170 likes | 302 Views
PCR Polymerase Chain Reaction. Marie Černá, Markéta Čimburová, Marianna Romžová. PCR (Polymerase Chain Reaction). Primers. short oligonucleotides (18 – 30 nucleotides) forward primer x reverse primer are complementary to the sequences at the 3´end of corresponding strand,
E N D
PCRPolymerase Chain Reaction Marie Černá, Markéta Čimburová, Marianna Romžová
Primers • short oligonucleotides (18 – 30 nucleotides) • forward primer x reverse primer • are complementary to the sequences at the 3´end of corresponding strand, delimited the target DNA region that will be amplified
Annealingtemperature • Tm = [4 x (G+C) + 2 x (A+T)] • Tanneal. = [4 x (G+C) + 2 x (A+T) -5] 5´ GAGACTCAAGAGAACCTA 3´ Tanneal. = ?
Number of copies of wanted DNA region A = 2n n = number of cycles
Modification of PCR • nested PCR • multiplex PCR with sequence specific primers
Analysis of the PCR product PCR with general primers: • Hybridization • Sequencing • RFLP (Restriction Fragment Length Polymorphism) • RT-PCR (quantitative PCR) RNA => cDNA