Double strand cDNA

1. AAAAA TTTTT AAAAA TTTTT AAAAA Oligo dT primer is bound to mRNA TTTTT RT Reverse transcriptase (RT) copies first cDNA strand RT Reverse transcriptase digests and displaces mRNA and copies second strand of cDNA RT Double strand cDNA Conversion of mRNA to cDNA by Reverse Transcription

2. B. Denature 96º 50º C. Anneal primers 72º D. Polymerase binds A. Double strand DNA 50º Taq Taq

3. 72º 1 96º 2 F. Denature 3 4 Taq Taq E. Copy strands Taq Taq First round of cDNA synthesis (4 strands)

4. 1 2 G. Anneal primers 50º 3 4

5. Taq Taq Taq Taq 1 72º 2 H. Polymerase binds 3 4

6. 1 Taq 72º 2 Taq I. Copy strands 3 Second round of cDNA synthesis (8 strands) Taq Taq 4

7. 1 J. Denature at 96º Anneal primers at 50º 2 3 4

8. 1 72º K. Bind polymerase (not shown) and copy strands 2 3 Third round of cDNA synthesis (16 strands) 4

9. 1 L. Denature at 96º Anneal primers at 50º 2 3 4

10. 1 M. Copy strands at 72º 72º 2 3 Fourth round of cDNA synthesis (32 strands) 4

11. 1 cDNA strands (32) are now shown as lines 2 3 4

12. 1 After 5 rounds there are 32 double strands of which 24 (75%) are are same size 2 3 4