Reaction buffer for microarray

Patent metadata

Field	Value
Publication number	US-9657337-B2
Application number	US-52098710-A
Country	US
Kind code	B2
Filing date	Dec 29, 2006
Priority date	Dec 29, 2006
Publication date	May 23, 2017
Grant date	May 23, 2017

Abstract

Embodiments of the present invention relate to a buffer composition for an integrated nucleic acid amplification and hybridization reaction. The buffer comprise about 50-200 mM of a salt, about 10-30 mM Tris-HCl, about 2-10M Water soluble magnesium salt, about 0.05-1.5% surfactant, about 0.05-0.15 mg/ml stabilizing protein about 50-300 nM of one or more primers, about 20-150 uM of one or more dNTPs, about 5-15% glycerine, about 0.5-1.5% formamide and at least about 5 unit/ml polymerase.

First claim

What is claimed is:

1. A method for the detection or quantification of a nucleic acid in an integrated nucleic acid amplification and hybridization reaction performed within a microarray, the method comprising:
selecting one or more primer(s), each with a length between 15 and 40 nucleotides and a melting temperature between about 55 degrees Celsius and about 65 degrees Celsius;
amplifying a nucleic acid with the one or more primer(s) in a buffer, where the amplifying comprises cycles of:
denaturing the nucleic acid in the buffer by increasing the temperature of the nucleic acid in the buffer to a first temperature of 94-98° C.;
annealing the nucleic acid and the one or more primer(s) in the buffer by decreasing the first temperature to a second temperature, the second temperature 5° C. below the melting point of the primer(s); and
elongating the annealed nucleic acid by increasing the second temperature to a third temperature of 72° C.; and

hybridizing the amplified nucleic acid in the buffer to probes within the microarray; and wherein the buffer comprises:
about 100 mM KCl;
about 20 mM Tris-HCl;
about 5 mM MgCl 2
about 0.1% triton X-100;
about 0.1 mg/ml BSA;
about 100-200 nM of the one or more primers;
about 100 μM of one or more dNTPs;
about 10% glycerine;
about 1% formamide; and
about 10 units/ml polymerase.

2. The method of claim 1 , further comprising analyzing the microarray.

3. The method of claim 1 , wherein the microarray is configured for expression profiling.

4. The method of claim 1 , wherein the one or more dNTPs comprise thymidine triphosphate (dTTP), deoxyadenosine (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) or combinations thereof.

5. The method of claim 1 , wherein the polymerase comprises Taq polymerase.

Assignees

• Xu Leon
• Sun Zhenhong
• Honeywell Int Inc

Inventors

• Xu Leon
• Sun Zhenhong

Classifications

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q2531/113

  PCR · CPC title

• C12Q2565/501

  being an array of oligonucleotides · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title