Cycle-Sequencing Reactions

Abstract

Capillary sequencing of DNA remains a mainstay in the toolkit of molecular biologists. Whether the technique is used to verify that a clone is constructed properly or to validate an interesting variant found by next-generation whole-genome sequencing, capillary sequencing is an extremely versatile tool. This method for cycle sequencing relies on the use of the ABI3730xl capillary sequencer. The template can be prepared from plasmid minipreps for direct sequencing of clones to identify a mutation or structure of interest, or amplified products can be sequenced individually with each of the two primers used in LongAmp amplification reactions. Both template preparations use primers designed specifically for “targets”—either for direct sequencing or for validation.