Reverse Transcription

Dr. Howard Temin

the enzyme reverse transcriptase was first reported by

Reverse transcription of RNA

conversion of the RNA template into its complementary DNA strand (cDNA)

cDNA

can be sequenced, cloned, and applied to estimate the copy number of specific genes in order to characterize and to validate gene expression.

cytoplasm

Begins when the viral particle enters the _______ of a target cell.

DNA Polymerase & Nuclease (Ribonuclease H/ RNase H)

Retroviral DNA synthesis is dependent on the what two distinct enzymatic activities of Reverse Transcriptase

Reverse transcriptase PCR

Developed to amplify RNA targets

1 step RT-PCR and 2 step RT-PCR

2 methods of RT-PCR

1 step rt-pcr

all are already included in one master mix

amplified cDNA molecule

end product of 1-step RT-PCR

2-Step procedure/2 step RT-PCR

method that provides flexibility

one-step procedure/ 1-step RT-PCR

It is a recommended used for large number of samples

Oligo dTs

Are synthetic DNA strands that can be represented as a sequence of ‘T’ (thymine)

Gene Specific primers

Can be designed to anneal to a specific gene.

Random hexamers

Consists of ten bases which anneal randomly to RNA molecules.

1. Linearization of RNA Template

2. Annealing of Oligonucleotides

3. Reverse Transcription by Reverse Transcriptase

4. Removal of mismatches by RNase H. (Proofreading)

5. Mismatch repair by Klenow Fragment DNA Polymerase

6. Downstream processing of cDNA via PCR

Take note: Process of RT-PCR

1. Research methods

2. Gene Insertion

3. Genetic Disease Diagnosis

4. Cancer Detection

Application for Reverse transcription

Polymerase Chain Reaction (PCR)

It is the standard method of coronavirus testing.

a tungsten halogen lamp

an excitation filter

mirrors

lens

mission filter

a charge-coupled device (CCD) camera

It is used for the measurement of the fluorescence signal

TaqMan probe

consists of a fluorophore covalently attached to the 5’ end of the oligonucleotide probe and a quencher at the 3’ end.