I am interested to know is it possible to directly sequence a single gene from total cDNA or gDNA without making genomic or cDNA libraries? Does anyone know about this?
Well, if it's from gDNA, you just need primers to flank your gene of interest and a gDNA extraction. You can sequence from the amplified PCR product. If you really want the cDNA, you will have to make a library and/or do an RNAseq run of the mRNA.
Hello, @ Katie A . Using primer for gene identification dose not mean sequencing which mentioned in question.
It depends on what he really wants. Sounds like they are unsure if they want the entire gene or just the transcript.
If you want the sequence of the gene from gDNA, then you can amplify by PCR, then clone and Sanger sequence the PCR product. This is a good first start, but won't properly show genetic diversity.
If you really want the sequence of the entire gene, including any genetic diversity, then you have to use a NextGen method.
Muhammad Shahzad Anjam what is your goal? Do you want to know the entire gene or the gene transcript?
Hi Thank for your answers. In fact I have sequence of a middle part of the transcript. Now I want to make sequencing primers in opposite directions to get the sequence of both ends using Sanger sequencing. So, probably PCR may not be the option in this case. However, RACE, could be one of the options. My question is whether, directly cDNA or gDNA cannot be used for Sanger sequencing to sequence the ends?
Hi, for your specific question it would be very probably indeed necessary to perform a RACE experiment. If you have already assembled whole genome sequence data for your object, you could try to find flanking regions / contigs / annotated genes that might be used for PCR primer design. But probably then the PCR fragments might be very large... too large for direct sequencing. If you have some time "perfect" primer to amplify your whole gene, you can use both cDNA or gDNA provided that the sequence including introns is not too long. We prefer cDNA and direct PCR amplicon sequencing. That makes sense if you expect not too much allelic variation within your gene. Otherwise, next generation amplicon sequencing is nowadays possible at a reasonably price (for instance: Genewiz Amplicon-EZ) .
You can't use gDNA directly for Sanger sequencing. It's not pure or concentrated enough (well, you might have some luck directly sequencing from a bacterial gene since they are so small, but I'm assuming you are not working with bacteria).
Honestly, given the low cost, I'd go for NextGen sequencing assuming that you can do the analysis yourself.
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