METHODS AND COMPOSITIONS FOR THE EXTRACTION AND AMPLIFICATION OF NUCLEIC ACID FROM A SAMPLE

• US 20100297710A1
• Filed: 05/30/2007
• Published: 11/25/2010
• Est. Priority Date: 05/31/2006
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Hoyal-Wrightson, Carolyn R., Schmidt, Karsten E., Braun, Andreas
• Granted Patent: US 8,679,741 B2
• Time in Patent Office: Days
• US Class Current: 435/91.52
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium

Abstract

Provided herein are methods, compositions and kits to extract and relatively enrich by physical separation or amplification short base pair nucleic acid in the presence of a high background of genomic material (e.g., host or maternal nucleic acids).

Claims

1-38. -38. (canceled)

39. A method for selectively amplifying a target nucleic acid from a biological sample containing a mixture of non-target nucleic acid, wherein the target nucleic acid is a double stranded, blunt-ended nucleic acid fragment with 5′

phosphorylated ends, comprising the steps of;

a) mixing said biological sample, a 5′

adapter and a 3′

adapter, wherein the 3′

adapter is complementary to the 5′

adapter at the 3′

end and thus capable of creating a double-stranded adapter complex;

b) introducing a ligase to the mixture of step a) and ligating the 5′

adapter of the double-stranded adapter complex to the target nucleic acid, thereby creating a ligated sample;

c) heating the ligated sample to release the 3′

adapter;

d) adding a polymerase to fill in the single-stranded 5′

protruding ends; and

e) adding 5′

adapter primers and amplifying the target nucleic acid.

40. The method of claim 39, wherein a dideoxy-nucleotide is incorporated into the 3′

position of the 3′

adapter.

41. The method of claim 39 that includes an additional amplification step after step e), wherein the amplification is performed using a target-specific PCR primer.

42. The method of claim 39, wherein the 5′

adapters of step a) are bound to a solid support.

43. The method of claim 42, wherein a spacer arm is introduced between the 5′

adapter at the 5′

end and the solid support.

44. (canceled)

45. (canceled)