Double strand cDNA

1
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
A. Double strand DNA
50º
Taq
Taq
3
Taq
Taq
E. Copy strands
Taq
Taq
First round of cDNA synthesis (4 strands)
4
1
2
G. Anneal primers
50º
3
4
5
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