dear all
please I need your help, I'am new in cloning stuff, I amplified my gene ( ITS 2) and I want to sequence it to study genetic variability, I don't understand why we have to pass through cloning the gene in order to sequence it (this step is mentionned in all papers that I'am based in my exprement). what's the advantage of this step (cloning), why I can't sequence my gene directly after PCR without cloning my gene.
thank you a lot for your help
leila bouazzi
A PCR product is a mix of several cDNAs which are more or less different (allelic variants, splice forms, inserts and deletions). Any sequencer likes homogenous material to sequence, a heterogenity makes it crazy. Cloning allows you to extract from this mix a single copy randomly. Sequence analysis of several clones gives you wanted knowledge on genetic variability.
So, if you want perfect sequences, clone the gene and forget about innovations.
A PCR product is a mix of several cDNAs which are more or less different (allelic variants, splice forms, inserts and deletions). Any sequencer likes homogenous material to sequence, a heterogenity makes it crazy. Cloning allows you to extract from this mix a single copy randomly. Sequence analysis of several clones gives you wanted knowledge on genetic variability.
So, if you want perfect sequences, clone the gene and forget about innovations.
Dear leila
as you may know, in most of organisms ribosomal DNA have multiple copy, up to 30 copy number in the genome of some species. the sequence of ITS in these copies are not exactly identical. so if you go seqencing whitout cloning you will get noises in loci that differ in different copy and you can not make any conclusion. So , you need cloning befor sequencing. Hope this be of helps for you
sequencing from PCR product have chances of lossing some initial saquences while through cloning u'll get sequence of your entire product......
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