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PCR APPLICATIONS. Dr Fiona Tanzer. & SPECIAL TECHNIQUES. Some applications:. 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
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PCR APPLICATIONS Dr Fiona Tanzer & SPECIAL TECHNIQUES
Some applications: • 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 40-base oligomers DNA template assembly: 30 - 50 cycles PCR 1 5' 3' 3' 5' add outside primers; gene amplification: 25 cycles PCR 2 clone into sequencing vector 3
40-base oligomers 20 20 20 40 1 60 80 2 add outside primers; gene amplification
SITE-DIRECTED MUTAGENESIS A .… ACTTGCAAATTGGTCGATCG…3’ …..TGAACGTTTAACCAGCTAGG…5’ 5’ – ACTTGCAAATTATCGATCG- 3’ T A SIGNAL OR RESTRICTION SITE ADDITION Hind III start 5’ - NNAAGCTTNNATGACTTGCAAATTGG – 3’ …..… ACTTGCAAATTGGTCGATCG…
COLONY PCR No need to extract DNA from bacteria
Reverse Transcription-PCR (RT-PCR) Amplified copies of specific mRNA 1. Extract total RNA 2. Reverse Transcription 3. PCR dNTPs primer Double stranded “Copy DNA” (PCR template) Reverse Transcriptase
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
