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de ifz

Member Since 08 May 2011
Offline Last Active Feb 14 2013 03:25 AM
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#139999 Pcr product size determination for a fusion gene

Posted bob1 on 26 August 2012 - 12:33 PM

Probably the easiest way to determine the full lenght will be to sequence the product you have and/or clone it, then sequence.

However, just found this paper:
Vaccination of patients with chronic myelogenous leukemia with bcr-abl oncogene breakpoint fusion peptides generates specific immune responses

J. Pinilla-Ibarz, K. Cathcart,T. Korontsvit,S. Soignet,M. Bocchia,J. Caggiano,L. Lai,J. Jimenez,J. Kolitz, andD. A. Scheinberg

Which contains the following paragraph (second one in the paper, immdeiately following the abstract, bolding mine):
"Chronic myeloid leukemia (CML) is a pluripotent stem cell disorder characterized by the presence of the Philadelphia chromosome (Ph1). Ph1 is the result of a translocation of the c-abl oncogene from chromosome 9 to the breakpoint cluster region (bcr), within the bcr gene on chromosome 22, forming a chimeric bcr-abl gene.1-3 The fused genes encode an 8.5-kb chimeric mRNA that is translated to a 210-kd protein.4-6 This p210 bcr-abl protein shows tyrosine kinase activity, is present in the leukemia cells of patients with CML, and is necessary and sufficient for transformation.7 In 95% of patients, the breakpoint in the bcr gene occurs either between bcr exon 2 (b2) and 3 (b3) or between bcr exon 3 (b3) and 4 (b4). Hence, 2 alternative chimeric p210 bcr-abl proteins, comprising either a b3a2 or a b2a2 junction, can result from this fusion gene.8"


#122348 Getting a smaller PCR product than required

Posted bob1 on 25 October 2011 - 06:07 PM

Are you doing exactly the same experiment (same species DNA and everything) as the one in the paper?

Have you looked at the specificity of your primers?  Try primer-blast on the NCBI website, if you haven't.

Small products will amplify more efficiently than bigger ones, if there is a potential smaller product in your mix, it will be amplified in preference over bigger ones.

Which stage of the nested PCR is causing the problem?


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