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

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

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

1 2 G. Anneal primers 50º 3 4

1 Taq 72º 2 H. Polymerase binds 3 4

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

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

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

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

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

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

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