Microarray system with improved sequence specificity

• US 8,945,928 B2
• Filed: 06/06/2013
• Issued: 02/03/2015
• Est. Priority Date: 08/12/2007
• Status: Expired due to Fees

• Current Assignee: Kerry B. Gunning, Mark Aaron Behlke
• Original Assignee: Kerry B. Gunning, Mark Aaron Behlke
• Inventors: Behlke, Mark Aaron, Gunning, Kerry B
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/911,971
• Publication Number:
  US 20130316927A1
• Time in Patent Office: 607 Days
• Field of Search: 436/18, 435/6.1
• US Class Current: 436/18
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent

Abstract

The invention provides a novel array method for nucleic acid sequence detection with improved specificity which allows for detection of genetic variation, from simple SNPs (where the variation occurs at a fixed position and is of limited allelic number) to more complex sequence variation patterns (such as with multigene families or multiple genetic strains of an organism where the sequence variation between the individual members is neither fixed nor consistent). The array is comprised of short, synthetic oligonucleotide probes attached to a solid surface which are hybridized to single-stranded targets. Single stranded targets can be produced using a method that employs primers modified on the 5′ end to prohibit degradation by a 5′-exonuclease that is introduced to degrade the unprotected strand. The invention further provides for printing buffers/solutions for the immobilization of oligonucleotide probes to an array surface. The invention also provides hybridization and wash buffers and conditions to maximize hybridization specificity and signal intensity, and reduce hybridization times.

Claims

1. A hybridization buffer comprising:

Tris, EDTA, an anionic surfactant, albumin, a cationic surfactant, polysucrose, polyvinylpyrrolidone 360 (PVP-360), a tertiary alkyl ammonium salt, formamide, and placental DNA that is predominantly 50 to 300 bp in size and enriched for repetitive DNA sequences.

2. The hybridization buffer of claim 1, wherein said Tris is at a pH of about 8.

3. The hybridization buffer of claim 1, wherein said anionic surfactant is Sarkosyl.

4. The hybridization buffer of claim 1, wherein said cationic surfactant is cetrimonium bromide (CTAB).

5. The hybridization buffer of claim 1, wherein said tertiary alkyl ammonium salt is tetramethyl ammonium chloride (TMAC).

6. The hybridization buffer of claim 1, wherein said hybridization buffer comprises:

Tris buffer at a pH of 7 to 8.5;

1 mM to 10 mM EDTA;

0.1% to 1% Sarkosyl;

0.1 mg/ml to 1 mg/ml ovalbumin;

0.1 mM to 5 mM CTAB;

0.1 mg/mL to 1.0 mg/mL polysucrose 400;

00.1 mg/mL to 1.0 mg/mL PVP-360;

2.0 M to 3.0 M TMAC;

0.20% to 10% formamide; and

1 μ

g/mL to 100 mg/mL placental DNA that is predominantly 50 to 300 bp in size and enriched for repetitive DNA sequences.

7. The hybridization buffer of claim 1, wherein said hybridization buffer comprises:

37.5 mM Tris pH 8;

3 mM EDTA;

0.25% Sarkosyl;

0.4 mg/mL Ovalbumin;

1 mM CTAB;

0.4 mg/mL polysucrose 400;

0.4 mg/mL PVP-360;

2.5M TMAC;

10% formamide, 10 μ

g/mL placental DNA that is predominantly 50 to 300 bp in size and enriched for repetitive DNA sequences.