6 public questions tagged with this topic.
Reverse transcriptase is used to synthesize complementary DNA (cDNA) from an mRNA template.
Ref: NCERT Class 12 Biology Chapter 10: Biotechnology and its Applications Agricultural Applications - Nutritional Enhancement - Golden Rice and GM Crops
Reverse transcriptase synthesizes complementary DNA (cDNA) from an mRNA template, a key step in cloning eukaryotic genes.
Ref: NCERT Class 12 Biology Chapter 9: Biotechnology: Principles and Processes Bioreactors and Downstream Processing
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.
3 prime RACE exploits the natural poly-adenine tail present at the 3 prime end of most eukaryotic messenger RNAs. First-strand complementary DNA synthesis uses an oligo-dT primer that includes an adapter sequence, anchoring at the poly-A junction. The second strand is not synthesized by random hexamers alone, which would generate non-specific products. Instead, amplification utilizes the adapter-tagged oligo-dT as reverse primer combined with a forward gene-specific primer located within known exonic sequence. This strategy ensures selective enrichment of the downstream region between known sequence and poly-A tail terminus.
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.
Rapid amplification of complementary DNA ends targeting the 5 prime terminus requires generation of a full-length first-strand copy of messenger RNA. After reverse transcription, the complementary DNA possesses an accessible 3 prime hydroxyl at the end corresponding to the messenger RNA 5 prime region. Terminal deoxynucleotidyl transferase, a template-independent polymerase, adds a homopolymeric tract, typically poly-adenine or poly-cytosine, to this 3 prime end. This artificial tail provides an anchor site for an oligo-dT adapter primer, enabling subsequent PCR amplification using gene-specific and adapter primers to capture unknown upstream 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.
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.
