lecture 3 - use of PCR

Question 1

reverse transcriptase PCR

Answer 1

convert RNA to cDNA using RT
the amplify DNA using PCR
source of RNA may be mRNA or RNA virus

Question 2

real time qPCR

Answer 2

start with clean material
need a thermos lightcycler (detects flourescence)
DYBR green or TaqMan (flourescent)
same as end point PCR but
each cycle cDNA amount is double
more flourescence

Question 3

flourescence

Answer 3

during elongation SYBR binds to groove of ds DNA - increases flourescence
more cDNA more flourescence

Question 4

TaqMan

Answer 4

uses probes with flourescence reporter and quencher
probe binds to template and cleaves probe and flourences reporter is activated

Question 5

Ct value is cycle threshold

Answer 5

the number of cycles required for the flourescence signal to cross the threshold
the lower the Ct threshold line the greater the amount of DNA in the sample (more starting material)

Question 6

reference genes

Answer 6

constant level of expression - not afected by exprimental factors
essential to support the validity of qPCR results
examples: beta actin, GAPDH, albumin, 18SrRNA, TATA sequence binding protein