Autumn ecology and systematic population mnmn

1. REVERSE TRANSCRIPTION
POLYMERASE CHAIN REACTION
In the reverse transcriptase PCR cDNA is
constructed from the RNA using a
reverse transcriptase enzyme to study
gene expression

2. • Rt PCR is one of many variants of polymerase
chain reaction .This technique is commonly used
in molecular biology to detect RNA expression.
• RT PCR is often confused with real time
polymerase chain reaction .
• They are seprate and distinct technique.
• RT PCR is used to qualitavely detect gene
expression through creationog cDNA transcript
RNA.

3. • RT Present in retirovirus like HIV convert RNA to
DNA .
• RNA dependent polymerase
• Enzyme used for RT is usually MMLV/RTAMV
• Moloney strain of murine leukemia virus reverse
transcriptase .
• RT is done at 37-42
• Using DNA primers like oligo dt or random
hexamersor gene specific primers.s

4. • Principle
• In RT-PCR the RNA template is first converted
intoa complementary DNA (cDNA)using a reverse
transcriptase . The cDNA is then used as a
template for expontial amplication using PCR.
• ONE-STEP RT-PCR VS TWO STEP RT-PCR
• In the one step approch the entire reaction
occurs in a single tube.
• In two step reaction the reverse transcriptase
reaction and PCR amplication be performed in
separate tubes.

5. ONE STEP Rt PCR
• In a single or single reaction reverse transcriptase
and amplication are performed therefore it is
namedasd one –step RT-PCR.
• It is widely used in repeat quantifcation assays
and high throughput screening due to its high
accuracy specificity and easy to use simple set up.
• In comparision to two step PCR the chance of
reaction failure and contamination are less
because we are performing the reaction in a
single tube here. S

6. Selection of primers
• Random primers
• Oligo Dt primers
• Sequence –specific primer
• Random primers are short stranded sequences of
hexamers or octamers . The random primer binds at
the complementary random location on the RNA .it can
bind to many types of RNA (tRNA,rRNA ,or mRNA)and
synthesizes the cDNA.
• THE RANDOM PRIMERS work finely for prokaryotic
DNA rRNA and smaller RNA but as it cannot bind to
poly-A tail it is less preferred for eukaryotic RNA
amplification.s

7. The oligo dT primers
• Are specially designed to amplify the mRNA as
we know that the mRNA has a poly –A tail the
oligo dT primers only bind to the poly A tail of
mRNA .hence it is used to amplify smaller mRNA
as well.
• The oligo dT primers are 12to 18 nucleotide long
single stranded DNA which contains one
additional nucleatide at the 3 end to anchor the
binding .the anchored primers prevent the
• Primer slippage and denaturation from the poly A
tail.ss