Size of genomic DNA is too large with many exons to amplify, so can it be done using a cDNA?
Hi Madhubala,
starting from mRNA, yes you can but the risk is to miss some regions and important points, since the cDNA production will not be perfect along the genome (high GC contents for example and poor representation of low expressed genes...). If you plan to analyze NGS, I would advice you to run an exome library preparation to limit your data to exons and splicing events.
fred
Well, you better have really good justification for ignoring most of the genome. Even if you are only interested in missense and nonsense mutations, you will not be able to detect any mutations that create a splice-site mutation or a truncated (and quickly degraded) mRNA. You could use cDNA as a pilot study or first look, but it's very limiting. Talk to your colleagues and choose wisely. Good luck!
Reverse transcriptase is very error prone. You should sequence deeply if transcript sequencing is your only choice.
Okk. Thank you Fred, Katie and Govinda for your valuable inputs. I would prefer going with genomic dna.
In my Opinion, the genomic DNA will give you a more accurate result and will compensate for the time and resources involved in carrying out numerous pilot test for cDNA. Your decision to go with the genomic DNA is well accepted by me. Silas
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