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Double strand cDNA - PowerPoint PPT Presentation


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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.

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PowerPoint Slideshow about 'Double strand cDNA' - bernad


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Presentation Transcript
slide1

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

slide2

B. Denature

96º

50º

C. Anneal primers

72º

D. Polymerase binds

A. Double strand DNA

50º

Taq

Taq

slide3

72º

1

96º

2

F. Denature

3

4

Taq

Taq

E. Copy strands

Taq

Taq

First round of cDNA synthesis (4 strands)

slide4

1

2

G. Anneal primers

50º

3

4

slide5

Taq

Taq

Taq

Taq

1

72º

2

H. Polymerase binds

3

4

slide6

1

Taq

72º

2

Taq

I. Copy strands

3

Second round of cDNA synthesis (8 strands)

Taq

Taq

4

slide7

1

J. Denature at 96º

Anneal primers at 50º

2

3

4

slide8

1

72º

K. Bind polymerase (not shown) and copy strands

2

3

Third round of cDNA synthesis (16 strands)

4

slide9

1

L. Denature at 96º

Anneal primers at 50º

2

3

4

slide10

1

M. Copy strands at 72º

72º

2

3

Fourth round of cDNA synthesis (32 strands)

4

slide11

1

cDNA strands (32) are now shown as lines

2

3

4

slide12

1

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

2

3

4