PCR - Polymerase Chain Reaction.ppt

PCR - Polymerase Chain Reaction PCR is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence. PCR amplification is achieved by using oligonucleotide primers. These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence. The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template. This results in the synthesis of new DNA strands which are complementary to the parent template strands. These new strands have defined 5 ends (the 5 ends of the oligonucleotide primers), whereas the 3 ends are potentially ambiguous in length. Primer selection Primer is an oligonucleotide sequence – will target a specific sequence of opposite base pairing (A-T, G-C only) of single-stranded nucleic acids For example, there is a ? chance (4-1) of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance (4-2) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every 416 bases (=4 294 967 296, or 4 billion): this is about the size of the human or maize genome, and 1000x greater than the genome size of E. coli. Primer Specificity Universal – amplifies ALL bacterial DNA for instance Group Specific – amplify all denitrifiers for instance Specific – amplify just a given sequence Forward and reverse primers If you know the sequence targeted for amplification, you know the size which the primers should be anealing across If you don’t know the sequence… What do you get? DNA Polymerase DNA Polymerase is the enzyme responsible for copying the sequence starting at the primer from the single DNA strand Commonly use Taq, an enzyme from the hyperthermophilic organisms Thermus aquaticus, isolated first at a thermal spring in Yellowstone National Park This enzyme is h