L2-4: PCR Flashcards
What enzyme is used in PCR?
DNA polymerase
What molecules are involved in PCR?
- The precursors dATP (dGTP, dCTP, dTTP)
- template DNA
- 3’ end dNTPs as building blocks of new DNA strand
- Forward & reverse primers
- Taq polymerase
- -Mg, critical co factor for DNA polymerase & its concentration affects stringency of primer binding
- MgCl2 is essential for Taq activity
- buffer, to maintain pH and provide necessary salt
What is the function of helicase
To unwind DNA
What is the function of primase
To initiate synthesis
What are the different polymerase forms and their functions?
Alpha: intiation
Delta: extends after RNA on lagging strand
Epsilon: replicates lead strand
What is the function of nucleases?
Removes DNA from 5’
What is the function of ligase?
Catalyzes the reaction to form phosphodiester bonds between two nucleotides juxtaposed with 5’-P and 3’-OH
Describe the process of test tube DNA synthesis
- Both primers added for simultaneous strand synthesis
- Boil DNA (95C)
- Anneal primers (55C)
- Extend with polymerase (72C)
How a primers for a sequence written?
In a 5’ to 3’ direction matching the DNA template
List ways PCR can go wrong
- wrong primers used
- reaction set up incorrectly
- PCR machine may have been misprogrammed
What is an indication of successful PCR?
Much dsDNA
How can successful PCR be detected?
Dye binding/ showing fluorescence e.g. ethidium bromide
Outline the mechanism to detect PCR products
- Run reaction products on agarose gel
- Use intercalating dye to stain DNA
- Determine size and yield
Name one alterntive to gel PCR
Real time/ quantitative PCR (qPCR)
Explain the qPCR process
- DNA is detected during the replication process
- PCR monitored every 7s (using fluorescence)
- doesn’t indicate product size/ specificity
- the more template held, the quicker the yield detected (lvls increase proportionate to dsDNA produced)
Define threshold cycle (ct)
The cycle at which a (small) amount of PCR is detected
List PCR applications in the wider world with examples
- genetic testing (prenatal, cancer)
- diagnosis (infection, cancer)
- personalised medicine (patients genomic DNA seq)
- these look for specific biomarkers
- forensics
How can PCR be used to measure RNA level?
By reverse transcriptase PCR (RTPCR)
- polydT primer (recgonises polyA tail) added, which binds to and allows amplification of all mRNA
- RT is added forming cDNA from mRNA
- from this point ssDNA occurs
How can PCR be used to manipulate DNA molecules
E.g transforming artificial PCR fragment into yeast genome
- hybrid primers amplify drug resistance cassette from plasmid
- introduce appropiate yeast DNA sequence at each end of PCR product
- transform PCR product into yeast
- yeast repairs broken fragment by homologous recombination using ends of PCR product (integrates DNA into RAD9 locus and replacwa in with Kanr cells)
- if DNA inserts into genome, cell becomes resistant to drug= genome altered
What is the purpose of using PCR to manipulate DNA molecules?
To better understand life (gene function, protein localisation, function and structure)
How can PCR be used to fuse protein with GFP (green fluorescent protein)?
- GFP taf (238aa) fuses to protein and is integrated into genome
- Allows fusion protein localiation to be determined in live organisms
Why is PCR clinically valuable?
- Sensitive: can amplify as little as one DNA molecule
- Specific: can amplify unique target sequence, with high stringency depending on method, temp & [Mg2+]
- Relatively cheap
- Rapid: results in a few hours
- Robust: DNA is v stable and can be amplified from old degraded samples
Name 3 common uses of PCR in diagnosis/ prognosis
- Genotyping patient: diagnosis of genetic traits & detection of specific carrier alleles. Used for tissue matching (HLA typing and predicting drug response)
- Genotyping pathogen: diagnosis of species/ strains
- Phenotyping disease:
What 2 PCR techniques are used to genotype patients?
PCR-RFLP and ARMS-PCR (amplificarion refractory mutation system PCR)
