RAPD,Random Amplification of Polymorphic DNA

목차

PCR

Genetic marker

RAPD

Another marker

본문내용

RAPD






PCR(Polymerase chain reaction)
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▶ PCR is a technique in molecular biology to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

▶ PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.


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PCR(Polymerase chain reaction)
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▶ 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; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Divalent 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 ions.

▶ PCR stages
Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present.
Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.
Plateau. No more product accumulates due to exhaustion of reagents and enzyme.

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Genetic marker
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▶ Gene or DNA sequence with a known location on a chromosome that can be used to identify cells, individuals or species. It can be described as a variation, which may arise due to mutation or alteration in the genomic loci, that can be observed.

▶ A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like minisatellites.

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키워드

RAPD.,   PCR(Polymerase chain reaction),   Genetic marker,   Random Amplification of Polymorphic DNA