NON-CLONING AMPLIFICATION OF SPECIFIC DNA FRAGMENTS FROM WHOLE GENOMIC DNA DIGESTS USING DNA INDEXERS

A highly systematic, non-cloning method of distinguishing and isolatin g every fragment in a class-IIS or interrupted palindrome restriction digest has been developed in our laboratory. These enzymes produce inf ormative, non-identical cohesive ends which can be selectively modifie d by ligation to individual synthetic oligodeoxyribonucleotides with t he corresponding complementary ends. In this way, polymerase chain rea ction and sequencing primer sites and labels can be introduced specifi cally into a single fragment in a total genomic digest. Known and unkn own fragments from genomes of the complexity of Escherichia coli can b e isolated directly in sequenceable form without the necessity of synt hesizing unique primers. Human DNA has also been accessed in this way. Problems intrinsic to cloning (selective fragment loss, mutation and sequence rearrangement) are avoided. Systematic characterization of DN A fragments by their cohesive ends and length provides tremendous powe r and flexibility for analysis of any DNA molecule without specific cl ones, probes or libraries. We report proof of principle of this remark able system and indicate potential applications in DNA sequence tagged site and restriction mapping, sequencing, restriction-fragment-length polymorphism analysis and DNA diagnostics.