PCR-based detection of a rare linear DNA in cell culture.
The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 10(7) or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.
Saveliev
Sergei V.
SV
Department of Biochemistry, University of Wisconsin-Madison. 433 Babcock Drive, Madison, WI 53706-1544. USA. saveliev@biochem.wisc.edu