amplification Flashcards
what are the three distinct events during PCR cycles?
- denature template
- primers anneal
- synthesis by thermostable polymerase
why is the denaturation during the first cycle lengthened?
to make sure all dsDNA dissociates into single stranded
what does heat change in PCR?
changes the conformation of polymerase to be active.
what is the temperature and purpose of the denaturing step?
95-98 C, breaking apart dsDNA so primers can anneal
why does the now ssDNA stay apart after cooling in the denaturing step?
because it would be a lot more energy to put them together than just binding to the random other stuff floating around
T/F: primers must be PERFECTLY split to anneal to the template.
true
when the reaction cools to 45-65 C what happens?
the primers hybridize to complementary single-stranded DNA.
why do primers preferentially hybridize to complementary sequences.
primers are in much greater concentration than DNA and much shorter
what is the last step of PCR?
DNA synthesis and DNA extension.
how is DNA extension preformed?
extension of primer by thermostable polymerase
how long is extension generally?
~1 min/kb
what happens if DNA synthesis is too hot?
the proteins will be irreversibly damaged
how many times is PCR repeated?
~30
how many copies are generally produced during PCR?
10^12 usually
what are the raw materials in PCR?
- target template DNA
- thermostable DNA polymerase
- oligonucleotide primers
- deoxynucleotide triphosphates
- reaction buffer
- magnesium ions
- optional additives
what two raw materials are paired together in PCR?
reaction buffers and magnesium ions
generally how much template DNA is needed for a mammalian genome?
up to 5 ng
T/F: for plasmid, mitochondrial, or chloroplast DNA you require less template DNA to get the same copies of template DNA.
true
what kind of DNA polymerase is required for TaqPCR?
thermus aquaticus
T/F: taq pol can incorporate errors.
true
when is the incorporation of errors in taqPCR a problem?
when there is low template DNA causing errors in early cycles of PCR to get propogated
what is the general primer length?
18-24
what bases have a higher melting temperature?
G-C because 3 h bonds
in an oligonucleotide primer how many G-C bonds should be in the sample
less than 50% of the primer
what are the target sequences of oligonucleotide primers?
<2000 bp
T/F: a primer should be extremely specific to one part of the genome
true
T/F: to get a super specific primer it should then be extremely long to increase specificity.
false, too long a primer means the melting point might be close to or above 72C (aka extension temp of PCR)
what can you do if there is too much primer?
slow down the reaction then test it on a gel seeing if you get one band
how are oligonucleotide primers used?
in excess
after 30 cycles of PCR most of the primers are where?
consumed by the reaction
what are dNTPs?
raw materials for DNA amplification
T/F: dNTPs are used in limited amounts to maximize PCR efficiency
false, they are used in non-limiting
most applications use what concentation of each dNTP?
0.2 mM
generating 10^12 copies of a 100 bp target sequence consumes how many dNTP molecules?
10^12*100 bp = 10^14 dNTP molecules
what is Mg for in PCR?
affects the performance of Taq and the stringency of primer annealing
Mg is the cofactor of what/in what?
enzymatic reaction of DNA polymerase
in the absence of adequate free magnesium what happens?
taq pol is inactive
in the excess of adequate free magnesium what happens?
increases level of nonspecific amplification
what is an optional additive that binds potential PCR inhibitors?
Bovine Serum Albumin (BSA)
a long initial denaturing step will do what?
dissociate high-molecular weight template strands
the final extension step helps to add what onto the end of PCR products? why?
additional adenosine which makes scoring easier than if a product is a mixture of fragements with and without extra adenosine
when should you keep going past 30 cycles of PCR?
in very low template DNA amounts available
what happens when you keep going past 30 cycles in a normal sample?
often results in the amplification of undesired regions
when should you stop PCR (on a graph)?
just before the plateau phase when you get a major and minor contributor
T/F: you normally get a pure sample in PCR amp.
false
what does the profiler plus cycling program do?
evens out locus specific variation.
what does the touchdown cycling program do?
~50 cycles run at incrementally lower temperatures annealing above suspected melting temp gradually declining
what does the touchdown cycling program ensure?
first primer-template hybridization events are stringent and keeping undesired amplification low.
what is hot start PCR?
adding taq at higher temps
T/F: taq pol is ALWAYS used.
false
what kind of taq does most humans use?
proofreading taqs
what 3 controls are needed in PCR?
- negative
- positive
- primer
what is a negative control?
there should be no DNA and no PCR products other than primer-dimers
what is a positive control?
a sample known to work properly and amplifies well at the given locus
what are the first controls to fail in PCR?
primer controls
what does it mean when the controls look good and most samples look good but some didn’t amplify?
some samples may have inhibitors or low templates
what does it mean when no amplification of template control but the primer control reaction worked?
primer died halfway through
what does it mean when neither the template control nor the primer control reaction worked?
you may have forgotten to add something important to your PCR
what does it mean when there is an amplification of a negative control?
evalutate based on negative at beginning or end, reagents or sample may have been contaminated.
what is a multiplex PCR?
amplifying many loci at once
what is difficult to avoid in multiplex PCR?
primer-dimers
what are the advantages and disadvantages of PCR?
- low amounts of DNA can provide product
- does not have to be high molecular weight
- contamination (can be an issue with low template samples)
- PCR inhibitors
- DNA mutations, primers will not anneal.
what does whole genome amplification use?
polymerase and random hexamers as primers to amp the entire genome.
what does the WGA enzyme do?
works by multiple displacement amplification or rolling circle amplification
