5 PCR Flashcards
(49 cards)
What is PCR
Polymerase chain reaction
It’s amplification of large amount of DNA from small amount of dna
What are the uses of PCR
DNA detection /diagnosis (genotyping)
DNA amplification from trace amounts (like crime scenes)
Clonging and manipulation of dNA (mutagenesis)
RNA detection /amplification (reverse transcriptase)
Radiolabelling DNA
What are the five components of PCR
Template : has to be DNA to give dsDNA
Primers: ssDNA primers, have orientation and have to point towards each other
dNTPS: need equal amount of them (Not NTP since making DNA)
Taq: dna polymerase
Buffer: needs mg2+ in it
What are the cycle conditions of PCR
- Initial denaturation
- 25-25 cycles of denaturation (94 deg, splits dsDNA), annealing (50-65 deg, primers bind), elongation ( 72 deg, poly activity to extend past primers) repeating
- A Final elongation (to make sure pol makes the full length of the dna)
Summarize the overall reaction of PCR
- Denaturation splits dna (94 deg)
- Annealing where primers bind with opposite directionally to top and bottom strand (50-65 deg)
- Elongation (72 deg)
What special about the first few rounds of PCR
How is it fixed
Want just a small region of the DNA but the dna is made extra past the end point you want
After you keeps doing PCR the ends only get made up to the point you want (now only the dna region between the two primers is made replicated)
After five round of PCR Whag is the number of DNA
32 duplexes
22 of them are the same size
Further cycles dilute out the ones of other sizes
What are the important criteria of PCR
template
Primers
dNTP
taq pol
Buffer
Cycle conditions
What is concerning about the template contamination in PCR
PCR is highly sensitive (meaning you can’t get your own DNA in there because it easy to amplify the contaminant dna rather than the sample)
It’s also easy to amplify junk due to non specific priming (primers bind to whatever)
What is the criteria of the template in PCR
Types of template:
- genomic, plasmid, or cDNA
Plasmid templates are low as 1pg (but ng more common)
Genomic templates need more (at least 5ng, bc your gene is one little piece in that whole sequence)
- the genomic dna are all coding so GC rich, meaning have high melting temp
- so you add DMSO (1-10%) to help lower the melting temp
Might need more template for some Taq polymareses (like pfu)
But too much template can inhibit PCR
What are the faint bands in the bottom of a PCR gel
Primer dimers
What do you take into account for primers criteria
Their synthesis
Primer dimers
Primer Tm
Primer binding and mg2+ concentration
What to consider for the synthesis of primers
Usually made with a 5’ OH (not 5’ phos)
Downside:
If using PCR to detect gene expression from cDNA : might need the 5’ phos for DNA ligase to work)
If using PCR to make an insert for cloning (also need the 5’ phos to ligation to the vector)
What to consider for the primer dimers
Primer dimers are secondary structures made due to micro homology between the primers (cross dimer) or in one primer (hairpin)
Once made, they get amplified really well because so small , so target gene not amplified
This happens in every PCR reaction it doing a lot of cycles
What to consider for the primer Tm
What is tm
Tm of the primer is the temp at which 50% of the primers are perfectly duplexes with their compliment and 50% are not
Primer Tm are what determine the annealing temp of the reaction (choose annealing temp that matches the Tm of the primers)
What to consider for the primer binding and Mg2+ concentration
The mg2+ ions stabilize the annealing of primers to the dna by reducing the electrostatic repulsion of the phosphate backbone
If mg2+ too low or too high: more non specific binding because have too much repulsion (not binding at all) or too little repulsion (so binds anywhere)
What to consider when designing primers
Primer specificity
3’ conplimentarity
Design of RE overhangs
When design primer What do you consider for primer specificity
Want primer to bind only one target in the template
So the primer sequence must be unique and shouldnt be able to anneal to any other site of the dna
- can’t be too small (binds anywhere) or too long (too high annealing temp)
What can you use to check if your primer is good
Primer 3
Blastn (NCBI)
Explain how non specific priming error gets amplified
Non specific priming error get easily amplified in PCR bc primer bind to wrong spot, taq elongates wrong dna template
During next round, If another primer binds downstream of the incorrect seqeunce, the newly made incorrect sequence will get amplified
When design primer What do you consider for 3’ complimentary
The primer complimentarity to the 3’ end is more important to than at the 5’ end
Meaning mismatches at the 5’ end have less problems
This is because dna pol extends at the 3’ end of the seqeunce, so if mismatched the pol won’t work , doesn’t care about 5’ end
When designing primer what do you consider for RE overhangs
What does this change
Since 5’ primer complimentary doesn’t matter as much, we can add RE sites to the 5’ end of the primer
But need to consider how many bp need to be added to the ends (usually 6) for the RE to work
Also need to consider that one you start adding more BP to the ends, tm, GC content, length changes and there’s potential for hairpins and cross dimers
What are the rules of thumb for primer design
Length: 18-30 nucleotides (longer increases annealing temp but give better specificity, shorter makes non specific binding)
GC content: 40-60% (too low means poor binding to template, too high means false binding to template)
Tm: 55-70 deg, both primer should have Tm within +/- 5 deg of each other
GC clamp: primer need 1-3 GC in the last five nucleotides (bc binding at ends matters more)
Primers shouldn’t have any complimentarity with each other
Primers should mint be able to pair with themselves or form secondary structures (like hairpin)
Primers need polarity (need to point toward each other) and need to be reverse compliments
