PCR artefacts - (Jul/28/2003 )
I carried-out an RT-PCR that has given, with the same set of primers, two main products of the waited length of approximatively 260 bp and 550bp, corresponding to two forms of the same mRNA differentially spliced (the longer product retaining intronic regions). Moreover two other,less intense,unexpected bands were present of intermediate length (approximately 400 and 500bp). I do not succeed to eliminate these two last bands performing RT-PCR with more stringent conditions.
Therefore I have supposed that these two bands are not non-specific products of amplification but probably two ulterior forms of the same one mRNA deriving from alternative splicing.
When i try to reamplify these bands, by band-stab or by agarose gel excision, in order to achieve a greater quantity of products for sequencing, i reobtained also stronger signals corresponding to the two main bands of 260 bp and 550 bp, and a little amount of the bands that i try to reamplify.So i have difficulties to obtain the quantity of DNA necessary for sequencing.
Have someone an explanation for this? Could these two intermediate length bands be an artefact of PCR?
Probably obvious but have you checked whether one of your primer binds to a repeated region in your sequence of interest?