2. REVERSE TRANSCRIPTION
SYNTHESIS OF cDNA FROM RNA
1-Technique used in molecular biology to detect RNA
expression by generation of complementary DNA
(cDNA) transcripts from single stranded RNA
2-Transcription: synthesis of RNA from DNA
3-Reverse transcription: transcription of
single stranded RNA into cDNA with the
help of the enzyme Reverse Transcriptase.
8. RT Buffer
TrisHCl: for maintaining the pH
MgCl2 (MnCl2), KCl salt: cofactor.
1- the polymerase uses it in the catalytic area to balance
the
negativley charged phosphate groups of RNA template
backbone.
2- stabilizes duplex’s structure because the negative
charges would
otherwise repel one another in the DNA strands
3- forms soluble complex with dNTPs
9. DTT:
loosen the secondary structure of RNA,
breaks disulfide bonds - reduces thermostability of the
bonds
10. 1-One-step RT-PCR
One-step RT-PCR combines first-strand cDNA synthesis
(RT) and subsequent
PCR in a single reaction tube
2- Two-step RT-PCR
Two-step RT-PCR entails two separate reactions,
beginning with first-strand
cDNA synthesis (RT), followed by amplification of a
portion of the resulting
cDNA by PCR in a separate tube.
12. Advantage:
1. Simple
2. Minimize possible contamination
3. Processing of large numbers of samples
4. Useful for detecting multiple genes in a single RNA
sample.
Disadvantage:
1. One-step RT-PCR uses gene-specific primers for
amplification,
limiting the analysis to one gene per RNA sample.
2. Include multiple steps for an extended workflow
3. Increasing the chance of contamination.
