prasher@HYATT.WHOI.EDU (Doug Prasher) (09/29/90)
Fellow Netters, If you are directly sequencing PCR products, I need your advice. We are trying to sequence PCR products of 400bp and 750bp in length and having almost no success. We have tried changing a number of variables none of which improve the situation. Our M13 control reactions done in parallel ALWAYS work. OUR STANDARD CONDITIONS are: 0.6 pmol ds template, 6 pmol primer (also used in PCR), boiled 10 minutes, snap frozen in liquid nitrogen. Thawed on ice and then sequenced using Sequenase. THE GENERAL RESULT is: No sequence ladder (or extremely faint) but there is a fairly intense band in all four lanes at a position in the sequencing gel approximating the full-length fragment. On some gels, a short sequence ladder is observed just below this intense band, which we intrepret as termination at the end of the fragment. We have assumed the problem results from a low concentration of the template-primer complex in the labelling reaction. We believe there is enough nucleotide present in the labelling reaction such that the Sequenase makes the complexes completely double-stranded before the dideoxys are added in the termination reaction. We have tried the following variables, individually of course, with no improvement: 1) 4 pmol ss template (via assymetric PCR) instead of 0.6 pmol ds template. 2) Increasing sequencing primer to 40 pmol. 3) Changing the thawing conditions: i) leave on ice 90 min ii) place in a -20C block, let warm to 7C or 15C. 4) Dilute labelling mix: 1:5 is normal, 1:20, 1:75, 1:300. 5) Shorten labelling reaction time at RT and at 0 C. PLEASE SEND YOUR COMMENTS. IF REQUESTED I WILL SUMMARIZE AND REPOST. Douglas Prasher Woods Hole Oceanographic Inst Woods Hole, MA prasher@hyatt.whoi.edu
BAFM1@cluster.sussex.ac.uk (10/01/90)
In reply to Doug Prashers query RE problems with sequencing PCR products, it is important (at least with klenow) to NOT have the buffer present (ie. TM) during the annealing step. It is added at the sequencing reaction stage. The presence of ss binding protein also helps. I must admit that I prefer to reclone into M13, but direct sequencing is useful if you just want to check a short region. Hope this helps, Jim Brannigan BAFM1@UK.AC.SUSSEX.CLUSTER
MSALMINEN%FINNPHI@PUCC.PRINCETON.EDU (MIKA SALMINEN) (10/02/90)
As I think this is of general interest I reply to the net: Doug, We at @FINNPHI.BITNET do a lot of PCR-sequencing with reasonable success. Here's our protocol in which you may find the answears you need: 1. denature 0.6-1.8 pmol of pcr product (purified) by heating 2 min at 100 C in water. 2. snap cool on ice for 5 min. 3. add 6 pmol primer. 4. add buffer. 5. anneal primer for 10 min at +37 C 6. add enzyme, label and do the labelling for 5 min at +37 C 7. divide to extension/termination reactions and keep at +37 C 8. stop reaction (loading buffer) There are a few more tricks: - if your DNA is dirty (as agarose purified) use double amount of enzyme, this countereffects the agarose inhibitory effect on enzyme activity. - if you want to read near the primer add manganese buffer from the newest Sequenase kit (one-tenth of total reaction volume) (Tabor et Richardson:PNAS 1989;86, 4076-4080). We do our sequencing using the Amersham Multiwell system in a 30 mikrol reactionvolume, but i know about people using Sequenase with similar success. Those of you using Klenow outhere.... Forget about ancient inferior systems, T7 rules OK! DISCLAIMER: I have none whatsoever commercial interests in Ame or USB corp:s, just forwarding my own personal opinions which do not represent any policies of this institute. Hope this helps, Mika salminen MSALMINEN@FINNPHI (BITNET)
ldow@biosys.UUCP (Leslie Johnston-Dow) (10/03/90)
Dear Doug, We are doing alot of PCR sequencing. In my own experience, sequencing symmetric PCR fragments can be difficult. The things that have really made a difference are 1) lowering PCR primer amount to 5 pm each (or removing PCR primers with centicon 100) and 2) using Taq polymerase for sequencing and to cycle the sequencing reaction using a labeled sequencing primer( in our case a fluorescent primer). By cycling the sequencing reaction, you will get a linear amplification of extension products which should give plenty of sequence data. Also, with this approach you can directly sequence symmetric PCR fragments without any purification! If you want more info just let me know....Good-luck. Sincerely, Sandy Koepf Applied Biosystems Inc. I can be contacted via ldow@apldbio.com