Bisulfte PCR and sequencing PCR and Primer Notes - Tips and hints on PCR for bisulfite converted DNA and bisulfite primer (Jan/08/2007 )
The following is an adaptation of my notes about bisulfite PCR and PCR primer design for bisulfite PCR and sequencing. Please note, these notes are only applicable to BSP or bisulfite pcr and sequencing and NOT MSP (methylation specific PCR).
Comments, additions and annotations are welcome.
Two rounds of PCR were used for bisulphite amplification.
1. Set up PCR to a final volume of 25?l. Promega master-mix and Sigma Taq polymerase was used for PCR.
2. Sigma Taq polymerase was used with a Mg titration of 0.5 – 3mM Mg.
3. Mastermixes were assembled with PCR dedicated pipettes.
4. PCR was run in 500?l reaction tubes (thin wall).
5. 1?l of each primer was used in the PCR. (10?M stock) so 10pmol of primer final concentration of 400nM of each primer.
6. 1?l of bisulphite reaction DNA was used in the first round of PCR (done in the “dirty” area).
7. For the second round of PCR, reactions were set up as for the first round of PCR.
8. 1?l of first round of PCR was used for the second round of PCR.
9. PCR was set up at ambient temperature!?!? No ice.
PCR cycling profile.
PCR was performed in an Eppendorf mastercycler gradient machine. This can take 500?l tubes for PCR.
Total 30 cycles for each round of PCR.
Hot start PCR. Machine was set at 95°C and tubes were placed into the block and the program was initiated.
95°C 4min (denaturation)
95°C 30 sec
52°C 90 sec (annealing. Temperature is set according to calculated Tm from primer express. 2°C lower than the lowest Tm primer, round one PCR the primer was 54°C, round 2 48°C).
72° 120 sec (extension time to ensure no PCR recombination)
× 5 cycles.
95°C 30 sec
52°C 90 sec
72°C 90 sec (ex tension time is still longer than usual as the product is about 300 bases.)
× 25 cycles.
72°C 4 minutes
Things to consider for the PCR.
Separate areas for gDNA work, PCR set up and post PCR work. Here in X's laboratory, there is a separate area with pipettes and centrifuges for each area. PCR set up is performed in a room with TC, but pipettes have no contact with any DNA. This is to avoid cross contamination. PCR machines are located in a fourth area.
Things to consider for primer design.
The nature of bisulphite is that there will be a homogenous mixture of highly converted templates to ones that are not converted at all. To favour amplification of the fully converted templates primers must be chosen with this taken into account.
The + and – strands are not complementary and thus primers are designed for one strand and it is usually the plus strand as given by the text editor.
The criteria for primer selection includes:
• Primer sets amplifying no more than a maximum of 450bp.
• At least 30% C within the primer.
• A primer that selectively amplifies bisulfite converted templates.
• An even distribution of bases prior to bisulfite conversion.
• As high GA content as possible.
• A lack of CpG residues and if this can not be avoided then degeneracy at the possibly methylated cytosine residue.
• A long primer (25-30mer) to ensure uniqueness of the primer.
• Have a nested set to do the second round of PCR. Nested or hemi-nested primer is fine.
• Tm calculations to be equivalent for primer pairs. Input primer sequence into Primer Express to check the Tm. Primer express uses nearest neighbour method to calculate Tm. Match primer Tm. If they can not be matched then that can not be helped. Set Tm of the PCR reaction to be 2°C lower than the lowest Tm of the primer set. Tm calculation between Primer Express and NetPrimer are different.
The bad thing about methprimer is that it does not take into account primers amplifying unconverted sequences. 5’ and 3’ ends should have sites where conversion has occurred (C to T). This is to avoid amplification bias toward the unconverted sequence. There are tracts of primers where there was no reason to preferentially bind to the converted sequence as opposed to the unconverted.
The nature of the PCR amplification step is very sensitive. Would we require a genomiphi step to enrich and then do one round of PCR? Depends on the population of DNA in the solution. If the conversion were efficient then yes that would be an option, though it seems that does not need to be the case here. X has done library amplification, digestion and ligation of a universal linker and then one round of PCR. It has worked in the past but she says as with libraries, you can lose some genes, as some genes especially after bisulfite conversion are not amenable to amplification. Contact amersham about it all.
thanks Methylnick for these informations, I have few questions :
Why is it important to set up the PCR at ambient temperature instead of ice as it is usually done ? I am setting up my reactions on ice and then transfer them directly to the block at 95°C.
You are using a quite long annealing time (90s). Is there a reason for that ? (I use 30s).
Why are you changing the extension time after the first 5 cycles ?
How important is the length of the extension time ?
Thanks in advance.
these are notes I took while visiting a collaborator's laboratory. It was the first time I saw that a PCR could be set up at ambient temperature with no effect on the amplicon!
The extended annealing time just allows for the proper binding of the primer to the template remembering that wiht your bisulfite converted DNA there is a mixture of templates of different sequences after conversion, the extended time just allows for the proper binding of the primer to sequence.
As for the longer extension time, BSP is a strand-specific amplification. Bisulfite conversion generates non-complementary strands of DNA, therefore primers are designed to amplify one or the other strand of DNA. if you don't allow for full extension of the amplicon in the first rounds, P
CR recombination may occur across a subsequence where there are not many cytosines (ie: where they are complementary). The initial 5 cycles allow for full extension of the amplicon of interest as only one of your primers recognises the DNA sequence, it is only after the extension from this primer, the sequence for the other primer is generated.
hope this helps.
great - everything is in here! Thanks a lot.
Just one short question - do you use the same protocol for round 2 of a nested PCR? I normally use shorter times (and lower extension temperature, but we have discussed about that) in the second round, as the strand specifity should be no problem there, right?
yes I use the same protocol, but you are right you can reduce the TM and times because of the increased levels of specificity.
i am new to this methylation area and i am quite confused by the bisulfite sequencing and MSP..I could understand some of MSP, but lots of doubts (most of them blunders, pls forgive me) ..
Fist of all why do we have to design 2 primers for sequencing. y do we have to seqence both strands?
will the primers amplify onlythe unmethylated DNA during PCR?
then how can we be sure that the bisulfite treatment we give is 100%effective? wont it give 2 peaks (C+T)
if the methylation is monoallelic, wont it give 2 peaks too?
Pls i am so sorry that i know nothing abt these..
i would be very greatfull if you could help me out or atleast refer me some papers or books to read about this..
Welcome to the methylation area (some call it Area 52 )!
I would suggest that you have a look at the pinned threats in this forum (for example "Difference between MSP and BSP"). I think, they can answer your question easily.
The main point is to understand the difference between MSP and BSP, afterwards allmost everything is easy to understand ... more or less ...
I have a problem with bisulfite treatment. I don't think it is a problem of the PCR, it must be before that. I'm using the unmodified protocol of Olek, and also some modified versions. I don't want to pool so many cells so I don't isolate gDNA, just want to rus the bisulfit conversion in the cell. So I use the agarose bead method. I have tried out so meny things, changing incubation times, NaOH concentration, wash steps according to the differet papers published - but no restult. The interesting is, that when I first did it with cells, it worked in one of my 3 samples but then I was in a hurry so I'm not sure I did everyting like it was in my protocol. Since then I can't reproduce it and I'm going mad with it. since a while I'm trying with isolated gDNA mixed into LMP agarose, but I got no result. De you have any hints that I should pay more attention on while doing bisulfite reaction? Thanks for any help!
First of all: thank you! I have been browsing the forum and your comments are extremely helpful.
Here's my question:
How are the primers designed for Round1 and Round2? Is it a nested setup? Could you drop a few lines to nested PCR Setup for BSP (and in general for that matter )
I am currently taking over a methylation project whose initiator has taken a different job and am very much overwhelmed. I have no lab experience at all (med student) and noone here has any knowledge of epigenetics, so please bear with me if I'm a bit slow.
Another question: I have been hearing that direct sequencing rarely works and cloning is usually necessary. Why is this so? The only reason that I can think of is allelic differences, or differences in sample population. Given a homogenous sample, why should cloning be necessary / preferable?
welcome to the world of DNA methylation and epigenetics.
Primer design is a nested straegy for two rounds of PCR. But I have been using a hemi nested approach because I look at many loci for methylation and I am saving on the cost of an extra primer.
Direct sequencing usually requires a primer from which you performed the original PCR from, there are pro's and con's to this method. Usually the reverse primer works if it does, but it reason why it doesn't work is that the primers used for bisulfite PCR are usually AT rich and not ideal as a sequencing primer, one way to get around this is to use a tailed PCR primer with either SP6 or T7 attached to your bisulfite primer.
One thing you have to note (I have just received some data from another group with direct sequencing) is that the basecaller of the sequencer will over compensate for the lack for your C signal (representing methylation) so it is quite difficult to quantitate the amount of methylation by simply measuring the peak heights unless you calibrate the assay with a standard (ie: several reactions with know methylation percentages).
Direct sequencing does cut the costs down but it does not give you clonal information in that you know the methylation pattern across your region of one allele from one cell. Direct sequencing only gives you an average of the population.
Good luck cyburn and please ask as many questions as you like, welcome to the world acording to epigenetics.