Title: Amplification and cloning of single DNA molecules using rolling circle amplification

Abstract

The present invention relates, e.g., to a method for amplifying a small number of copies (e.g. a single copy) of a single-stranded circular DNA molecule (e.g. having a size of about 5-6 kb) by an isothermal rolling circle mechanism, using random or partially random primers and a F29-type DNA polymerase. The method, which can also be used for amplifying DNAs by non-rolling types of multiple displacement amplification, comprises incubating the reaction components in a small volume, e.g. about 10 μl or less, such as about 0.6 μl or less. The degree of amplification can be about 109 fold, or higher. A method for cell-free cloning of DNA, using the rolling circle amplification method of the invention, is described.

Inventors:

Hutchison, III, Clyde A.; Smith, Hamilton O.

Issue Date:

Tue Jul 30 00:00:00 EDT 2013

Research Org.:

Synthetic Genomics, Inc., San Diego, CA (United States)

Sponsoring Org.:

USDOE

OSTI Identifier:

1531861

Patent Number(s):

8497069

Application Number:

11/919,515

Assignee:

Synthetic Genomics, Inc. (San Diego, CA)

Patent Classifications (CPCs):

C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

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

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C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C12N15/1075 - {by coupling phenotype to genotype, not provided for in other groups of this subclass}
C12N15/64 - General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

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

C12Q1/6848 - characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
C12Q2525/204 - specific length of the oligonucleotides
C12Q2527/143 - Concentration of primer or probe
C12Q2531/119 - Strand displacement amplification [SDA]
C12Q2531/125 - Rolling circle

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DOE Contract Number:  

FG02-02ER63453

Resource Type:

Patent

Resource Relation:

Patent File Date: 2006-05-01

Country of Publication:

United States

Language:

English