cDNA Calculators

0 calculators tagged with “cDNA”

Complementary DNA (cDNA) is a double-stranded DNA molecule synthesized from a mature mRNA template using the enzyme reverse transcriptase. Because it is derived from processed mRNA (which has already had introns removed by splicing), cDNA represents only the protein-coding regions of the genome — it contains exons only, with no introns or non-coding sequences. cDNA is used to: express eukaryotic genes in bacteria (which cannot splice introns); perform RT-PCR to measure gene expression; generate cDNA libraries for cloning; and produce recombinant proteins. First-strand cDNA synthesis uses an oligo-dT primer (anneals to the poly-A tail of mRNA) or random hexamers.

All Calculators

No calculators found for this topic.

cDNA Synthesis

  1. Anneal primer: oligo-dT (anneals to poly-A tail) or random hexamers + mRNA + RNase inhibitor
  2. Reverse transcriptase extends primer → synthesizes first-strand cDNA (RNA:DNA hybrid)
  3. RNase H degrades RNA template; DNA polymerase synthesizes second strand → dsDNA

RT-PCR Using cDNA

RT-qPCR: mRNA → cDNA (reverse transcription) → qPCR amplification with gene-specific primers → Ct value → 2^(−ΔΔCt) quantification. Used to measure gene expression levels. Distinguish from genomic DNA: design primers that span an intron → genomic DNA gives larger band (intron included); cDNA gives expected small band (intron absent).

cDNA Library

A collection of cDNA clones representing the expressed genes in a cell or tissue at a specific time. Steps: isolate mRNA → synthesize cDNA → insert into expression vector → transform E. coli → resulting library covers the expressed transcriptome. Advantage over genomic library: no introns → can be expressed in bacteria; enriched for expressed genes.

Key Applications

  • RT-qPCR: gene expression measurement
  • Heterologous protein expression: express eukaryotic proteins in E. coli
  • FISH probes: labeled cDNA probes for detecting specific transcripts
  • RNA-seq: cDNA library sequenced to map the transcriptome

Glossary

cDNA (Complementary DNA)
DNA synthesized from mRNA by reverse transcriptase; contains only exons (no introns); used to express eukaryotic genes in bacteria; the substrate for RT-qPCR and RNA-seq library construction.
Reverse Transcriptase
RNA-dependent DNA polymerase that synthesizes cDNA from mRNA; used in RT-PCR and RNA-seq; examples: MMLV RT, AMV RT; requires primer (oligo-dT or random hexamers) and dNTPs.
Oligo-dT Primer
A short sequence of deoxythymidines (typically 15–25) that anneals to the poly-A tail of mRNA; used in cDNA synthesis to prime reverse transcriptase; selects mRNA over rRNA and tRNA.

Frequently Asked Questions

cDNA (complementary DNA) is synthesized from mature mRNA using reverse transcriptase. Key differences from genomic DNA: cDNA: contains only exons (protein-coding sequences); no introns; no promoters, enhancers, or other regulatory regions; starts at the 5' cap of the mRNA and ends at the poly-A tail. Genomic DNA: contains both exons and introns; has regulatory regions; entire chromosome sequence. Why cDNA is useful: bacteria cannot splice eukaryotic introns → expressing a human gene in E. coli requires intron-free cDNA, not the genomic sequence. cDNA represents only the expressed genes at the time of extraction → reflects cellular gene expression state.

Reverse transcriptase (RT) is an RNA-dependent DNA polymerase (from retroviruses: HIV reverse transcriptase, MMLV RT, AMV RT). First-strand synthesis: bind primer to mRNA template; RT synthesizes complementary DNA strand (first strand) from 3'→5' mRNA → 5'→3' cDNA. Primer types: oligo-dT: anneals to poly-A tail → selects mRNA over rRNA and tRNA; gives full-length cDNA if RT is processive. Random hexamers: anneal throughout mRNA → multiple priming sites; better for secondary structure regions; produces shorter fragments. Gene-specific primer: anneals to specific mRNA → targeted cDNA for one gene. Second strand: RNase H degrades RNA template; DNA polymerase I uses remaining RNA fragments as primers → second-strand cDNA → double-stranded cDNA (dscDNA).

RT-PCR (reverse transcription PCR) measures mRNA levels by: (1) Extracting RNA from cells or tissue (with RNase-free technique). (2) Converting mRNA to cDNA using reverse transcriptase (RT step). (3) Amplifying the cDNA with gene-specific primers using PCR. In RT-qPCR: use fluorescent detection (SYBR Green or TaqMan) to measure amplification in real time → Ct (cycle threshold) inversely proportional to starting mRNA amount. Quantification: ΔΔCt method: ΔCt = Ct(target) − Ct(reference gene). ΔΔCt = ΔCt(treated) − ΔCt(control). Fold change = 2^(−ΔΔCt). Primer design tip: span an intron-exon junction → primers only amplify cDNA (no intron) not genomic DNA contamination.

A cDNA library is a collection of cDNA inserts cloned into a vector that represents all the mRNAs expressed in a cell or tissue at a given time. Construction: extract total mRNA → synthesize cDNA → ligate into expression vector → transform E. coli → store as individual clones. Uses: Expression cloning: screen library by functional activity or antibody binding → identify cDNA encoding a specific protein. Protein production: express library clones to produce recombinant proteins. Gene discovery: screen with labeled probe → identify clones expressing gene of interest. EST (expressed sequence tag): partial cDNA sequences from a library → catalogue expressed genes. Modern replacement: RNA-seq has largely replaced cDNA library screening for gene discovery — next-generation sequencing of cDNA (RNA-seq) provides transcriptome-wide coverage without building physical libraries.