Reagent kit producing shortened target DNA segments usable in sequencing large DNA segments

Abstract

Abstract A process is provided for producing an ordered series of cloned, circular, DNA molecules containing shortened target DNA segments derived from a long target DNA segment, which are suitable for use in determining the nucleotide sequence of the long target DNA segment, or for targeting specific regions within the target DNA segment. The process includes producing, by molecular cloning, a plurality of double-stranded recombinant DNA molecules each containing: (i) vector DNA; (ii) a sequencing primer binding site; and, (iii) a DNA region having unique endonuclease sites and a long target DNA segment. The sequencing primer binding site is spaced from the long target DNA segment by at least a portion of said DNA region having the unique endonuclease sites. The plurality of double-stranded circular recombinant DNA molecules are cleared using two restriction endonucleases. The cleavage occurs in the portion of the DNA having unique endonuclease sites lying between the long target DNA segment and the sequencing primer binding site. The cleavage and, if necessary, subsequent processing steps, produces double-stranded linear DNA molecules having an end containing a long target DNA segment that is susceptible to exonuclease digestion and a sequencing primer binding site end that is not susceptible to exonuclease digestion. Next, the target DNA segment end is subjected to exonuclease digestion. At timed intervals, portions of the exonuclease digested linear double-stranded DNA molecules are removed. Since part of the target DNA segment has been deleted by the exonuclease, the removed linear double-stranded DNA molecules include shortened target DNA segments. Next, the removed linear double-stranded DNA molecules are blunted and then recircularized to reform circular recombinant DNA molecules. The reformed recombinant DNA molecules are cloned to produce a plurality of circular DNA molecules containing shortened target DNA segments that differ in length by the amount of target DNA that was removed by the exonuclease. The reformed circular DNA molecules are suitable for use in DNA sequencing other molecular manipulations of genetic material.

Inventors

• 👤 Steven Henikoff
• 👤 Richard E. Gelinas

Bibliographic Details