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BS PCR..please help


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#1 PSUMike

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Posted 01 September 2009 - 04:54 PM

Hello everyone. I'm looking for some help with BS PCR. I am looking to analyze two genes for BS PCR. I am first starting with what I thought was the easier gene. I converted and isolated DNA using the Zymo Research EZ Methylation Direct Kit. I searched for CpG islands in my genes using Methyl Primer Expression from Applied Biosystems.

Below is my converted sequence from Methyl Primer express:
TTTTTGTATTATGTTTGTTTTTGTGTTTTGTTATTTATTATTTGTTTTTTTTATAGTAGCGGGTAAGTTTCGTTAAAGGA
GTATTGTTAATTTTTTTTATTTTTGTATTAATTATTAGTATTTAGTATAGTAGGAGTTTGGCGAATATTTGTATAATGTAT
GAGGGCGGTATGTAATTAGAGTGGGAGGTGAGGGGGTGGATGAAGTGTTGTTTTTTTTTTTTGGTTTTGGTTATAGAGTAG
ATATTAATGTTTTTTTTGGGTTCGGGAGTTTATAGGGGAGGGTAGGTGAAGGTGTTAGTTAGGAATAGGTTGAGTTAGATA
CGTTAAGTTTAGATGTTTTTTTTATATTAATTTTATTTTGGGAGTATTTTTTAGGGTTAGAGAGTATTTGTAAAATAGTTA
GTTAGGCGTGTCGGGTGGTGTTGCGTGTTTTGTTATTAAAAATGTGGATATACGTTGTTTAGGTTTTTGTTTTTAGGTAGA
AGTCGAGTATCGTACGTAAATTCGGTTTTTTGTATCGTTGGCGTTGTTTTGTCGCGTTTTTGTAAAGTATAGATTATTTTT
ATTTTTTAAATTTTATTTTAACGTTTTAGGAGAGTTGGTTTTTTAAGAGTTTTATTTACGGTTTTTTTTTTTTAGTTAGCG
TGATAGTATTGGGATTCGCGTTCGGTTGTGAGTTGGGTAGTTGGGTGGTTGCGCGGGTTTTTAGGTTTTTTTTACGTAATT
TCGGGGTATTTTGTTTTTAGTTAGGTGGGGCGGAGATAGGTAGTTCGATTTTTGTTTTTAGAGGATGGAGTAGGTTTACGT
ACGTTAGTTTTAGGAATTTGTGTGCGTGTTAAGTATTATTTTTTGTAGTTTTAGATTTTTTTTTGTTGTTTCGCGTGGATT
TTTTTTTTATTTTTTTTTTTATTATATTTAGATAGTTTTGTATTCGTCGTTCGATTTTTTTGTGTTGTTTGTTTTAGTATG
TATGTATAATATTAGTAAGTTTCGGGAGTTTATAGGGAGTTGTAAGCGGGGATTTTCGGGTTTGTAGTTTGTTTAAGGTGA
TTTTTAGAGGTGAGATTTGTAAGTTTGTTATTTATTTTTATTTTTTAGATGAAGTAGCGCGAATTTCGGTCGATATTTAAT
TTGTGGGGTATAGAGTTAGTTTAATGGCGTTATTGGTTTTTTTGTTTTGTGATTTTTGGGTTGGGGTCGTTGCGTTTTTTA
CGCGAGTTTGGATTTAGTAATTTAGGGAAGTAAAGTTATTATTTAGTTGTTTTTTTTTTATTAGTTTTTTTCGGGTATTTA
TTGGTTTGTAGTAGGTAAGAGGATTTTATATATTGAATGTTTTAGGGGTGTAAAAATAGTGAAAAGGAATTTTTATGATTC
GGAATGGAGGTTTTAGTATTTATTTTTTAGGTTATTTTAGAATTTTTTTTTAAATTTTTTTTTTAGTGGGATTATGTATTG
TTTATGGAGTATTTTGAAAGTGTGGGGGGTGGTGATTTTAATTTTTATTTTTTTTTTTTTTTTATGTTTTTTTGTGTTTGT
ATTTATATTAGGTATTCGGTATGGTTTTTTGTTTATTTTTTTTTTTTTTTTTGTTTAGAGTATTTTTTAAGGTTGTTTTTT
TTTCGGTTTTATTATTGGGTTTAGATAAGGAGGCGTGGTTAATAGATATAGAGTTTTATAAAGGTGGTGGTGTTTTTATTT
TAATTTTGAAGGTGGTAGTTTTTGGAGTTTTTTCGATTTTTTTTAGGGTTTTAGAGAATATTTTTTATTTTAGTTTTTTTT
TATAGTTTAAGGTGAGTGTTTGGGGGGGTTTTTGGATTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAGAAA
AGGAAATTTTTGGTGAAGAGTTATTGGGTAGTTTTTTTAAAATAGGGAATTATATTTTTATTTATTTATTTATTTATTTAT
TTAGTGATTAGGTAAATTTTTTAAGGGAGAAATTGAGGTTAGAGGTATTAGTTATTTAGATAGAAATGAGATATTTTTTTA
TTTTAGGTTTTTTTTTTTTTTTATTGATTTTTTAGGGCGTTTTTTTTGTATTTTTAGTTTTTTTTTTAGGATTGTTAGTGG
GGGTGAGGGTTTTTTTTTGGATGTGTAGGGTGGATAGTGAAGATGTATTGAGTTTTTTTGGTGTTTTTTGTTTTATTTAAA
AATTATTTTTAATAGTTTTTATTTTTTTAAAGGGTAGTATGTTAGAGTTGTTTTTGATTTAGGTTAGAGAGGTTTTTTATT
TTATTGATTGTTTTTTTTATTTTTGTTGGGATATTATTAGTTTTATAAATTGAGAGTTTTAGGGAATTGGAAATATTAGTT
ATATTTTTTTCGGTTTTTTTATTTTTTAAAGGTTAGGGTTTTTTAGGGATATTAATTATGAAGTGTGTTTTTAGTTTTTTT
AGGAAGTTTTTTTGTTTTAAGTTGATTGTAGTATTTGGGAATTGTATATTTTTTTATCGTATGTGTTTTA

The program gave me the following primers:
F1: TTTTTTGGTTTTGGTTATAGAGTAGATATTAA
F2: GTTTTGGTTATAGAGTAGATATTAATGTTTTT
F3: TTATAGAGTAGATATTAATGTTTTTTTTGGGTT
R1: CTTACAAATCTCACCTCTAAAAATCACCTT
R2: TAAAATCCTCTTACCTACTACAAACCA
R3: TTAAACTAACTCTATACCCCACAAATT

These primer sets were designed outside my putative island (720 bp), and the primers give products ranging from 800-1000 basepairs.

I set up all the PCR primer pair combinations, so I had 9 different primer set reactions set up. Below is my PCR conditions:

25 ng bisulfite isolated DNA (quantified as ssDNA by Nanodrop analyzer)
2.5 mM MgCl2
0.5 mM dNTPs
1 uM primers
2 units of Takara Ex Taq polymerase

My PCR reaction was the following:
95 C for 5 min

40 cycles of:
95 C for 30 sec
Gradient (55-65 C) for 45 sec
72 C for 3 minutes

72 C for 5 minutes before 4 C storage.

After the PCR, I loaded 50% of the PCR product on a gel, and I saw absolutely no product. I did not even get a smear to suggest the PCR was partially working.

I am looking for any help in trying to get my PCR to work. I read these forums religiously before begining this project because I wanted to avoid a lot of the commons problems. But it appears I am still having problems getting it to work. I have posted as much information as I could so that you forum experts can hopefully give me some specific suggestions on how to get my PCR working. I am concerned about the size of the PCR products (800-1000 bp), but I have read multiple papers were sizes like this have been analyzed as I would like to do. I would appreciate any help that you experts could provide. Thanks a lot.

PSUMike

#2 methylnick

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Posted 01 September 2009 - 07:43 PM

Hi PSUMike,

welcome to the forums, looking at your experiment I have a couple of questions for you:

1. sample DNA, is this from fresh tissue? If so then 800bp-1Kbp as an amplicon is doable. If it's poor quality DNA. then I would suggest shorter amplicons (300-500bp).
2. nested PCR strategy, can you do this with your current primer designs?
3. you can restrict methyl primer express to only design primers to your region of interest and of a certain amplicon size.
4. Is takara EX taq have exo activity/proof reading ability? It was hard to determine on the website, but a standard run of the mill taq would be fine for PCR, ex/prrofreading does not work on bisulfite converted DNA in our hands.

good luck.

nick

#3 PSUMike

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Posted 02 September 2009 - 03:59 AM

Nick,

The DNA was isolated from cell culture samples. I testing this in a cell culture system before going into tissues.

I am considering a nested strategy by using my largest primer set first, then using the smaller amplicon primer sets as the nested set. What is your opinion of that??

I did restrict the region of Methyl Primer Express as close to my region of interest as I could. I wanted some buffer region (50-100 bp) around the region of interest to keep my island sequencing as accurate as possible (Our sequencing facility generally has the first 20-40 bases come up as "junk" sequence).

Yes, Ex Taq has proof reading ability. I was using this because I thought proofreading would be more accurate in keeping the fidelity of the bisulfite conversion. I will try PCR again using our regular Taq (I got the Ex taq especially for this experiment).

Thanks for the help nick. I'll try a different Taq first and see how if that changes my results. If that doesn't work, then I'll try the nested PCR strategy. Thanks for the help, and I'll post my results.

Mike

#4 methylnick

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Posted 05 September 2009 - 01:45 PM

good luck mike and yes nested PCR strategy is as you said it, you get enhanced specificity of your bisulphite converted template that way. To save costs (of primer) you can do a heminested PCR.

Look forward to hearing of your results.

Nick

#5 PSUMike

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Posted 25 September 2009 - 06:41 AM

I finally got back to testing all the suggestions, and it worked. I didn't use hemi-nested PCR, I just changed the Taq. I never thought that a high fidelity ex-taq would affect the amplification. But I am getting pretty clean bands now with my PCR reaction. I can now optimize another gene primer set and actually run the experiment on the first primer set. Thanks for the help Nick...........much appreciated!

Mike

#6 epimaster

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Posted 30 March 2010 - 09:56 AM

Hello everyone. I'm looking for some help with BS PCR. I am looking to analyze two genes for BS PCR. I am first starting with what I thought was the easier gene. I converted and isolated DNA using the Zymo Research EZ Methylation Direct Kit. I searched for CpG islands in my genes using Methyl Primer Expression from Applied Biosystems.



The program gave me the following primers:
F1: TTTTTTGGTTTTGGTTATAGAGTAGATATTAA
F2: GTTTTGGTTATAGAGTAGATATTAATGTTTTT
F3: TTATAGAGTAGATATTAATGTTTTTTTTGGGTT
R1: CTTACAAATCTCACCTCTAAAAATCACCTT
R2: TAAAATCCTCTTACCTACTACAAACCA
R3: TTAAACTAACTCTATACCCCACAAATT

These primer sets were designed outside my putative island (720 bp), and the primers give products ranging from 800-1000 basepairs.

I set up all the PCR primer pair combinations, so I had 9 different primer set reactions set up. Below is my PCR conditions:

25 ng bisulfite isolated DNA (quantified as ssDNA by Nanodrop analyzer)
2.5 mM MgCl2
0.5 mM dNTPs
1 uM primers
2 units of Takara Ex Taq polymerase

My PCR reaction was the following:
95 C for 5 min

40 cycles of:
95 C for 30 sec
Gradient (55-65 C) for 45 sec
72 C for 3 minutes

72 C for 5 minutes before 4 C storage.

After the PCR, I loaded 50% of the PCR product on a gel, and I saw absolutely no product. I did not even get a smear to suggest the PCR was partially working.

I am looking for any help in trying to get my PCR to work. I read these forums religiously before begining this project because I wanted to avoid a lot of the commons problems. But it appears I am still having problems getting it to work. I have posted as much information as I could so that you forum experts can hopefully give me some specific suggestions on how to get my PCR working. I am concerned about the size of the PCR products (800-1000 bp), but I have read multiple papers were sizes like this have been analyzed as I would like to do. I would appreciate any help that you experts could provide. Thanks a lot.

PSUMike

Hi PSUMike,
I use the same kit which you used Zymo Research and also I used Methyle prime expression that you used, All of the conditions are that same, my primers are long and the give product is less than 200bp but I see very faint smear always. I used the extracted DNA from brain tissue, by Qiagen kit , also I use Rnase for purifying DNA, but

I don't cuase the DNA to be ss DNA, how I have to do that, I think converting by bisulfate cause breaking of double DNA, so what do you think? if doing ssDNA is necessary before the procedure start? how I can do that by NaoH ?

and If You clean up ypur DNA after treatment? More than 5 months I'm busy with this project but Could not find any product of PCR yet. I just find this forum yesterday, and I read so many of the comments and posts. I think finally by looking at this forum, I 'll be get good result,, can you guide me Plz?

and what do U mean by telling a gradiant 55-65 C for 45 sec during PCR reaction?

Thanks In advance..

Edited by epimaster, 30 March 2010 - 10:08 AM.


#7 epimaster

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Posted 30 March 2010 - 10:06 AM

good luck mike and yes nested PCR strategy is as you said it, you get enhanced specificity of your bisulphite converted template that way. To save costs (of primer) you can do a heminested PCR.

Look forward to hearing of your results.

Nick

Nike, Hello
I read this forum several times and I saw your advises most the times work, can you guide me please?

I use the same kit which PSU, used Zymo Research and also I used Methyle prime expression that he used, All of the conditions are that same, my primers are long(>25 mer) and the give product is less than 200bp but I see very faint smear always. I used the extracted DNA from brain tissue, by Qiagen kit , also I use Rnase for purifying DNA after extracting, but I don't cuase the DNA to be ss DNA before treatment by bisulfate, how I have to do that, I think converting by bisulfate cause breaking of double strand DNA, so what do you think? if doing ssDNA is necessary before the procedure start? how I can do that by NaoH ? More than 5 months I'm busy with this project but Could not find any product of PCR yet. I just find this forum yesterday, and I read so many of the comments and posts. I think finally by looking at this forum, I 'll be get good result,, can you guide me Plz?

#8 epigenius

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Posted 30 March 2010 - 10:30 AM

good luck mike and yes nested PCR strategy is as you said it, you get enhanced specificity of your bisulphite converted template that way. To save costs (of primer) you can do a heminested PCR.

Look forward to hearing of your results.

Nick

Nike, Hello
I read this forum several times and I saw your advises most the times work, can you guide me please?

I use the same kit which PSU, used Zymo Research and also I used Methyle prime expression that he used, All of the conditions are that same, my primers are long(>25 mer) and the give product is less than 200bp but I see very faint smear always. I used the extracted DNA from brain tissue, by Qiagen kit , also I use Rnase for purifying DNA after extracting, but I don't cuase the DNA to be ss DNA before treatment by bisulfate, how I have to do that, I think converting by bisulfate cause breaking of double strand DNA, so what do you think? if doing ssDNA is necessary before the procedure start? how I can do that by NaoH ? More than 5 months I'm busy with this project but Could not find any product of PCR yet. I just find this forum yesterday, and I read so many of the comments and posts. I think finally by looking at this forum, I 'll be get good result,, can you guide me Plz?


I believe the zymo kit uses denaturing by heat. The purpose of the DNA denaturation by PCR is to convert to ssDNA. When you achieve 90+ degrees, your DNA should become ssDNA -- just follow the protocol's PCR programming conditions.

#9 epimaster

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Posted 31 March 2010 - 07:41 AM

Ok, Thanks for your reply but as you know +90 is the first step of the kit, this is the same protocol which I used exactly

1. extracting DNA from cerebellum tissue by qiagen kit(during extraction I Used RNAse for avoiding any RNA)
2. reading UV, that shows me the concentration of THe DNA is great always.
3. treatment and clean up by the same kit, EZ DNA methylation gold kit, Zymo research
4. doing PCR with TaKARA prime star tag polymerase,

always I do normal PCR withe designing primers from methyl primer express saftware
PCR condition as TAKARA kit said:
38 cycles for, below
98 for 10 sec
55 for 5 sec
72 for 1 min
at the end gel

my result always is very faint smear for my interested samples but very nice band for control group, I mean un-bisulfite treated DNA

I'm soo appreciate if any body here can guide me , please.

Farimah

Edited by epimaster, 31 March 2010 - 07:47 AM.


#10 epimaster

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Posted 31 March 2010 - 07:42 AM

Ok, Thanks for your reply but as you know +90 is the first step of the kit, this is the same protocol which I used exactly

1. extracting DNA from cerebellum tissue by qiagen kit(during extraction I Used RNAse for avoiding any RNA)
2. reading UV, that shows me the concentration of THe DNA is great always.
3. treatment and clean up by the same kit, EZ DNA methylation gold kit, Zymo research
4. doing PCR with TaKARA prime star tag polymerase,

always I do normal PCR withe designing primers from methyl primer express saftware
PCR condition as TAKARA kit said:
38 cycles for
98 for 10 sec
55 for 5 sec
72 for 1 min
at the end gel

my result always is very faint smear for my interested samples but very nice band for control group, I mean un-bisulfite treated DNA

I'm soo appreciate if any body here can guide me , please.

Farimah
[/quote]

Edited by epimaster, 31 March 2010 - 07:50 AM.


#11 epigenius

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Posted 31 March 2010 - 10:51 AM

Ok, Thanks for your reply but as you know +90 is the first step of the kit, this is the same protocol which I used exactly

1. extracting DNA from cerebellum tissue by qiagen kit(during extraction I Used RNAse for avoiding any RNA)
2. reading UV, that shows me the concentration of THe DNA is great always.
3. treatment and clean up by the same kit, EZ DNA methylation gold kit, Zymo research
4. doing PCR with TaKARA prime star tag polymerase,

always I do normal PCR withe designing primers from methyl primer express saftware
PCR condition as TAKARA kit said:
38 cycles for
98 for 10 sec
55 for 5 sec
72 for 1 min
at the end gel

my result always is very faint smear for my interested samples but very nice band for control group, I mean un-bisulfite treated DNA

I'm soo appreciate if any body here can guide me , please.

Farimah


How much input DNA are you using per reaction and how much converted DNA are you getting? Are you denaturing with the zymo kit with PCR prior to working with the Takara polymerase? what type of carrier are you using?

Here are some preliminary suggestions:

1. Check for full conversion
2. Check for DNA impurities/improve purification

Edited by epigenius, 31 March 2010 - 10:58 AM.


#12 epimaster

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Posted 31 March 2010 - 11:58 AM

Hello again epigenius,

After extracting DNA by qiagen kit, I take 2μg of DNA as 20μl volum(az the Zymo kit has said) for treatment process and washing, and at the end I have 10μl of DNA(the last step of the kit) but I don't know if it is converted completely or not? how I can check it? and then I use 3μl of this (10μl) for PCR using TAKARA prime star,

and if you think how I have to purify my DNA after treatment (the kit said this DNA is ready for PCR), If I have to purify my DNA after each step? I mean after extracting and after treatment? If you suggest any purification way for purifying my DNA?

Thank you so much for your attention.

Edited by epimaster, 31 March 2010 - 11:59 AM.


#13 methylnick

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Posted 14 April 2010 - 04:10 AM

it is not necessary to repurify your samples after conversion, the kit does this for you.

One of the major problems is the primers you are using for PCR amplification are not properly designed, what primers are you using? are they specific for bisulphite converted DNA?

N

#14 epimaster

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Posted 14 April 2010 - 09:01 AM

it is not necessary to repurify your samples after conversion, the kit does this for you.

One of the major problems is the primers you are using for PCR amplification are not properly designed, what primers are you using? are they specific for bisulphite converted DNA?

N

Hey Nick, Thanks for reply
I used Prime star TAKARA for PCR, without any results, but I changed it to TFI, finally I got band but with a very regular polymerase, Tfi,, my advisor is worried about that because he belives this polymeraze has verry errors during polymerization!! and interesting part is that I got very nice bands with both of my primers specific and non-specific for bisulphate. :(

#15 methylnick

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Posted 18 May 2010 - 06:21 PM

the error rate with standard Taq is unlikely to affect your amplicon of interest because they are short (less then 1kbp).

The proof reading taq's fall over because it is copying off a hybrid DNA containing U's that is why standard Taq works fine for bisulphite converted DNA.

Nick




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