is cDNA sequencing tricky? - (Jun/16/2008 )

Hi everybody,

I've just sent 2 cDNA library for sequencing and they told me that in a number of clones there is a homopolymer A tract that can mess up the TAQ activity...
do you know if there is any "special" way to sequence clones that are supposed to have a polyA tail as they come from mRNA?

thanks a lot
Raffaella

[b]Observation: The above figure shows a drop-off in sequencing quality after a polyT region(Pic 11-1) and a polyG region(Pic 11-2). Sequence data up to and including the polynucleotide region may be fine, but the last base of the poly region and all peaks following it may show a wave-like, stuttering pattern of double peaks that cannot be interpreted. This tends to be more problematic in PCR products, but can also occur when sequencing plasmids, especially when trying to sequence the polyA region of cDNA.

Cause:This difficulty is thought to arise due to enzyme "slippage" when the growing strand does not stay paired correctly with the template DNA during polymerization through the homopolymer region, thus giving rise to fragments of varying lengths that have the same sequence after this area.

Solutions: When sequencing cloned DNA with a homopolymer region, several options can be tried. Sequencing the opposite strand can sometimes be more successful, especially when going through a polyG region as the polyC strand is often easier to get through. An oligo dT(15-20T) primer that contains a wobble base (A, G or C) on the 3' end can be used to anchor the primer in place at the end of the polyA region and give clean sequence following. This primer (T20V) is provided by us to you free of charge. It anneals at the 3' end of the poly T and continues sequencing downstream. Sometimes designing a new primer that is closer to the homopolymeric region can help, as nucleotide concentration and enzyme activity will be in a more optimal range when extending the smaller fragments in the cycle sequencing reaction. And lastly, we can try adjusting our cycle sequencing conditions as higher annealing temperatures and longer extension times can sometimes be useful in cases like this. Similar approaches can be used when trying to sequence PCR products with homopolymeric regions, but, in the end, it may sometimes be necessary to clone the PCR product in order to read through the repetitive stretch.