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primer dimers


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#1 prime

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Posted 16 November 2004 - 06:21 PM

What are primer dimers and thymine dimers exactly? How do they affect PCR and gel scoring, and why do they occur?

Edited by prime, 16 November 2004 - 06:24 PM.


#2 nabla

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Posted 16 November 2004 - 10:57 PM

Primerdimer occur, when the Primer are -or parts- are complementary (3' of the FOR- and 3' of the REV-Primer). While PCR both oligos hybridizate and are elongated. The Product contains both primer sequences. Primerdimers reduce the avaiable ammount of primers for the pcr-reaction. Therefore the pcr effectivity is reduced because of this non-specific reaction. By using realtime pcr (SYBR-Green) you can detect them with a melting curve analysis. (http://www.roche-app...tcycler-online/ the last note) Perhaps you can see the dimers on a 3% agarosege, too.

FOR 5' aattggcc'3 ->
REV <- 3'ggatct '5

Result/Amplicon: 5' aattggccatct

Thymine dimers are pyrimidin dimers. They are photoproduct (UV-light) and abort the amplifcation of the template. UV-light stimulates the formation of a four-membered cyclobutyl ring between two adjacent pyrimidines on the same DNA strand by acting on the 5,6 double bonds [modern genetic analysis (2nd) griffiths, p 323]. A 6-4 photoproduct is also possible. This principle is used to prevent possible contamination by "old" amplicons/templates while pcr-reaction by treating puffers,tubes etc. with UV light before amplification step.

#3 prime

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Posted 17 November 2004 - 05:27 AM

Thanks




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