Topic 15.2: PCR

Question 1

what does PCR involve

Answer 1

amplify target DNA
denature - break H bonds between base pairs
to form two single stranded molecules
add small primers (reverse and forward primers, one for each strand)
rapidly cool and primers will anneal to either of the strands
heat up slightly for TAQ polymerase (thermostable) to act and make copies of the strands and REPEAT

Question 2

what is the product of PCR

Answer 2

PRIMERS INCLUDED - important for cloning

Question 3

why use PCR

Answer 3

amplify specific DNA fragment which helps in diagnostic techniques - single base mutation (sickle cell), small deletions or insertions (CF), variation (dna profiling_

Question 4

how do we check PCR works

Answer 4

run sample on agarose gel = check band of correct size
use positive and negative controls
sequence the PCR to check for errors

Question 5

how do analyse PCR

Answer 5

in mutant allele - restricition site…amplify around and do PCR to see if restriction site is present, if so = mutant
if carrier when recessive: depends on allele being sequenced, if B then no site if b then present, so if Bb will contain restriction site and normal base pairs

Question 6

what is reverse transcriptase PCR used for

Answer 6

to determine if gene expression - detects mRNA

Question 7

how does RT-PCR work

Answer 7

mRNA -> cDNA due to reverse transctiptase

Question 8

what is oligonucleotide directed mutagenesis

Answer 8

uses PCR to make defined mutations into cloned gene
modified primers with mutation and mix with clone, amplify using PCR of original…copies of plasmid sequence which contains one normal strand and one mutated strand. digested normal strand….results in mutated plasmid introduced into bacteria….produce plasmids with mutated gene