Amplification and cloning of single dna molecules using rolling circle amplification

• US 20090176280A1
• Filed: 05/01/2006
• Published: 07/09/2009
• Est. Priority Date: 04/29/2005
• Status: Active Grant

• Current Assignee: Telesis Bio, Inc.
• Original Assignee: J. Craig Venter Institute
• Inventors: Hutchison III, Clyde A., Smith, Hamilton O.
• Granted Patent: US 8,497,069 B2
• Time in Patent Office: Days
• US Class Current: 435/91.200
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2531/125 Rolling circle

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.

Claims

1. A method for amplifying part or all of a single copy of a DNA template, comprising contacting the DNA with a strand-displacing, processive DNA polymerase and a set of random or partially random primers, in a volume of 10 μ

l or less, under conditions that are effective for promoting DNA strand displacement, at a substantially isothermal temperature, wherein the amplification is performed

a) with a primer concentration of about 50 μ

M or less; and

/or

b) under conditions such that the amplified DNA is sufficiently free from background that it can be used directly, without further purification, for sequencing, restriction enzyme analysis, hybridization analysis, and/or in vitro recombination with other DNA molecules to assemble portions of a gene or genome.

2-62. -62. (canceled)