cDNA sequencing

Islam Khan ikhan at BGSM.EDU
Mon Aug 26 16:03:06 EST 1996


Dear readers:
I am sequencing PCR product by using sequenase version 2.0 DNA sequencing 
kit (USB, Cleveland, OH). The PCR product is obtained by amplifying the 
cDNA (from human T cells). In a particular segment of gene of interest 
the findings are as follows- when I used forward primer for manual 
sequencing, sequence is completely identical in healthy control and 
patient samples and matches to the published cDNA sequences. On the 
contrary, when I use reverse primer for seuqencing, two 'T's are missing 
in patient sample while the healthy control sequence matches to published 
sequence. The sequencing autoradiograms are very crisp and clear and I 
can read about 200 bases with ease.
I am using the alkaline denaturation method and following the 
manufacturer's protocol.  
I am just curious that these missing bases are just PCR artifact or 
something else. Please advise me.

Sincerely
Islam Khan, Ph.D.
ikhan at bgsm.edu



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