, but not all of the
   fingerprint of each of its parents, giving it a new, unique
   fingerprint.

   Although these resulting 'fingerprints' are unique (except for
   identical twins), genetic relationships, for example, parent-child or
   siblings, can be determined from two or more genetic fingerprints,
   which can be used for paternity tests (Fig. 4). A variation of this
   technique can also be used to determine evolutionary relationships
   between organisms.

Detection of hereditary diseases

   The detection of hereditary diseases in a given genome is a long and
   difficult process, which can be shortened significantly by using PCR.
   Each gene in question can easily be amplified through PCR by using the
   appropriate primers and then sequenced to detect mutations.

   Viral diseases, too, can be detected using PCR through amplification of
   the viral DNA. This analysis is possible right after infection, which
   can be from several days to several months before actual symptoms
   occur. Such early diagnoses give physicians a significant lead in
   treatment.

Cloning genes

   Cloning a gene, not to be confused with cloning a whole organism,
   describes the process of isolating a gene from one organism and then
   inserting it into another organism (now termed a genetically modified
   organism (GMO)). PCR is often used to amplify the gene, which can then
   be inserted into a vector (a vector is a piece of DNA which 'carries'
   the gene into the GMO) such as a plasmid (a circular DNA molecule)
   (Fig. 5). The DNA can then be transferred into an organism (the GMO)
   where the gene and its product can be studied more closely. Expressing
   a cloned gene (when a gene is expressed the gene product (usually
   protein or RNA) is produced by the GMO) can also be a way of
   mass-producing useful proteins, for example medicines or the enzymes in
   biological washing powders. The incorporation of an affinity tag on a
   recombinant protein will generate a fusion protein which can be more
   easily purified by affinity chromatography.
   Figure 5: Cloning a gene using a plasmid.(1) Chromosomal DNA of
   organism A. (2) PCR. (3) Multiple copies of a single gene from organism
   A. (4) Insertion of the gene into a plasmid. (5) Plasmid with gene from
   organism A. (6) Insertion of the plasmid in organism B. (7)
   Multiplication or expression of the gene, originally from organism A,
   occurring in organism B.
   Figure 5: Cloning a gene using a plasmid.
   (1) Chromosomal DNA of organism A. (2) PCR. (3) Multiple copies of a
   single gene from organism A. (4) Insertion of the gene into a plasmid.
   (5) Plasmid with gene from organism A. (6) Insertion of the plasmid in
   organism B. (7) Multiplication or expression of the gene, originally
   from organism A, occurring in organism B.

Mutagenesis

   Mutagenesis is a way of making changes to the sequence of nucleotides
   in the DNA. There are situations in which one is interested in mutated
   (changed) copies of a given DNA strand, for example, when trying to
   assess the function of a gene or in in-vitro protein evolution (also
   known as Directed evolution). Mutations can be introduced into copied
   DNA sequences in two fundamentally different ways in the PCR process.
   Site-directed mutagenesis allows the experimenter to introduce a
   mutation at a specific location on the DNA strand. Usually, the desired
   mutation is incorporated in the primers used for the PCR program.
   Random mutagenesis, on the other hand, is based on the use of
   error-prone polymerases in the PCR process. In the case of random
   mutagenesis, the location and nature of the mutations cannot be
   controlled. One application of random mutagenesis is to analyze
   structure-function relationships of a protein. By randomly altering a
   DNA sequence, one can compare the resulting protein with the original
   and determine the function of each part of the protein.

Analysis of ancient DNA

   Using PCR, it becomes possible to analyze DNA that is thousands of
   years old. PCR 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.

Genotyping of specific mutations

   Through the use of allele-specific PCR, one can easily determine which
   allele of a mutation or polymorphism an individual has. Here, one of
   the two primers is common, and would anneal a short distance away from
   the mutation, while the other anneals right on the variation. The 3'
   end of the allele-specific primer is modified, to only anneal if it
   matches one of the alleles. If the mutation of interest is a T or C
   single nucleotide polymorphism (T/C SNP), one would use two reactions,
   one containing a primer ending in T, and the other ending in C. The
   common primer would be the same. Following PCR, these two sets of
   reactions would be run out on an agarose gel, and the band pattern will
   tell you if the individual is homozygous T, homozygous C, or
   heterozygous T/C. This methodology has several applications, such as
   amplifying certain haplotypes (when certain alleles at 2 or more SNPs
   occur together on the same chromosome Linkage Disequilibrium) or
   detection of recombinant chromosomes and the study of meiotic
   recombination.

Comparison of gene expression

   Researchers have used traditional PCR as a way to estimate changes in
   the amount of a gene's expression. Ribonucleic acid (RNA) is the
   molecule into which DNA is transcribed prior to making a protein, and
   those strands of RNA that hold the instructions for protein sequence
   are known as messenger RNA (mRNA). Once RNA is isolated it can be
   reverse transcribed back into DNA (complementary DNA to be precise,
   known as cDNA), at which point traditional PCR can be applied to
   amplify the gene, this methodology is called RT-PCR. In most cases if
   there is more starting material (mRNA) of a gene then during PCR more
   copies of the gene will be generated. When the products of the PCR
   process are run on an agarose gel (see Figure 3 above) a band,
   corresponding to a gene, will appear larger on the gel (note that the
   band remains in the same location relative to the ladder, it will just
   appear fatter or brighter). By running samples of amplified cDNA from
   differently treated organisms one can get a general idea of which
   sample expressed more of the gene of interest. A quantative RT-PCR
   method has been developed, it is called Real-time PCR .

History

   Polymerase chain reaction was invented by Kary Mullis. He was awarded
   the Nobel Prize in Chemistry in 1993 for his invention, only seven
   years after he and his colleagues at Cetus first reduced his proposal
   to practice. The idea was to develop a process by which DNA could be
   artificially multiplied through repeated cycles of duplication driven
   by an enzyme called DNA polymerase.

   DNA polymerase occurs naturally in living organisms. In cells it
   functions to duplicate DNA when cells divide in mitosis and meiosis.
   Polymerase works by binding to a single DNA strand and creating the
   complementary strand. In the first of many original processes, the
   enzyme was used in vitro (in a controlled environment outside an
   organism). The double-stranded DNA was separated into two single
   strands by heating it to 94°C (201°F). At this temperature, however,
   the DNA polymerase used at the time were destroyed, so the enzyme had
   to be replenished after the heating stage of each cycle. The original
   procedure was very inefficient, since it required a great deal of time,
   large amounts of DNA polymerase, and continual attention throughout the
   process.

   Later, this original PCR process was greatly improved by the use of DNA
   polymerase taken from thermophilic bacteria grown in geysers at a
   temperature of over 110°C (230°F). The DNA polymerase taken from these
   organisms is stable at high temperatures and, when used in PCR, does
   not break down when the mixture was heated to separate the DNA strands.
   Since there was no longer a need to add new DNA polymerase for each
   cycle, the process of copying a given DNA strand could be simplified
   and automated.

   One of the first thermostable DNA polymerases was obtained from Thermus
   aquaticus and was called "Taq." Taq polymerase is widely used in
   current PCR practice. A disadvantage of Taq is that it sometimes makes
   mistakes when copying DNA, leading to mutations (errors) in the DNA
   sequence, since it lacks 3'→5' proofreading exonuclease activity.
   Polymerases such as Pwo or Pfu, obtained from Archaea, have
   proofreading mechanisms (mechanisms that check for errors) and can
   significantly reduce the number of mutations that occur in the copied
   DNA sequence. However these enzymes polymerise DNA at a much slower
   rate than Taq. Combinations of both Taq and Pfu are available nowadays
   that provide both high processivity (fast polymerisation) and high
   fidelity (accurate duplication of DNA).

   PCR has been performed on DNA larger than 10 kilobases, but the average
   PCR is only several hundred to a few thousand bases of DNA. The problem
   with long PCR is that there is a balance between accuracy and
   processivity of the enzyme. Usually, the longer the fragment, the
   greater the probability of errors.

Patent wars

   The PCR technique was patented by Cetus Corporation, where Mullis
   worked when he invented the technique in 1983. The Taq polymerase
   enzyme is 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.
   Interestingly, it seems possible that the legal arguments will extend
   beyond the life of the original PCR and Taq polymerase patents, which
   expire in 2006.
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