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Primer design for BSP - (Sep/08/2006 )

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Hi everyone, I am new in this group. My project is to analyze the methylation status of transgene in few of our transgenic plant lines. Im quite new in DNA methylation analysis, so seeking help for primer design. Plant DNA can be methylated in any sequence context of CG, CNG or CNN. I have also studied few about primer design. I am quite confused about sequencing of sense and antisense strand. Suppose at first I need to sequence the sense strand (the sequence which is visible in database). Is these OK if I design primer (as i do for normal PCR) for my sequence of interest and change the C/T residues of forward primer to Y and A/G residues of reverse primer to R. I will design the primer in such a way that PCR product should not be larger than 400bp and Tm should be low (45-55). For exmple, my sequence of interest is 70-270 of the following seq. :

1 ATCCTTCGCA AGACCCTTCC TCTATATAAG GAAGTTCATT TCATTTGGAG
51 AG
AACACGGG GGACTCTAGA TCTAGTGAAC GTACTGAATT CAAAGATGCG
101 GGAGCGAACC CTCCAGCCCC TAAGCCTCAG AATATCCCTC CACCACCCAC
151 AATAACTGAG GTTACTGATC CAGAAGACCC AAAGCAGGCA GCTTTGAGAG
201 CTGCACGAGC TAAGCAACCC GCAACCATTC CAGAATCATA TGGACGAGAC
251 ACTAGCAAGG AGAAGGAATC ATTTCCCCGA TCGTTCAAAC ATTTGGCAAT
301 AAAGTT
TCTT AAGATTGAAT CCTGTTGCCG GTCTTGCGAT GA

My designed primers to sequence the sense strands are (underlined in the seq.):
F primer: ATATAAGGAAGTTYATTTYATTTGGAGAG
R primer: AACTTTATTRCCAAATRTTTRAACRAT

Plese suggest me if the designed primers are ok or not and how to design the primers to sequence the lower strand.

Thanks in advance.
I am anxiously waiting for the kind reply.

Dnanlyzer

-dnanalyzer-

I would suggest to make ur primers atleast 30bases from the sequence of interest as the sequencing info that u get might have jumbled sequences in the very beginning. So if ur sequence of interest is from 70bp, then get a primer which will end by 35-40bps.

Also I would just order 21 bases of the gene to sequence. It works.

I didnt understand the part of changing ur C/T to Y , and same for the reverse ?

-scolix-

Hi, Scolix,
Thanks for your mail. After bisulphite treatment of DNA, it is assumed that all the C are converted to T, thats why degenerate primers were chosen to amplify the bisulphite treated DNA.
Somebody please tell me whether my primers are ok or not and how to deternine DNA methylation of the lower strand.

Dnalyzer

-dnanalyzer-

dnanalyzer,

your primers are not efficent for bisulfite modified DNA. As the 3' end of the primer is the most sequence specific part, if you have mismatches here Taq will not extend. so the 3' end should end in T's that were converted from C;s prior to bisulfite.

30mers are more ideal because if you are picking such primers it would be geared to be richer in AT.

to design the antisense strand I would reverse and complement your sequence and pick primers accordingly. if you are working in human you can almost say that methylation is symmetric, ie if you see it in one strand it would be on the other.

Nick

-methylnick-

Thanks Nick, for your valuable comments. At first I will explain my job in more detail.
I am working with transgenic plant, in which Cs can me methylated in any sequence contexts. I have already used these primers described before. I found Cs are highly methylated in silenced plants (transgene is silenced post-transriptionally, confirmed by detection of siRNA). But paradox was that, I found significant amount of Cs are also mehylated in non-silenced plant (which is not supposed to be). I can,t sure whether these was problem in my technique to assess DNA methylation.

The bisulphite sequencing technique which I used is summerized here:

1.500ng of plant genomic DNA was digested with EcoRV (to chop genomic DNA, no recognition site in transgene)

2. DNA was phenol-chlorophorm purified.

3. Purified DNA was bisuphite treated and cleaned by EZ DNA methylation kit (Zymo Res.)

4. Subjected to PCR using those primers with Jump start Taq (Sigma).

5. DNA fragment was gel purified by gel extraction kit (Qiagen).

6. DNA fragment was cloned to pGEM-T vector (Promega) and sequenced.

I assessed 6 transgenic plant lines (3 each for silenced and non-silenced.) In all the silenced lines Cs are highly methylated. In two non-silenced line no significant DNA methylation was observed. But in one non-silenced line DNA methylation was found in significant amount (not as much as in silenced one). Usually transgenes in non-silenced plants are not methylated. Have you any idea what's the explanation in my finding?

Thanks in advance.

Dnalyzer

-dnanalyzer-

plants can be a tricky beast.

can you test by another method to see the methylation status of your transgene? it's more difficult I assess conversion efficiency by bisulfite on plant DNA bcuase of non-cpg methylation of cytosines.

can you test the methylation status using methylsensitive restriction enzymes such as HpaII followed by a PCR?

N

-methylnick-

I have already used this strategy. I used Hpa II or MspI. In both the cases gel electrophoresis showed partial methylation at CG and CNG, respectively, (the band intensity was lower compared to undigested one).

Method applied was:

1.300ng of genomic DNA was digested with Eco RV and HpaII or Eco RV and Msp I (no sites of Eco RV within transgene, 5 units of each enzymes) for O/N.

2. After phenol-chlorophorm purification, 100 ng of DNA was used for PCR.

3. As a control, undigested DNA was used for PCR at the same reaction condition.

Please comment on my techniques. Do you think the BSP technique I used was alright? There is no one around me to discuss about BSP. I am depening on journals and online protocols for all of my research. Any evaluation and suggestion on these lab techniques will be highly appreciated.

Thanks in advance.

Dnalyzer

-dnanalyzer-

if band intensity was lower this could suggest incomplete digestion or that you have a mixed population of cells where that particular loci is methylated in some cells and not in others.

Nick

-methylnick-

Have you experience any problem using Aci I? In few cases it could't digest non-methylated DNA. I used even 10units of enzyme for 300 ng of genomic DNA (kept O/N at 37C). Where was the problem..I cann't find.

Dnalyzer

-dnanalyzer-

QUOTE (dnanalyzer @ Sep 9 2006, 09:27 PM)
Thanks Nick, for your valuable comments. At first I will explain my job in more detail.
I am working with transgenic plant, in which Cs can me methylated in any sequence contexts. I have already used these primers described before. I found Cs are highly methylated in silenced plants (transgene is silenced post-transriptionally, confirmed by detection of siRNA). But paradox was that, I found significant amount of Cs are also mehylated in non-silenced plant (which is not supposed to be). I can,t sure whether these was problem in my technique to assess DNA methylation.

The bisulphite sequencing technique which I used is summerized here:

1.500ng of plant genomic DNA was digested with EcoRV (to chop genomic DNA, no recognition site in transgene)

2. DNA was phenol-chlorophorm purified.

3. Purified DNA was bisuphite treated and cleaned by EZ DNA methylation kit (Zymo Res.)

4. Subjected to PCR using those primers with Jump start Taq (Sigma).

5. DNA fragment was gel purified by gel extraction kit (Qiagen).

6. DNA fragment was cloned to pGEM-T vector (Promega) and sequenced.

I assessed 6 transgenic plant lines (3 each for silenced and non-silenced.) In all the silenced lines Cs are highly methylated. In two non-silenced line no significant DNA methylation was observed. But in one non-silenced line DNA methylation was found in significant amount (not as much as in silenced one). Usually transgenes in non-silenced plants are not methylated. Have you any idea what's the explanation in my finding?

Thanks in advance.

Dnalyzer



I have no experience in this area yet. But I know that Sigma JumpStart, while very robust, generates high percentage of PCR mutation. Is this a possible problem?

-mushroom-

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