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Polymerase chain reaction

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Polymerase Chain Reaction (PCR) is a molecular biological method for amplifying (creating multiple copies of) DNA without using a living organism, such as E. coli or yeast. 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 cloning of genes, and paternity testing.

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History PCR was invented by Kary Mullis, who was awarded the Nobel Prize in Chemistry in October 1993 for this achievement, only seven years after he first published his ideas. Mullis's 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, where it functions to duplicate DNA when cells divide. It works by binding to a single DNA strand and creating the complementary strand. In Mullis's original PCR process, 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 96°C. At this temperature, however, DNA-Polymerase was destroyed so that the enzyme had to be replenished after the heating stage of each cycle. Mullis's original PCR process was very inefficient since it required a great deal of time, vast amounts of DNA-Polymerase, and continual attention throughout the PCR process.

Later, this original PCR process was improved by the use of DNA-Polymerase taken from thermophilic (heat-loving) bacteria that grow in geysers at a temperature of over 110°C. The DNA-Polymerase taken from these organisms is thermostable (stable at high temperatures) and, when used in PCR, did 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 called Taq. Taq polymerase is widely used in current PCR practice (May 2001). A disadvantage of Taq is that it sometimes makes mistakes when copying DNA, leading to mutations (errors) in the DNA sequence. Polymerases such as Pwo or Pfu, obtained from Archea[?], have proofreading mechanisms (mechanisms that check for errors) and can significantly reduce the number of mutations that occur in the copied DNA sequence.

PCR in Practice PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy only short DNA fragments, usually up to 10 kb (kb=kilo base pairs=1000 base pairs). DNA is double-stranded, and therefore, it is measured in complementary DNA building blocks (nucleic acids) called base pairs.' Certain methods can copy fragments up to 40 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)
  • DNA-Polymerase, which copies the region to be amplified
  • Nucleotides, from which the DNA-Polymerase builds the new DNA
  • Buffer, which provides a suitable chemical environment for the DNA-Polymerase

The PCR reaction is carried out in a thermocycler. 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, 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.

Primers

The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial DNA strands--not more than fifty nucleotides Since DNA is usually double-stranded, its length is measured in base pairs. The length of single-stranded DNA is measured in bases or nucleotides.--that exactly match the beginning and end of the DNA fragment to be amplified. They anneal (adhere) 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 depends on a number of considerations. The melting temperature of a primer--not to be confused with the melting temperature of the DNA in the first step of the PCR process--is defined as the temperature below which the primer will anneal to the DNA template and above which the primer will dissociate (break apart) from the DNA template. The melting temperature increases with the length of the primer. 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 temperature required to melt it. Melting temperatures that are too high, i.e., above 80°C, can also cause problems since the DNA-Polymerase is less active at such temperatures. The optimum length of a primer is generally from thirty to forty nucleotides with a melting temperature between 60°C and 75°C.

Procedure

The PCR process consists of a series of twenty to thirty cycles. Each cycle consists of three steps (Fig. 2). First, the double-stranded DNA has to be heated to 96°C in order to separate the strands. This step is called melting; it breaks apart the hydrogen bonds that connect the two DNA strands. Prior to the first cycle, the DNA is often melted for an extended time to ensure that both the template DNA and the primers have completely separated and are now single-strand only.

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. A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random.

Finally, the DNA-Polymerase has to fill in the missing 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. The time for this step depends both on the DNA-Polymerase itself and on the length of the DNA fragment to be amplified.


Figure 2 : Schematic drawing of the PCR cycle.
(1) Melting at 96C. (2) Annealing at 68C. (3) Elongation at 72C (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.

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).

The reaction mixture consists of :

  • 1.0 l DNA template (100 ng/l)
  • 2.5 l of primer, 1.25 l per primer (100 ng/l)
  • 1.0 l Pfu-Polymerase
  • 1.0 l nucleotides
  • 5.0 l buffer
  • 89.5 l H2O

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

The PCR process consists of the following steps:

Step 1
Initialization. Heat the mixture 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.
Step 2
Melting. Heat at 96°C for 30 seconds. For each cycle, this is usually enough time for the DNA to melt.
Step 3
Annealing. Heat at 68°C for 30 seconds.
Step 4
Elongation. Heat at 72°C for 45 seconds.
Step 5
Steps 2-4 are repeated 25 times, but with good primers and fresh polymerase, 15 to 20 cycles is sufficient.
Step 6
Hold mixture 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).


Figure 3 : PCR product compared with DNA ladder in agarose gel.
Image published with permission of Helmut W. Klein, Institute of Biochemistry, University of Cologne, Germany
DNA ladder (lane 1), the PCR product in low concentration (lane 2), and high concentration (lane 3).

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, e.g., blood from a crime scene can be 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, etc. 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.

Paternity Testing

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.


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.

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. PCR is often used to amplify the gene, which can then be inserted into a vector A vector is a means of inserting a gene into an organism. such as a plasmid (a circular DNA molecule) (Fig. 5). The DNA can then be transferred into a different organism where the gene and its product can be studied more closely. Expressing a cloned gene To express a gene means to produce the protein that it determines the production of. can also be a way of mass-producing useful proteins--for example, medicines.


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. 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 czar.



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