Method to produce DNA of defined length and sequence and DNA probes produced thereby

US8795968B2 · kind B2 · utility

Assignee

• The Board of Trustees of the Leland Stanford Junior University · US

Inventors

• Michael N. Mindrinos · Somerville, US
• Sujatha Krishnakumar · Palo Alto, US
• Ronald W. Davis · Menlo Park, US
• Peidong Shen · Union City, US
• Curt Scharfe · Oakland, US

Key dates

• Filing date: Dec 17, 2010
• Grant date: Aug 5, 2014
• Expiry date: Mar 13, 2032

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q2600/16
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

A method for producing a single stranded DNA (ssDNA) molecule of a defined length and sequence is disclosed. This method enables the preparation of, inter alia, probes of greater length than can be chemically synthesized. The method starts with a double stranded molecule, such as genomic, double stranded DNA (dsDNA) from any organism. A fragment of the starting molecule (dsDNA) is amplified by specific primers engineered to introduce cleavage sites on either side of the desired sequence. Cleavage steps on the amplified, engineered fragment are combined with a phosphate removal step, thereby creating a construct that can be digested with an exonuclease without damage to the desired ssDNA. Probes, which hybridize with large gaps between the ends of the probes, are also disclosed.