5 public questions tagged with this topic.
Reverse transcription is the process of making complementary DNA (cDNA) from an RNA template.
Ref: NCERT Class 12 Biology Chapter 10: Biotechnology and its Applications Ethical Issues - GEAC and Animal Ethics
Reverse transcription describes synthesis of DNA complementary to RNA template, reversal of conventional transcription flow. Enzyme reverse transcriptase discovered in retroviruses carries RNA-dependent DNA polymerase domain polymerizing deoxynucleotides using RNA, RNase H domain degrading RNA strand of hybrid intermediate, and DNA-dependent DNA polymerase producing double-stranded proviral DNA. Host tRNA primes reaction by annealing to primer binding site. Resulting provirus integrates into chromosome via integrase establishing latency. Mechanistically distinct from RNA synthesis, reverse transcription requires deoxyribonucleoside triphosphates and exhibits low fidelity due to absence of proofreading, generating retroviral variation.
Ref: Baltimore & Temin Nobel Lecture 1975 Reverse transcription DNA from RNA; Lodish Chapter 8 Reverse transcriptase activities; Alberts Chapter 5
Processed or retrotransposed pseudogenes originate via retrotransposition of mature mRNA intermediate. Line-1 encoded endonuclease and reverse transcriptase recognize polyadenylated transcript, generating cDNA copy integrated into genome at new location. Hallmarks include absence of introns, presence of 3' polyA tract remnant, flanking direct repeats and lack of promoter, reflecting derivation from spliced message rather than DNA duplication. They are typically transcriptionally silent due to missing regulatory elements, accumulate mutations and serve as molecular fossils of ancestral gene expression patterns, abundant in mammalian genomes.
Ref: Vanin EF, Annu Rev Genet 1985: Processed Pseudogenes Formed by Reverse Transcription of mRNA
RNA-seq platforms sequence DNA, so RNA must be reverse transcribed. Reverse transcriptase, an RNA-dependent DNA polymerase derived from retroviruses such as M-MLV or AMV, synthesizes complementary DNA, cDNA, from RNA template using oligo-dT, random hexamer or gene-specific primers. RNase H or second-strand synthesis then generates double-stranded cDNA suitable for adapter ligation and PCR amplification. RNA polymerase synthesizes RNA from DNA, DNA polymerase requires DNA template, and ligase joins nicks. Only reverse transcriptase possesses RNA to DNA polymerase activity essential for initial conversion.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.
Eukaryotic messenger RNAs possess a post-transcriptional poly(A) tail of 100-250 adenine residues at the 3' end, essential for stability, nuclear export, and efficient translation. Oligo(dT) primers consisting of 12-18 thymidylates anneal specifically to this poly(A) stretch through A-T base pairing, providing a free 3' hydroxyl for reverse transcriptase to initiate complementary DNA synthesis. This ensures selective reverse transcription of mRNA rather than abundant rRNA or tRNA contaminants. Random hexamers prime throughout transcripts, while oligo(dT) yields full-length, 3'-biased cDNA libraries enriched for eukaryotic coding sequences.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.
