Biomedical Engineering Reference
In-Depth Information
5'
AAAAAAAAAAAA 3'
mRNA
Reverse
Transcriptase
oligo-dT-SR
SR
T
TT
TTTTTT
TT
T
5'
3'
AAAAAAAAAAAA 3'
T
TT
TTTTTT
TT
T
5'
SR
cDNA
Exonuclease
RNase H
TdT
dATP
3'AAAAAAAAAAAA
5'
T
TT
TTTTTT
TT
T
SR
Ta q
Polymerase
oligo-dT-SR
T
TT
TTTTTT
TT
T
SR
3'
T
TT
TTTTTT
TT
T
T
TT
TTTTTT
TT
T
5'
3'
SR
SR'
ds-cDNA
5'
AAAAAAAAAAAA
SR
AAAAAAAAAAAA
oligo-dT-SR
Taq Polymerase
PCR Amplification
SR
T
TT
TTTTTT
TT
T
3'
SR'
T
TT
TTTTTT
TT
T
T
TT
TTTTTT
TT
T
SR
5'
5'
3'
SR
AAAAAAAAAAAA
AAAAAAAAAAAA
SR'
3'
SR'
T
TT
TTTTTT
TT
T
T
TT
TTTTTT
TT
T
SR
5'
5'
3'
SR
AAAAAAAAAAAA
AAAAAAAAAAAA
SR'
3'
SR'
T
TT
TTTTTT
TT
T
T
TT
TTTTTT
TT
T
SR
5'
5'
3'
SR
AAAAAAAAAAAA
AAAAAAAAAAAA
SR'
3'
SR'
T
TT
TTTTTT
TT
T
T
TT
TTTTTT
TT
T
SR
5'
5'
3'
SR
AAAAAAAAAAAA
AAAAAAAAAAAA
SR'
1:100 Dilution
Taq Polymerase
PCR Amplification
oligo-dT-SR
SR
T
TT
TTTTTT
TT
T
+
aa - dUTP
3'
T
TT
TTTTTT
TT
T
T
TT
TTTTTT
TT
T
5'
SR'
SR
5'
SR
AAAAAAAAAAAA
AAAAAAAAAAAA
SR'
3'
3'
SR'
T
TT
TTTTTT
TT
T
T
TT
TTTTTT
TT
T
SR
5'
5'
3'
SR
AAAAAAAAAAAA
AAAAAAAAAAAA
SR'
aa-dsDNA
3'
T
TT
TTTTTT
TT
T
T
TT
TTTTTT
TT
T
5'
SR'
SR
5'
SR
AAAAAAAAAAAA
AAAAAAAAAAAA
SR'
3'
3'
T
TT
TTTTTT
TT
T
T
TT
TTTTTT
TT
T
5'
SR'
SR
5'
SR
AAAAAAAAAAAA
AAAAAAAAAAAA
SR'
3'
Figure 5.3
Global PCR amplification of mRNA. An oligo-dT primer appended to a unique
sequence (SR) is used to reverse transcribe mRNA. Exonuclease treatment removes the excess
primer and the cDNA products are tailed using terminal deoxynucleotidyl transferase. The same
SR-oligo-dT primer is used to prime a second-strand synthesis and the resultant ds-DNA is then
amplified via PCR using the oligo-dT-SR primer.
