TdT with DNA sequencing

David Micklem drm21 at mole.bio.cam.ac.uk
Fri Sep 22 14:37:08 EST 1995


In article <DFB8v2.9t1 at cc.umontreal.ca>, "Benoît Grondin"
<GrondiB at IRCM.UMontreal.CA> wrote:

>Hi!
>
>We had also some sequencing problem with GC rich regions. We used the tDt 
>step according to DeShazer et al Biotechniques 17, 288-289. At some piont 
>we had also to separate the samples on highly denaturing gel that 
>included formamide according to Kapelner et al BioTechniques 17,64-67. 
>Beware of these urea/formamide gel: they have to be loaded warm (so it 
>does't let you a lot of time before polymerization) and they are very 
>hard to dry (for that cover them after drying with baby powder).

I too have had good results with urea/formamide gels, but I don't think
they address the same type of sequencing problem that TdT does.  AFAIK,
TdT is useful for getting rid of bands across all four lanes.  These are
supposed to be caused by secondary structure in the TEMPLATE causing the
polymerase to fall off.  Because under these circumstances the truncated
products do not have a terminal dideoxy nucleotide, TdT can extend them to
a length where they are not a problem.

Formamide in the gel is useful in resolving compressions, where the bands
run irregularly due to secondary structure in the PRODUCT.  I haven't seen
the reference referred to, but I was taught to add 10% de-ionised
formamide to my normal (gradient) gel mixes, and this seems to work very
well.  I do this after filtration, as formamide attacks most syringe-end
filters.  I'm not sure  I understand the last sentence above - but I've
had no problem pouring these gels at room temperature, nor do I do
anything special loading samples.

The baby powder really helps - my continuing thanks to the net community
for that tip!


Cheers,

David

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
Fax: [+44] (0)1223 334089               
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