Real time PCR

pcr limitations

low sens, short range, low res, manual, not quantitative, same end point reults diff template, diff end point same initial amount

Baseline

signal level little change in signal

threshold

level of signal that reflects sig increase

C threshold

threshold cycle cycle number where signal crosses threshold. used to calculate initial DNA copy number

traditional pcr

gel for detection of PCR amp

Real time PCR

fluorescent chem for detection

real time pcr info

probe or dye added to signal

after each cycle DNA is measured signal directly proportional to PCR molecules

detection of signal allows quantitation of starting material

Observed signal creates exponential curve with lag phase lin and stat phase

length of lag inversely proportional to amount of starting material

3 phases of pcr

exponential doubling every cycle

linear components are being consumed reaction slowing

plateau no more products made

sybr green

binds minor groove of and dsdna

binds any amplified product target or non target

more vulnerable to specificity problems

taqman

based on fret

nucleotide seq with 5’ fluor and 3’ quencher

red supresses green

nucleotide binds between target primers

5’-3’ exonuclease cleaves taqman probe

sybr vs taq

sybr detects all amp dsdna non specific as well taq amp products only

sybr enables amp and monitoring of any gene or target of interest

sybr requires no probes

taqman requires probes and targets to signal allows for multiplex pcr and post pcr eliminated

qpcr analysis

end point pcr is measured in plateau phase

qpcr measured during exponential phase amplification can plateau at same place even though exponential shows difference

plateau do not truly represent starting target mat

input qpcr

inversely proportional to threshold cycle

lower ct more na available

qpcr app

t9;22

post therapy monitoring

can detect 1 cml cell in 100,000 normal cells

peripheral blood can be used instead of marrow