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Human mtDNA amplification problems


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#1 cla.sav

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Posted 22 January 2010 - 06:54 AM

Hello

I am having trouble amplifying a fragment of the control region of human mitochondrial DNA. The fragment is 1300 bp. The PCR reaction works with control DNA we have in the laboratory and with some old samples, but not with the samples that I want to study. These samples that fail to amplify worked well for the amplification of other fragments of smaller size (around 280 bp). All samples and control DNA were extracted in the same way (Chelex) from bloodstains on filter paper, but the extraction was done at different times.
The picture is the electrophoresis of this PCR reaction. Cases 3 and 4 are the oldest samples, and besides these cases I also put two Chelex controls and a control extracted by salting out from blood in EDTA.

I thought of using BSA, but I'm not sure if it is the appropriate solution to this problem. I'm afraid to use my DNA samples, because I can not get more of them.
Does anyone have suggestions?

Attached Thumbnails

  • mtDNA_contr_region_PCR.jpg


#2 Maddie

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Posted 22 January 2010 - 01:48 PM

Hard to tell from here but here are a few ideas/suggestions/questions.
1] Any chance your DNA is simply degraded? 1300bp is big for ancient DNA
2] What part of the mt genome are you working on? If it's the CR, you may simply have a binding issue with one of your primers.
If the amplification works with 280bp, I doubt inhibition is the problem, but, hey, BSA never hurts (as long as it is non acetylated).

Hope that helps a bit.

Maddie
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#3 cla.sav

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Posted 04 February 2010 - 10:27 AM

Hello

Thanks for the suggestions.
We study congenital malformations, and our samples are DNA extractions from blood spots on filter paper. Our oldest sample was extracted in 2005.
We discovered that we had some wrong information about our samples. The only two samples of cases that amplified in our PCR was not extracted by Chelex, as we thought. They were extracted by a kit from Quiagen. However, still our lab control DNA, extracted by Chelex, amplified.

We quantified the samples and we do not notice major differences between cases and control of our lab.

I think that the mitochondrial DNA of these samples must be degraded, and therefore does not amplify a fragment of 1300bp. But my supervisor thinks that mitochondrial DNA does not degrade in such a short time (5 years) in frozen samples.

We're trying to amplify the CR. I don't think the problem is with a primer because they worked for some samples (extracted by the kit), and the controls of the reaction (one control extracted by Chelex and another by salting out) also amplified. These were sequenced and the fragment is correct.

I'm out of ideas now... ;)

#4 phage434

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Posted 04 February 2010 - 04:53 PM

Dilute your template and try again, or repurify with a phenol/chloroform extraction. You might also try the pre-pcr enzymes from Epicentre or NEB, which attempt to fix up degraded DNA.




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