PCR

PCR

The polymerase chain reaction (PCR) is a biomedical technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. 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.

The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase, after which the method is named, are key components to enable selective and repeated amplification.

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, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis.

A basic PCR set up requires several components and reagents. These components include:

  • DNA template that contains the DNA region (target) to be amplified.
  • Two primers that are complementary to the 3′ (three prime) ends of each of the sense and anti-sense strand of the DNA target.
  • Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C.
  • Deoxynucleoside triphosphates (dNTPs, sometimes called “deoxynucleotide triphosphates”; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.
  • Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
  • Bivalent 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
  • Monovalent cation potassium

Procedure

Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps. The cycling is often preceded by a single temperature step at a high temperature (>90°C).

Initialization step(Only required for DNA polymerases that require heat activation by hot-start PCR): 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.

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 DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

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. This temperature needs to be low enough to allow for hybridization of the primer to the strand, but high enough in order for the hybridization to be specific, i.e. the primer should only bind to a perfectly complementary part of the template. If the temperature is too low, the primer could bind imperfectly. If it is too high, the primer might not bind. Typically the annealing temperature is about 3–5°C 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 formation.

 

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, 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.

Final elongation: This single step is occasionally performed at a temperature of 70–74°C (this is the temperature needed for optimal activity for most polymerases used in PCR) 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.

PCR

 

 

PCR-1

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