Success with Direct Sequening of Bisulfite Pcr Products - (Mar/24/2006 )
Hi gaodaxia, thanks for the information. However, I am not able to find the software on the HEP site. Can you provide the link? Thanks, Krümel
the link to ESMS software
this link is in bottom of the <publication> page, so it is not surprising you can not find.
because i can not try this software (i do not have linux), so please let me if this software works well in your hand. thanks.
Thanks, I will give it a try!
(Several hours later ...)
I just remeber that everybody can use Linux, even on a Windows platform. I paste a fool-proof guide here.
... I haven't tried ESME yet, but it may be a problem, that I am using a ABI 3100 Avant Sequencer. The software suppprts only the 3730 series.
Do I understand it well if I say that both the outer and inner PCR reaction is a linear PCR? In mind mind it follows like this:
I have the BS treated DNA
I design a primer pair for example for the sense strand; forward is the same as the treated antisense (of course I'll transform U-s to T-s on the ordering form), and the antisens primer is is the backward sequence of the antisense BS terated strand, too. So both strands of my product will be antisense! Which will give me a single-straded DNA product at the and, the number of molecules are multiplied by the cycle nr.
Next round, the inner PCR: I have to desing a primer set complementary to the previous antisense products, so both primers will have the "sense" sequence, of course they are directing from different directions. but in this case this PCR also will be a linear PCR, producing a sigle-stranded population of DNA! Or did I misunderstand something? Pls help me, I'm really confused by now... So far I was makind several PCRs with primers based on a paper. But there the sequence of the forward primer is the same as the bisulfite treated sense 5'->3', and the reverse is the reverse sequence of the same strand. but because we don't have anymore a complementer sequence to the treated sense - for the antisense, after the bisulfite treatment has changed to another - I guess that primer will do nothing in my reaction! I need teh information, it is urgent, any help is appreciated...
there is a false assumption in your thought - bsp amplicons are double-stranded. It is explained in this topic.
Thank you, Krümel, on the way home I managed to find out, how it works... unfortunately I felt in panic at the end of the day so I was unable to think logically :-P
searching for an answer... I made BSP of 450 Bp long amplicons followed by direct sequencing with sense primers. To clean the products I used the Qiagen PCR purification Kit. The results were mediocre: the first 60-80 bases not evaluable followed by an acceptable sequence. I thought that primer-dimers could be the reason for such results: the products showed always a consistent, smeared band on bp 40-80 on agarose-gel too. So I extracted with the Gel-extraction Kit (Qiagen) again and sequenced with se + as primer. Now i´m wondering about the results: sequencing with as primer works well but with se primers i get the same results. So, if not primer-dimers, what could it be?
For some reasons (something about the base composition, pcrman has written something about it in one thread...) in many casesz only the reverse sequencing primer works well. So your observation is not uncommon. Porbably it will also work without the painful gel extraction. Just use the as primer and the PCR purification Kit (or maybe better a Dye Terminator Kit).
I have a problem with direct sequencing: my amplicon is 500 bp long and for direct sequencing I concentrate 75ul of the PCR probe by ethanol precipitation to 15 ul. Then I clean the Probe with the gel extraction Kit of Qiagen (Qiaquick), the concentration after gel-purification is always high. For sequencing I choose the As primer because i get better results with it. Now, sometimes I get a wonderful full-length sequence, but sometimes the sequence gives a reliable signal up to base 280-300 and after that no more correct and very weak signal. I observed that every time that i get such kind of sequences, the probe on the gel shows a weak smear on the gel, but on the larger site (from bp 500 to ~bp 750)! Is it possible that the probe degrades in only 4-5 days? (stored at -20°C) I observed this phenomena always when several days (5-10) passed between the PCR and sequencing.
Have somebody an Idea?