polymerase chain reaction Flashcards
1
Q
principles of PCR
A
- rapid method for detection of target DNA
- DNA polymerase & primers
- prone to technical errors e.g. pipetting
2
Q
main steps of PCR
A
- dNTPs are added in excess
- denaturation at 94-96C
- PCR buffer (8-9.5 pH) contains MG2+ for primer annealing
- annealing at -68C
- Taq polymerase (DNA polymerase) & primers to synthesize new strands of DNA
- elongation at 72C
- results in exponential amplicons (copies) of target sequence
- visualised by agarose gel electrophoresis
3
Q
principles of reverse-transcription PCR
A
- to detect RNA
4
Q
main steps of reverse-transcription PCR
A
- reverse transcriptase makes DNA/RNA
- then makes DNA/DNA double helix
- reverse transcriptase -> RNA to cDNA reverse transcription
5
Q
principles of quantitative PCR
A
- economical
- rapid time to results
- well-established technology
- requires normalisations to controls for relative quantification of nucleic acids
- uses a standard curve to
6
Q
main steps of quantitative PCR
A
- can quantify different expression levels with the housekeeping gene
- uses fluorescence with DNA-binding dyes (e.g. SYBR green). increases up to 1000x when bound to dsDNA
- uses fluorescence with fluorescent-specific probes: hydrolysis (e.g. TaqMan) and hybridisation probes
- hydrolysis has 5’ reporter and 3’ quencher, 5’ -> 3’ exonuclease activity -> Taq polymerase frees 5’ report
- hybridisation contains donor and acceptor + they’re brought together in annealing
7
Q
principles of digital PCR
A
- ratio of positive fluorescence to negative
- quantifies rare targets in complex
- rapid, simple optimization
- not reliant on a standard curve
- accurate and absolute quantification of nucleic acids
8
Q
main steps of digital PCR
A
- division of each sample into subunits
- each of 1000s partitions should contain 0/1 template molecules
- qPCR of individual partitions using fluorescent probes
- analysis of fluorescent signal
- amplified product =1 (fluorescent), if not then =0
9
Q
applications of PCR
A
- amplification of gene fragments
- the modification of DNA fragments
- detection of pathogenic microorganisms
- DNA analysis of archaeological specimens.
10
Q
applications of rt-PCR
A
- distinguish exons from introns
- identify changes in a gene or chromosome to diagnose diseases
- monitor response to cancer therapy and improve prognosis
11
Q
applications of qPCR
A
- gene expression
- pathogen detection
- environmental studies
- siRNA, miRNA, lncRNA detection
12
Q
applications of dPCR
A
- mutation detection
- genome edit detection
- copy number variation
- standard validation
13
Q
what are primers?
A
- short sequences (20-25) of single-stranded DNA that are target sequence complementary
- starting point for DNA synthesis
- a pair of forward and reverse primers are used
