I am working on a gene which is 2.24kb long. I have already cloned its shorter form of 1700 bp. I have prepared cdna through Hek293 cell line, but when I put this gene's pcr I always get shorter form of this gene. So, when full form of a gene is not getting amplified, I can't proceed to next step. Kindly advice me how can I overcome this problem of cloning full length of this gene (i.e.,2.24kb). What are the major steps I should focus on to clone more than 2 kb gene?
Thanks.
Do you know the long form of this gene is expressed in your cells. Do you use the same primers for both isoforms? If so, the shorter form may outcompete the longer version, e.g. due to higher expression of the short gene or regions in the long version that are difficult to ampify like GC-rich regions.
Yes sir, it do express in these cell lines. It has been reported in literature too. I am using different primers for both forms because there is variation in length i.e., Full length = 2244 bp and its isoform= 1700bp. do i need to redesign primers for full form of this gene?
Is the long isoform always expressed? Sometimes alternative spliced is induced under specific conditions. Since you are using cDNA maybe the condition you are using leads to short isoform expression and that is why you can get the longer isoform. I worked with a gene that suffered different splicing when cells were stimulated with IFNgamma or IL12. So I had to isolate three different cDNAs one from IFNgamma treated cells (to isolate the short version), one from IL12 treated cells (to isolate the mid version) and one from untreated cells (to isolate full lenght).
If you are absolutely sure the longer version of your cDNA exists in cells, just do splicing in vitro or amplify missing part from genomic DNA and add it to your shorter version.
I would suggest you to find cell line in which just long form is expressed if it is possible. If not try to design your primers for long form to have higher Tm then for short form, and be careful about extension time in PCR.
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