I have encounter countless problems along the methylation analysis way. Most current is with sequencing of plasmids after GW cloning of PCR product. The PCR product comes from bisulfite converted DNA (genomic - cancer cells). After miniprep I performed PCR to chech whether the plasmids contain my desired insert - they did. I sent the samples for sequencing and the chromatograms looked afwul. I prepared samples for sequencing in a standard way according to instruction of the firm (plasmid DNA up to 400 ng, I tried also lowering it to 200 ng) + pDONR221 sequencing primers M13-R and M13-F (no I did´t mix them together into one sample).
I am trying to analyse 465 bp long promoter part where are 7 CpG sites.
I have got back all sorts of bad results
- looked good but then died after roughly 100 bases into my insert
- no familiar sequence at all
- no readable sequence at all (multiple peaks - complete mess)
- relatively nice sequence but a plasmid with one original clone should be cleaner - there were smaller peaks/noise
Now to my question finally - have you ever had any of those problems? If yes how did you solve them? I am fairly new to cloning and every advice will be deeply appreciated.
Can you attach some of the original .abi sequence files please? They often contain useful diagnostic information
Can I ask what way you performed your bisulfite conversion (kits etc)
If I understand you right you did the following
1) You have a plasmid that was bisulfite converted then underwent amplification through PCR (or was it amplified by PCR then converted?)
2)You then GW cloned this product
It may be that the bisulfite conversion was excessively harsh on the DNA and broken it up a little too much for the following steps. I have no experience with GW cloning but I know myself sometimes the bisulfite conversions themselves can be very intensive on the DNA.
The other option I can think of atm is that the transition from Cytosine to Thymine following PCR isn't taken into account in the GW cloning phase for the regions that were unmethylated and unprotected during bisulfite conversion causing the issues you described. Methylation isnt 100% after all
Hopefully you find an answer for it
Stephen Reynolds I used Sigma-Aldrich Imprint DNA modification kit. I converted genomic DNA from cancer cells acc. to their instructions. Afterwards I did PCR for the desired region. After this I used the product in two rounds of GW PCR (to achieve the overhangs needed for cloning). Afterwards I cleaned the product on agarose gell and performed BP cloning reaction followed by standard cloning steps. After measuring c of clean plasmid from miniprep I did PCR with primers specific for my insert to check whether they contain the insert. All of them did.
From 9 sequenced samples only 2 looked well however only with reverse primer. Other were well se for yourself. Paul Rutland
Legend:
1 - forward = sample 1 with M13-F
1- reverse = sample 1 with M13-R (correct one)
2 = sample 2 which showed on gel that it contains the insert but from the chromatogram it doesnt
3 = sample 3 which dies after few bases into my insert
4 = sample 4 which is just mess
Another interesting fact is that I originaly tried direct sequencing without cloning. I amplified converted DNA by nested PCR, cleaned the product on agarose gell and send for sequencing. Of course there was some noise and I know this method is not the best for quantification. But the true problem with this method was that only the first half of the sequence was readable then there were just peaks one through another.
I am thinking some troubling secondary structures resulting from long stretches of polyT and polyA? But the again why does it sometimes work and sometimes not? Becuase a few sequences were again readable.
Thanks for the reply, im afraid ill be of little help. Im not sure of your budget but it may be worth sequencing each step, may give you insight to where things start going wrong or maybe one round of GW PCR may be sufficient?
Dear Andrea
About the individual sequences:
1forward Strong signal above 600 and falling to below 400 signal strength after an AT rich almost repeat like sequence. My feeling is that this early termination could be caused by enzyme slippage or more likely the AT rich sequence forming a loop and at the sequencing temperature sometimes the enzyme reads the loop sequence and gives a correct sequence and sometimes it reads across the double stranded section of the neck of the loop and appears to read a deleted sequence from that point onwards.. This is a secondary structure issue and If I were sequencing Iwould either sequence in presence of 4% DMSO to remove secondary structure issues in the same way as sequencing GC rich regions or linearise the vector with any restriction enzyme that does not interfere with the sequence needed. As you are sequencing commercially contact them and ask that your sequences be treated as high GC even though you know that they are not.
1 reverse Good sequence….if all of the other sequences are with the forward primer then sequence all with the reverse primer
2 I wish this was PCR product then I could blame the first 140 bases which are clearly 2 sequences as either primer dimer or non specific product but it is plasmid so the best that I can come up with is the forward primer annealing in 2 places but it is hard to account for the good sequence between bases 170-310, I struggle with this result. Possibly sequencing at a higher annealing temperature might remove bot the inappropriate primer binding and the sequences ability to form a loop
4 Again strong peaks so the sequencing reaction is working well but a total mess. This is much more like the sequencing primer annealing in 2 places. Sequence at a higher temperature ( tricky commercially) or with the other primer.
3forward Strong peaks with an obvious background ( is this BG the same as the early part of seq 2 I wonder?. Good sequence up to the polyAT region then it fails. Try linearise vector,high GC sequencing or sequencing with DMSO as sample 1forward
Hopefully something here will help you
paul
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