amplification
No description yet. This curated deck is ready for daily study.
Card preview (24)
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