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Biochem pcr (29.6.10)

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Biochem pcr (29.6.10)

  1. 1. Double strand cDNA AAAAA TTTTT RT RT RT Oligo dT primer is bound to mRNA Reverse transcriptase (RT) copies first cDNA strand Reverse transcriptase digests and displaces mRNA and copies second strand of cDNA Conversion of mRNA to cDNA by Reverse Transcription AAAAA TTTTT AAAAA TTTTT
  2. 2. A. Double strand DNA 50 º Taq Taq B. Denature 96 º C. Anneal primers 50 º D. Polymerase binds 72 º
  3. 3. Taq Taq E. Copy strands First round of cDNA synthesis (4 strands) Taq Taq 72 º 1 2 3 4 F. Denature 96 º
  4. 4. 1 2 3 4 50 º G. Anneal primers
  5. 5. 1 2 3 4 72 º H. Polymerase binds Taq Taq Taq Taq
  6. 6. 1 2 3 4 Taq Taq Taq Taq I. Copy strands 72 º Second round of cDNA synthesis (8 strands)
  7. 7. 1 2 3 4 J. Denature at 96 º Anneal primers at 50 º
  8. 8. 1 2 3 4 72 º K. Bind polymerase (not shown) and copy strands Third round of cDNA synthesis (16 strands)
  9. 9. 1 2 3 4 L. Denature at 96 º Anneal primers at 50 º
  10. 10. 1 2 3 4 M. Copy strands at 72º Fourth round of cDNA synthesis (32 strands) 72 º
  11. 11. 1 2 3 4 cDNA strands (32) are now shown as lines
  12. 12. 1 2 3 4 After 5 rounds there are 32 double strands of which 24 (75%) are are same size

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