firststrand

Firststrand, in molecular biology, refers to the first-strand complementary DNA (cDNA) synthesized from an RNA template during reverse transcription. The enzyme reverse transcriptase uses an RNA template to produce a DNA strand that is complementary to the RNA sequence. This first-strand cDNA is typically single-stranded and serves as the starting material for subsequent steps such as second-strand synthesis, PCR amplification, cloning, or sequencing. Priming strategies for first-strand synthesis include oligo(dT) primers that bind to polyadenylated mRNA, random hexamers that provide broad coverage, or gene-specific primers for targeted reverse transcription.

In common workflows, first-strand cDNA is generated from total RNA or mRNA, then used to create double-stranded cDNA by synthesizing the second strand. The resulting dsDNA can be inserted into vectors for cloning, used to create cDNA libraries, or employed in downstream applications such as reverse-trans transcription PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), and RNA sequencing library preparation. The choice of primers, reverse transcriptase enzyme, and reaction conditions affects the representation of transcripts, with biases possible toward 5′ or 3′ ends depending on the method.

Key considerations for first-strand synthesis include RNA integrity, the choice of priming strategy, the activity and fidelity of the reverse transcriptase, and potential biases introduced during cDNA synthesis. First-strand cDNA remains a foundational step in many molecular biology workflows that convert RNA into a DNA template suitable for amplification, cloning, or sequencing.