DNA-SEQUENCE ANALYSIS OF PRINKER-MODIFIED RESTRICTION FRAGMENTS AFTERCOLLECTION FROM CAPILLARY ELECTROPHORESIS WITH REPLACEABLE MATRICES

This paper demonstrates the procedure of sequencing DNA restriction fr agments isolated by a recently developed fraction collector after CE s eparation. In particular, using pBr 322 plasmid as a model system, a d ouble digest was performed with Eco RI and Pst I restriction enzymes t o produce two fragments of 749 base pairs (bp) and 3612 bp, both with cohesive ends. Prinkers, specific linkers complementary to the cohesiv e ends, were then ligated to both fragments (increasing the size by 59 bp each). These Prinker-modified fragments were separated by CE and c ollected. The success of the collection was demonstrated by reinjectio n of each isolated fraction with laser-induced fluorescence detection, using ethidium bromide as intercalater. The 808 bp isolated fragment was then polymerase chain reaction-amplified with appropriate primers for the Prinker ends, followed by cycle sequencing. Both strands of th e fragment were run on an ABI 373, sequencing 427 bases and 450 bases, respectively, with a read accuracy of 99.3%. This approach with Prink er-modified restriction fragment and automated CE fraction collection can be used as a general procedure for sequencing unknown genomic DNA as well as mutated DNA mixtures.