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microsatellite PCR from poor quality/trace amounts of DNA


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3 replies to this topic

#1 zakirG

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Posted 26 January 2005 - 04:08 AM

Hello to everyone here. Im a new user. Im having huge problems with my PCR. im trying to amplify a microsatellite marker using DNA extracted from paraffin wax-embedded tissue (human oesophageal tissue). Because the wax blocks are old, and because of the destructive procedures using in fixing the tissue, any DNA that can be extracted will be of poor quality(low molecular weight) and low yield. Ive tried a MgCl titration, different annealing temps, etc...nothing has worked. Its really frustrating. If anyone out there has any suggestions for PCR using poor quality DNA, microsatellite PCR, etc, please respond to this...and help to ease my pain!! :) It would be most appreciated...if yu could provide any helpful hints or advice, il definitely take it into consideration. Thanks!

#2 Georgiana1977

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Posted 26 January 2005 - 06:48 AM

I have read recentlly a new article about that.... I have the same problems...

do u use a kit for PCR? because if u do... you may want to design your own primer set, more closer to the marker, or even within the STR and to reduse at min the dimention of your PCR product.

succes

#3 zakirG

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Posted 27 January 2005 - 12:49 AM

Thanks for your reply. Its not feasible for me to redesign my primer sets at the moment...besides...im not too sure how to go about doing that?...anywayz...could you..or anyone else...provide a reference to that paper you were talking about? Its sort of comforting to know someone else is experiencing the same problems as me!:-)

#4 bob1

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Posted 27 January 2005 - 02:50 PM

I suspect that the DNA you are getting is just too damaged and possibly you are not getting enough DNA to amplify. Extraction from fixed tissue is always a problem, there are a few papers out on methods for extracting from formaldehyde fixed tissue that you could try (sorry no refs off the top of my head). I havn't done these myself, but others that I work with say that these proceedures work.

Good luck! Msats are hard enough as it is!
Bob

Edited by bob1, 27 January 2005 - 02:51 PM.





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