DNA separating, fractionating and analyzing method and system therefor


Ontology type: sgo:Patent     


Patent Info

DATE

1996-03-13T00:00

AUTHORS

KAMBARA, HIDEKI , OKANO, KAZUNORI

ABSTRACT

A long DNA is digested into small fragments to be sequenced without subcloning using a small library of 16 primers. Each DNA fragment can be isolated by the combined technique of gel electrophoresis separation and hybridization coupled with DNA polymerase reaction, which does not use subcloning, therefore, is good for full automation of the process. The present invention comprises: a process of creating a plurality of DNA fragments by digesting the target DNA with restriction enzyme or the like and introducing the oligomer having known sequence to their termini through ligation reaction or terminal base addition reaction by terminal transferase; a process of preparing the DNA probe comprising the oligomers which include the known sequence and the sequence for selecting fragments where 1 to 3 bases of the 3' terminal side of the DNA probe can take every sequences but they are held separately for each sequence selecting species; a process of hybridizing the DNA fragment with the DNA probe on the solid; a process of stabilizing hybrid by extending only the DNA probes in the hybrid for which the selective sequence of two bases (1-3 bases) at 3' terminus of DNA probe is perfectly complementary to the DNA fragments; and a process of removing the DNA fragments hybridized with the DNA probes which were not subjected to complementary strand extension, and of selecting and fractionating the specific DNA fragment. Addition of a process of analyzing the DNA sequence of the DNA fragments separated and fractionated by this separation and fractionation method provides a DNA analysis method according to the present invention. Whenever required, the separation and fractionation process by gel electrophoresis may be used in combination. Said DNA separating, fractionating and analyzing method according to the present invention uses the DNA separation and fractionation system comprising the vessel incorporating the solid immobilizing said DNA probes and a robot system comprising a means for loading substances required for said vessel, a means for temperature control, a sample transfer means and a means for fractionating the separated target DNA fragment. More... »

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