Transcript PCR APPLICATIONS - University of Cape Town

Some applications:
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Specific sequence targeting
Fishing for unknown sequences (uncloned)
Assembling artificial sequences
Site-directed mutagenesis
Adding enzyme sites, start and stop codons
Detecting clones – colony PCR
Detecting mRNA – Reverse transcriptase PCR
Assaying copy number – real-time pcr
ASSEMBLING
ARTIFICIAL
SEQUENCE
DNA template assembly:
30 - 50 cycles PCR
1
5'
3'
2
3
40-base
oligomers
3'
5'
add outside primers;
gene amplification: 25 cycles PCR
clone into sequencing vector
40-base
oligomers
40
1
60
20
20
20
80
2
add outside primers; gene amplification
SITE-DIRECTED MUTAGENESIS
A
.… ACTTGCAAATTGGTCGATCG…3’
T
…..TGAACGTTTAACCAGCTAGG…5’
A
5’ – ACTTGCAAATTATCGATCG- 3’
SIGNAL OR RESTRICTION SITE ADDITION
Hind III
start
5’ - NNAAGCTTNNATGACTTGCAAATTGG – 3’
…..… ACTTGCAAATTGGTCGATCG…
COLONY PCR
No need to
extract DNA
from
bacteria
Reverse Transcription-PCR
Amplified copies
of specific mRNA
(RT-PCR)
1. Extract total RNA
2. Reverse Transcription
3. PCR
dNTPs
primer
Reverse
Transcriptase
Double stranded
“Copy DNA”
(PCR template)
ASSAYING COPY NUMBER:
REAL-TIME PCR
Agarose Gel
Traditional
PCR detection
Area for Real-time
PCR detection
10 copies
50 copies
Problems with traditional
end-point detection:
Low sensitivity
Low resolution
Poor precision
SYBR Green Dye Assay
Binds to any double-stranded DNA
- Specific product
- Non-specific product
- Primer dimers
FRET =
Fluorescent
Resonance
Energy
Transfer