cDNA Synthesis – Principle, Steps
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cDNA Synthesis – Principle, Steps
- 1. cDNA Synthesis – Principle, Steps Microbiologynote.com Table of content What is cDNA? cDNA synthesis principle cDNA synthesis steps cDNA synthesis protocol Troubleshooting tips cDNA synthesis ppt References Don't miss a post What is cDNA? • A cDNA is a functional part of DNA made from an RNA molecule. It is created using a special enzyme called reverse transcriptase. • cDNA sequences as complementary DNA is different from the genomic DNA sequence, which only contain coding DNA sequences. • cDNAsynthesis is an important first step for many molecular applications. • Next generation sequencing (NGS), gene expression analysis, pathogen identification, and genetic testing by qPCR or NGS are just some examples of applications that require the conversion of RNA into cDNA as an early step. • To get accurate and precise results from these applications requires high fidelity in cDNA library construction so that the cDNA libraries accurately represent the original RNA transcripts. • When selecting a cDNA synthesis kit, two important considerations are the reverse transcriptase (RT) enzyme and the primer design strategy. • Enzyme selection influences the transcription rate and reaction fidelity. If priming strategies are not optimized, they may result in a biased cDNA library that does not accurately reflect the entire transcriptome.
- 2. cDNA synthesis principle • cDNA synthesis is an enzymatic process that involves the use of reverse transcriptase (RT) and forms complementary DNA (cDNA) from messenger RNA (mRNA). The whole process is technically known as reverse genetics. • Naturally, it has existed in viruses like lentiviruses, human immunodeficiency virus 1 (HIV-1), murine leukemia virus, feline immunodeficiency viruses, etc., broadly classified as retroviruses. • The polymerization property allows the reverse transcriptase enzyme to identify each nucleotide from the mRNA and form the complementary cDNA (cDNA) at the same time, by using the primer and incoming nucleotides. • However, the in vivo process of cDNA synthesis is somewhat similar to the natural one, but differs in some ways. It is divided into two parts: First strand synthesis and second strand synthesis. • We will learn about the in vitro process of complementing DNA synthesis in the next section. cDNA synthesis steps There are different steps for synthesis of cdna such as; 1.Prepare sample 2.Remove genomic DNA 3.Select reverse transcriptase 4.Prepare reaction mix 5.Perform cDNA synthesis