PCR Polymerase Chain Reaction PCR Polymerase Chain Reaction Marie Černá, Markéta Čimburová, Marianna Romžová.

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Presentation transcript:

PCR Polymerase Chain Reaction PCR Polymerase Chain Reaction Marie Černá, Markéta Čimburová, Marianna Romžová

PCR (Polymerase Chain Reaction)

Primers short oligonucleotides short oligonucleotides (18 – 30 nucleotides) forward primer x reverse primer forward primer x reverse primer are complementary to the sequences are complementary to the sequences at the 3´end of corresponding strand, delimited the target DNA region that will be amplified

Annealing temperature T m = [4 x (G+C) + 2 x (A+T)] T m = [4 x (G+C) + 2 x (A+T)] T anneal. = [4 x (G+C) + 2 x (A+T) -5] T anneal. = [4 x (G+C) + 2 x (A+T) -5] 5´ GAGACTCAAGAGAACCTA 3´ T anneal. = ?

Number of copies of wanted DNA region A = 2 n n = number of cycles

Modification of PCR nested PCR nested PCR multiplex PCR multiplex PCR with sequence specific primers with sequence specific primers

Analysis of the PCR product PCR with general primers : Hybridization Hybridization Sequencing Sequencing RFLP (Restriction Fragment Length RFLP (Restriction Fragment LengthPolymorphism) RT-PCR (quantitative PCR) RT-PCR (quantitative PCR) RNA=> cDNA