cDNA Synthesis – Principle, Steps https://microbiologynote.com/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

3. https://microbiologynote.com/cdna-synthesis-principle-steps/