Process for producing shortened target DNA fragments usable in sequencing large DNA segments
This patent describes a process for producing cloned, circular DNA molecules containing shortened target DNA fragments, the fragments derived from a long target DNA segment. The cloned, circular DNA molecules suitable for use in determining the nucleotide sequence of the long target DNA segment. The process consists the steps of: producing, by molecular cloning, double-stranded circular recombinant DNA molecules, each molecule containing vector DNA, a sequencing primer binding site, and a DNA region comprising a long target DNA segment, a first restriction site adjacent to the long target DNA segment adapted to be cut by a first restriction endonuclease in a manner that creates a first terminus on the DNA molecules adjacent the long target DNA segment that is susceptible to digestion by an exonuclease, and a second restriction site located between the first restriction site and the sequencing primer binding site adapted to be cut by a second restriction endonuclease in a manner that creates, without additional terminus blocking or digestion, a second terminus on the DNA molecules that is not susceptible to digestion by an exonuclease; cutting the double-stranded circular recombinant DNA molecules at the first restriction site using a first restriction endonuclease and at the second restriction site using a second restriction endonuclease to form double-stranded linear recombinant DNA molecules having a first terminus that is susceptible to digestion by an exonuclease and a second terminus that is not susceptible to digestion by an exonuclease.
- Assignee:
- Fred Hutchinson Cancer Research Center, Seattle, WA
- Patent Number(s):
- US 4843003
- OSTI ID:
- 5547838
- Resource Relation:
- Patent File Date: 17 Feb 1984
- Country of Publication:
- United States
- Language:
- English
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