Title: Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

Abstract

A method of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths.

Inventors:

Gardner, Shea N; Mariella, Jr., Raymond P; Christian, Allen T; Young, Jennifer A; Clague, David S

Issue Date:

Tue Jun 25 00:00:00 EDT 2013

Research Org.:

Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)

Sponsoring Org.:

USDOE

OSTI Identifier:

1086952

Patent Number(s):

8470537

Application Number:

12/959,789

Assignee:

Lawrence Livermore National Security, LLC (Livermore, CA)

Patent Classifications (CPCs):

C - CHEMISTRY C07 - ORGANIC CHEMISTRY C07H - SUGARS

C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

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C - CHEMISTRY C07 - ORGANIC CHEMISTRY C07H - SUGARS

C07H21/00 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C12N15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N15/1027 - {by DNA shuffling, e.g. RSR, STEP, RPR}
C12N15/1031 - {mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR}

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

C12Q1/6813 - Hybridisation assays
C12Q1/6844 - Nucleic acid amplification reactions
C12Q1/686 - Polymerase chain reaction [PCR]
C12Q2521/101 - DNA polymerase
C12Q2525/143 - incorporating a promoter sequence
C12Q2533/107 - Probe or oligonucleotide ligation

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DOE Contract Number:  

AC52-07NA27344

Resource Type:

Patent

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES