Week 10 MolBio

PCR

Polymerase Chain Reaction:

Denaturation, Annealing, Extension

Tells us if a certain sequence is there in the DNA + conc measured thru nanodrop

qPCR

quantitative REAL TIME PCR

DNA isolation, Denaturation, Annealing, Extension, DNA Synthesis/Fluorescence

Tells us if a certain sequence is there in the DNA + conc measured thru the Fluorescence part of PCR

Useful if you can’t measure conc thru nanodrop

Measure gene copy number

Detect DNA from pathogenic organisms

qPCR mechanism

Ct

Ct is the cycle number at which the fluorescent signal crosses the fluorescence threshold

Lower Ct = more target molecules because fewer cycles needed to amplify to reach maximum fluorescence threshold

qPCR math normalization

RT PCR

REVERSE TRANSCRIPTASE PCR

instead of DNA we isolate mRNA

make complementary DNA with reverse transcriptase

do PCR to amplify

Used to:

• Detect expressed genes

• Examine transcript variants

• Generate of cDNA templates for cloning and sequencing

RT PCR mechanism

RT-qPCR

REVERSE TRANSCRIPTASE real time quantitative PCR

RT-PCR isolation + extension and then we do qPCR for the PCR part

RT-qPCR mechanism