cDNA, or complementary DNA, is a form of DNA synthesized from a messenger RNA (mRNA) template through a process known as reverse transcription. This process is catalyzed by the enzyme reverse transcriptase. The primary function of cDNA is to represent the coding sequences of genes, effectively reflecting the mRNA present in a cell at a specific time.

Technical Overview

Reverse Transcription:

• Enzyme: Reverse transcriptase
• Template: mRNA
• Product: cDNA

Reverse transcriptase synthesizes cDNA by using mRNA as a template. The enzyme adds complementary DNA nucleotides to the mRNA strand, producing a cDNA strand that is complementary to the mRNA sequence.

cDNA Synthesis Steps:

• mRNA Isolation: Extraction of mRNA from cells or tissues.
• Priming: Use of oligo(dT) primers or random hexamers to initiate the synthesis of cDNA. Oligo(dT) primers bind to the poly-A tail of mRNA, while random hexamers bind to random sequences.
• Synthesis: Reverse transcriptase enzyme extends the primer, synthesizing a complementary DNA strand.
• Second Strand Synthesis: The single-stranded cDNA is then converted into double-stranded DNA using DNA polymerase.

Applications:

• Gene Cloning: cDNA can be inserted into plasmid vectors for cloning.
• Expression Studies: Enables analysis of gene expression by creating cDNA libraries and quantifying gene expression levels.
• Functional Genomics: Facilitates functional analysis of genes by expressing them in various systems.

Advantages of cDNA:

• Representation of Expressed Genes: Only the coding sequences expressed in the cell are captured.
• Stability: cDNA is more stable than RNA, making it easier to handle and analyze.

Considerations:

• Transcript Variants: cDNA libraries may represent only certain isoforms of genes.
• Quantitative Analysis: The quantity of cDNA can reflect the mRNA abundance but may require normalization and validation