Selecting and amplifying one fragment from a DNA fragment mixture by polymerase chain reaction with a pair of...

A new method for selecting and amplifying a single DNA fragment from a mixture is proposed. This method is applicable for the rapid classification of DNA fragments from a mixture and for preparation of sequencing templates. DNAs of several to tens of kilobases (kb) are digested with a four-base recognition restriction enzyme to produce smaller fragments. The complementary strand extension reactions are then carried out to produce fluorophore-labeled DNA fragments from the digestion products. These fragments can be rapidly classified according to their terminal-base sequences and their sizes are analyzed by capillary-array gel electrophoresis (CAGE). Electropherograms are used to characterize the fragments and to select polymerase chain reaction (PCR) primers. Any fragment in a digestion mixture can be amplified by PCR with a pair of primers selected from a primer pool by referring to the electropherograms of the fragments. This method was successfully used to compare the electropherograms of two different DNA strands and to sequence a several-kb DNA fragment without subcloning. Combined with CAGE, this method could be used to dramatically simplify DNA fragment analysis.