SELECTING AND AMPLIFYING ONE FRAGMENT FROM A DNA FRAGMENT MIXTURE BY POLYMERASE CHAIN-REACTION WITH A PAIR OF SELECTIVE PRIMERS

A new method for selecting and amplifying a single DNA fragment from a mixture is proposed. This method is applicable for the rapid classifi cation of DNA fragments from a mixture and for preparation of sequenci ng templates. DNAs of several to tens of kilobases (kb) are digested w ith a four-base recognition restriction enzyme to produce smaller frag ments. The complementary strand extension reactions are then carried o ut to produce fluorophore-labeled DNA fragments from the digestion pro ducts. These fragments can be rapidly classified according to their te rminal-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 pai r of primers selected from a primer pool by referring to the electroph erograms of the fragments. This method was successfully used to compar e 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.