Rtpcr Using

RT-PCR --Reverse Transcriptase PCR Zhang-HaiFeng Department of Biochemistry

What is RT-PCR? It refers to the process in which DNA is synthesized from RNA template by reverse transcriptase (RTase).

Why RT? • mRNA tied to gene expression / protein synthesis DNA

transcription

mRNA

translation

 however, RNA difficult to work with • unstable; RNases ubiquitous • no way of amplifying

tRNA/protein

RTase enzymes discovered by Temin and Baltimore in the early 1960s. used by retroviruses (such as HIV) to catalyze synthesis of cDNA from viral RNA template

RTase Multifunction of RTase:  RNA-dependent DNA polymerase (RDDP)  RNase  DNA-dependent DNA polymerase (RDDP)  No proofreading function

Intron and Exon in Eukaryotic Cells exon promotor

3’

intron

exon

intron

exon

DNA stop codon

start codon

5’

Transcription

3’ mRNA

5’ Processing

cap poly A tail

Splicing Intron deleted Take place in nucleus

mature mRNA

To cytoplasm Juang RH (2004) BCbasics

The Process of RT from mRNA Baltimore, Dulbecco, Temin (1975)

5’ Reverse transcription

3’

mature mRNA

RNA

hydrolysis

3’ TTTT 5’

3’

5’ DNA

3’ CCC 5’ GGG

poly A tail

polymerase

5’ 3’ Juang RH (2004) BCbasics

 Reverse Transcriptase PCR • make a cDNA strand from RNA • cDNA more stable • cDNA can be amplified using PCR

U A C U G G U U C A G A C AG A TGA C C AAGT C T G C TU A

Issues related to RNA isolation

 protection of RNA against degradation by RNases

• avoid touching samples • RNase inhibitors such Guanidinium Thiocyanate

Bye-bye !