Problems purifying DGGE bands - PCR/DGGE (Dec/21/2003 )

Dear members,

I have the problem of having more than a single band in a lane, after reamplification of the sequence I picked up from a previous gel. I pick up the bands using steril tips (the ones for 2 to 10 microlitres), piercing slightly the band and placing this DNA in PCR quality water overnight. Then I reamplify the DNA and run a DGGE to check for purity of the DNA sequence. From a mix of sequences in a single lane I usually can purify the ones that are retained at a higher denaturant concentration, but not the ones that appear in the lower denaturing part of the gel. In the second round of DGGE to check for purity they come together with the bands that were at the higher gradient. I have tried picking again the band several times and reapeating all the procedure, making dilutions of the picked up DNA to make sure that only the sequence of interest is amplified, then running a DGGE and picking up the bands again and nothing, all my efforts come with the same results, I still have this multibanding in the gels. To me it's very strange that this happens only when I want to purify the bands at the lower gradient and not when I purify the bands at the higher gradient. It seems that some DNA from the bands that migrate further keep behind and contaminate other bands in the same lane. I am using primers 338Fgc-530R to amplify partial 16S rDNA. I would like to know if this is a problem that I have since my PCR reaction, or my technique of DGGE and if someone knows I really appreciate advice. Thanks a lot.

Monica

Did you find the solution of your DGGE band purifying problem?

I have a same problem as you. So I tried to make a clone to isolate the one sequence. If you want to get one sequence, just cloning it.

It's more effective.

Hans

what is your template of PCR amplification, mixture (such as environmental sample) or a purified strain,

when i use DGGE to analysis PCR products from purified strains, there are many bands in one lane, i think ther probolem come from PCR, but i don't know how to resolve it ,did you purify you PCR products, if you analysis environmental samples, wheather the purification could lost some bands.

let's help eatch other.
thanks

stoneliu

In my case, environmental DNA was used as a template. If you used PCR-DGGE product amplied from purified strain, it should be shown one band. However, in some case, several bands can be shown, since some strains have more than one operon of rRNA gene. Therefore, PCR could be amplified the different sequence(one point base pair change=wobble base). So you can see more than two bands on your dgge. However, it should not be over two or three bands. If you have more than three bands, you should doubt your PCR product by multi-strains. Although you have isolated strain by pure culture, you could get the mixed strains.
You can excise the bands and check the sequence. You can easily know the sequence information by BLAST search and determine the mixed strains or not.
If you have multiple band pattern before purification, the purification will not help to reduce it. Which primer set for 16S rDNA amplification?
Or you can modified the PCR annealing step to increasing the temperature getting more annealing specificity.

Good luck.

In my experiment, I mean all the pure strains had two bands, one of the bands is strong, and close the button of the gel (high denaturant concentration), i think it's what i want,
the second band was faint, and run slowly, close the top of the gel (low denaturant concentration), i don't know wheather it was single-strand DNA, i use the EB to stain the gel.
if the strains have more than one operon of rRNA gene, i think there should not have so big difference between the two bands

Hello again,

Someone gave me the clue that perhaps the low-denaturing bands may have been heteroduplex from the homoduplex in the bottom of the gel, which makes sense because everytime I tried to purified the low-denaturing bands, PCR reproduced the bands at the bottom of the gel but it never happened when I purified the high-denaturing bands. I always got 1 band in each lane in the last case. So what I did is that I discarded the bands that seemed heteroduplex at the top and worked only with the bands that more likely were the homoduplex at the bottom.

I amplified the sequences with the primers 338F-530R.

I would like to know if there is a more 'scientific' way to test for heteroduplex that this way of trial and error. Hope someone could make a comment on this respect.

Cheers

Monica

Hi Monica
Thanks for your information.
Actually, we using a GC-clamped primer (GC clamp is hang on at one side). So, heteroduplex and homodeuplex can not be formed in theoretically. But sometimes GC-clamp(or GC-clamped primer) can be released from the primer(or PCR products). That is the why we can get a different sequence from one DGGE band.
There are some other reasons. If we load the high DNA concentration into DGGE well, denaturing condition is not enough to denature the DNA molecules. So some DNA band can be different migration even though DNA has a same sequence.

Hans

Monica, I would say you may have found the answer, probably there's contaminating molecules in the rear part of the run; I'd say cloning the extracted molecules from the bands is the solution.

Additionaly, a single (pure) species may have up to 10 bands in a gel from the 16S gene (see Muyzer and Smalla, 1998).

There's a previous reply that says the GC clamp detaches from the amplified products, what are the facts proving such conclusion?

Carlos

I'm not sure what happened to me was what happened to you, but I found out the primers I received had less than 50% purity if I didn't request for purification (PAGE) after primer synthesis. The company said this is due to the high GC content of the primer (from the GC clamp) that cuases the difficulty for synthesis. I guess, be sure to spend more money on primers with GC clamps.