PCR Flashcards
why is PCR useful?
removes the need for cloning vectors or transformation as able to amplify the amount of DNA in a short amount of time
what things are required for a PCR?
source DNA, supply of nucleotides, heat resistant polymerase, primers for DNA synthesis
what are oligonucleotides?
DNA sequences of only 10-20 bps
what do the DNA primers do?
complementary to the 3’ end and initiate DNA synthesis and ‘flank’ the sequence
what is Taq polymerase?
heat stable DNA polymerase
where was Taq polymerase found?
isolated from a bacterium that lives in hot springs
what is pfu polymerase?
a more accurate and stable heat resistant polymerase yet more expensive
how many cycles usually occur in PCR?
30-40 cycles
what occurs in denaturation?
reaction mixture heated to 94 degrees causing the DNA to become ss
what occurs in annealing?
temperature reduced to 54-60 degrees to allow annealing of primers
what happens in extension?
temperature raised to 72 degrees so the polymerase begins to add bases and both strands are copied
why is an extra 5 minutes allowed at the end of the final cycle?
to allow for any completion for any unfinished template strands
what causes the synthesised strands to be too long?
polymerase has no stop signals
when do the strands become to be exact?
after 3 cycles
why does PCR not fully replace gene cloning?
some errors in cloning so instead PCR amplifies the DNA to then be inserted into a vector
how can PCR strands used for implant?
primers can be used to add restriction sites into the DNA so they can be cleaved
what are the advantages of PCR?
only need a small amount of DNA, variety of sources, can detect genetic disorders, only need around 20 cells
what kinds of regions are used for PCR in fingerprinting?
a highly polymorphic region so that no two individuals will have the same sized fragments
how are microarrays prepared?
make cDNA which is fluorescently labelled and apply to the array with each gene in a different well
how can different tissue states be tested on the same array?
use a different coloured label for different tissues
what is RNA sequencing?
cDNA is made from mRNA and is then sequenced and then mapped by computer into a genome sequence
what does cas-9 protein do?
acts with a guide strand of RNA that has been made by CRISPR
what does CRISPR do when the guide strand enters?
cuts both strands of the DNA that is complementary to the guide RNA
what causes the gene to be inactivated?
when the DNA is repaired, nucleotides may be introduced or removed causing deactivation
