Types of Polymerase Chain Reaction

Only available on StudyMode
  • Download(s) : 85
  • Published : April 24, 2013
Open Document
Text Preview
Apeh Daniel O. TYPES OF POLYMERASE CHAIN REACTION
DNA Replication which forms the basis of biological evolution and inheritance [1] is a "semi conservative" process in that each (one) strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Hence, following DNA replication, two identical DNA molecules are been produced from a single double-stranded DNA molecule [2]. The need to amplify genes for various purposes among which are forensic application, genome studies, medical applications have led to the development of various techniques now known as polymerase chain reaction (PCR) as a more convenient alternative of gene cloning via recombinant DNA technology. The idea of Polymerase chain reaction came up in 1983 when Kary Mullis a scientist working for Cletus cooperation was driving along US route 101 in Northern California; it was then introduced into the scientific community in 1985 at a conference in October where Cetus also rewarded kary Mullis with $10,000 bonus for his invention. Later, during a corporate reorganization, Cetus sold the patent for the PCR process to a pharmaceutical company Hoffmann-LaRoche for $300 million [3]. PCR technique which is also called a DNA photocopier, is an in vitro technique that uses a few basic everyday molecular biology reagents to make large numbers of copies of a specific DNA fragment or a specific region of a DNA strand in a test-tube. The process which is carried out in a PCR machine requires DNA template, primer(s), Taq or other polymerase(s), deoxynucleoside triphosphates (dNTPs), buffer solution and divalent cations (eg.Mg2+ ) to run [2]. The basic steps in conducting a conventional PCR involves; Denaturation achieved by heating the reaction mixture to a temperature between 90-98° C such that the dsDNA is denatured into single strands by disrupting the hydrogen bonds between complementary bases, Annealing achieved by cooling the reaction mixture to a temperature of 45-60° C such that the primers base pair with the complementary sequence in the DNA and the hydrogen bonds reform and Elongation achieved by adjusting the temperature to 72° C which is ideal for polymerase allowing primers extension by joining the bases complementary to DNA strands, the polymerase continually adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template. This completes a first cycle another cycle is continued. As PCR machine is automated thermocycler the same cycle is repeated upto 30-40 times [1]. This review attempts to summarize as many types of PCR as possible including the principles on which they work, their applications and in some cases their advantages and disadvantages as well as experimental procedures where necessary. The emphasis is neither placed on the PCR machine level of sophiscation nor time of its use (old or new) but on technical difference basically brought about by different applications of PCR.

TYPES OF POLYMERASE CHAIN REACTION

INVERSE PCR
The inverse PCR method includes a series of digestions and self-ligations with the DNA being cut by a restriction endonuclease. This cut results in a known sequence at either end of unknown sequences [4]. Inverse PCR Steps

1) Target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion. 2) Self-ligation is induced under low concentrations causing the phosphate backbone to reform. This gives a circular DNA ligation product. 3) Target DNA is then restriction digested with a known endonuclease. This generates a cut within the known internal sequence generating a linear product with known terminal sequences. This can now be used for PCR (polymerase chain reaction). 4) Standard PCR is conducted with primers complementary to the now known internal sequences. Inverse PCR uses standard PCR (polymerase chain reaction), however it has the primers oriented in the...
tracking img