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PCR Polymerase Chain Reaction

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,

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PCR Polymerase Chain Reaction

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  1. PCRPolymerase Chain Reaction Marie Černá, Markéta Čimburová, Marianna Romžová

  2. PCR (Polymerase Chain Reaction)

  3. 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

  4. Annealingtemperature • Tm = [4 x (G+C) + 2 x (A+T)] • Tanneal. = [4 x (G+C) + 2 x (A+T) -5] 5´ GAGACTCAAGAGAACCTA 3´ Tanneal. = ?

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

  6. Modification of PCR • nested PCR • multiplex PCR with sequence specific primers

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

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