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

transcription translation
DNA mRNA tRNA/protein

 however, RNA difficult to work with

• unstable; RNases ubiquitous
• no way of amplifying
 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 exon exon

promotor intron intron DNA
3’ 5’
start codon stop codon
Transcription
5’ 3’ mRNA
Processing

cap
poly A
Splicing
tail
Intron deleted

mature mRNA To cytoplasm

Take place in nucleus
Juang RH (2004) BCbasics
 The Process of RT from mRNA
Baltimore, Dulbecco, Temin (1975)

mature mRNA
5’ 3’
poly A tail
Reverse
transcription TTTT
3’ 5’
RNA hydrolysis

3’ 5’
DNA polymerase

3’ CCC 5’
5’ GGG 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 !