Molecular Biology Lab - 06 Reverse Transcription

MOLECULAR BIOLOGY AND DIAGNOSTICS LABORATORY REVERSE TRANSCRIPTION OUTLINE • Introduction o Principle of the Test o Procedure: Process of Reverse Transcriptase • Reverse Transcription o Reverse Transcription of RNA o Reverse Transcriptases in Viruses o Reverse Transcription in Cells: Overview o Reverse Transcriptase - PCR REVERSE TRANSCRIPTION: INTRODUCTION • A reverse transcriptase is an enzyme used to generate complementary DNA from an RNA template, a process termed reverse transcription. PRINCIPLE OF THE TEST • RNA synthesis involves separation of the DNA strands and synthesis of an RNA molecule in the 5' to 3' direction by RNA polymerase, using one of the DNA strands as a template. In complementary base pairing, A, T, G, and C on the template DNA strand specify U, A, C, and G, respectively, on the RNA strand being synthesized. PROCEDURE: PROCESS OF REVERSE TRANSCIPTASE 1. Lysyl-tRNA acts as a primer and hybridizes to a complementary part of the virus RNA genome called the primer binding site or PBS. 2. Reverse transcriptase then adds DNA nucleotides onto the 3' end of the primer, synthesizing DNA-complementary to the U5 (non-coding region) and R region (a direct repeat found at both ends of the RNA molecule) of the viral RNA. 3. A domain on the reverse transcriptase enzyme called the RNAse H degrades the U5 and R regions on the 5’ end of the RNA. 4. The tRNA primer then "jumps" to the 3’ end of the viral genome, and the newly synthesized DNA strands hybridizes to the complementary R region on the RNA. 5. The complementary DNA (cDNA) added in (2) is further extended. 6. The majority of viral RNA is degraded by RNAse-H, leaving only the PP sequence. 7. Synthesis of the second DNA strand begins, using the remaining PP fragment of viral RNA as a primer. 8. The tRNA primer leaves and a "jump" happens. The PBS from the second strand hybridizes with the complementary PBS on the first strand. 9. Both strands are extended to form a complete double- stranded DNA copy of the original viral RNA genome, which can then be incorporated into the host's genome by the enzyme integrase. REVERSE TRANSCRIPTION REVERSE TRANSCRIPTION OF RNA • Refers to the conversion of the RNA template into its complementary DNA strand (cDNA) is an essential step in the analysis of gene transcripts. • cDNA can be sequenced, cloned and applied to estimate the copy number of specific genes in order to characterize and to validate gene expression. • Reverse Transcriptases are RNA dependent DNA polymerases which bind to RNA templates and convert them to their complementary DNA sequence in the presence of dNTPS and other essential cofactors. REVERSE TRANSCRIPTASES IN VIRUSES • The enzyme Reverse Transcriptase was first reported by Dr. Howard Temin. • It was discovered in reverse transcribing viruses. • These viruses contain a genome which is composed of RNA but which is reverse transcribed to DNA in the host cells. REVERSE TRANSCRIPTION IN CELLS: OVERVIEW • • begins when the viral particle enters the cytoplasm of a target cell • viral RNA genome enters the cytoplasm • process of reverse transcription generates a linear DNA duplex •

• Retroviral DNA synthesis is dependent on: the two distinct enzymatic activities of RT: o DNA polymerase o Nuclease (ribonuclease H/RNase H) REVERSE TRANSCRIPTION IN CELLS 1. Minus- strand DNA synthesis is initiated using the 3′end of a partially unwound transfer RNA which is annealed to the primer-binding site (PBS) in genomic RNA, as a primer. 2. Following RNase-H-mediated degradation of the RNA strand of the RNA: –sssDNA duplex, the first strand transfer causes –sssDNA to be annealed to the 3′end of a viral genomic RNA. 3. Once the –sssDNA has been transferred to the 3′R segment on viral RNA, minus-strand DNA synthesis resumes, accompanied by RNase H digestion of the template strand. 4. The RNA genome contains a short polypurine tract (PPT) that is relatively resistant to RNase H degradation. A defined RNA segment derived from the PPT primes plus-strand DNA synthesis. 5. RNase H removes the primer tRNA, exposing sequences in +sssDNA that are complementary to sequences at or near the 3′end of plus-strand DNA.

6. Annealing of the complementary PBS segments in +sssDNA and minusstrand DNA constitutes the second strand transfer. 7. Plus- and minus-strand syntheses are then completed, with the plus and minus strands of DNA each serving as a template for the other strand. REVERSE TRANSCIPTASE - PCR • developed to amplify RNA targets (RNA viruses such as HIV, HCV, and influenza are key examples). • the method entails an initial step of transcribing a portion of the RNA genome into complementary DNA (cDNA) which is then amplified through PCR. PRIMERS • Three kinds of primers : o Oligo dTs are synthetic DNA strands which can be represented as a sequence of ‘T’ nucleotides as in: 5’ – G T T TT T TT TT T TT TT TT -3’ o Gene specific primers can be designed to anneal to a specific gene. o Random hexamers consist of a ten bases which anneal randomly to RNA molecules. RNA TEMPLATE • The RNA template comprises the messenger RNA. • RNA is single stranded and tends to form secondary structures via Watson- Crick base pairing. • Heating the template is essential in order for it to be linearized prior to annealing of the primers. REVERSE TRANSCRIPTASES • RNA dependent RNA polymerase is derived from reverse transcribing viruses. • Mooloney Murine Leukemia Virus • Avian Myeblastoma Virus (AMV) THE PROCESS OF RT-PCR • Linearization of RNA template. • Annealing of Oligonucleotides. • Reverse transcription by Reverse Transcriptase. • Removal of mismatches by RNase H. • Mismatch repair by Klenow Fragment DNA Polymerase. • Downstream processing of cDNA • . •