METHODS AND COMPOSITIONS FOR ANALYZING NUCLEIC ACID MOLECULES UTILIZING SIZING TECHNIQUES

• US 20060057566A1
• Filed: 06/19/2003
• Published: 03/16/2006
• Est. Priority Date: 01/23/1996
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Qiagen Genomics, Inc.
• Inventors: Van Ness, Jeffrey, Mulligan, John T., Tabone, John C., Howbert, J. Jeffry
• Granted Patent: US 7,052,846 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

Tags and linkers specifically designed for a wide variety of nucleic acid reactions are disclosed, which are suitable for a wide variety of nucleic acid reactions wherein separation of nucleic acid molecules based upon size is required.

Claims

1-45. -45. (canceled)

46. A kit for genotyping, comprising a plurality of amplification primer pairs, wherein at least one of the primers has the formula:

T^ms-L-X

wherein,

T^ms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine;

L is an organic group which allows a T^ms-containing moiety to be cleaved from the remainder of the compound, wherein the T^ms-containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; and

X is a nucleic acid fragment wherein L is conjugated to X at a location other than the 3′

end of X; and

each primer pair associates with a different locus;

the compound also having the formula;

wherein

G is (CH₂)_1-6 wherein a hydrogen on one and only one of the CH₂ groups is replaced with —

(CH₂)_c-Amide-T⁴;

T² and T⁴ are organic moieties of the formula C_1-25N_0-9O_0-9 H_α

F_β

wherein the sum of α and

β

is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms,

Amide is

R¹ is hydrogen or C_1-10 alkyl;

c is an integer ranging from 0 to 4, and

n is an integer ranging from 1 to 50 such that when n is greater than 1, then G, c, Amide, R¹ and T⁴ are independently selected.

47. The kit according to claim 46 wherein the plurality is at least 3.

48. The kit according to claim 46 wherein the plurality is at least 5.

49-59. -59. (canceled)

60. A kit for genotyping, comprising a plurality of amplification primer pairs, wherein at least one of the primers has the formula:

T^ms-L-X

wherein,

T^ms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine;

L is an organic group which allows a T^ms-containing moiety to be cleaved from the remainder of the compound, wherein the T^ms-containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; and

X is a nucleic acid fragment wherein L is conjugated to X at a location other than the 3′

end of X; and

each primer pair associates with a different locus;

the compound also having the formula;

wherein

G is (CH₂)_1-6 wherein a hydrogen on one and only one of the CH₂ groups is replaced with —

(CH₂)_c-Amide-T⁴;

T² and T⁴ are organic moieties of the formula C_1-25N_0-9O_0-9H_α

F_β

wherein the sum of α and

β

is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms;

Amide is

R¹ is hydrogen or C_1-10 alkyl;

c is an integer ranging from 0 to 4; and

T⁵ is an organic moiety of the formula C_1-25N_0-9O_0-9H_α

F_β

wherein the sum of α and

β

is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms; and

T⁵ includes a tertiary or quaternary amine or an organic acid; and

m is an integer ranging from 0-49 where when m is greater than 1, then G, c, Amide, R¹ and T⁴ are independently selected.

61. The kit according to claim 60 wherein the -Amide-T⁵ is selected from:

62. The kit according to claim 60 wherein -Amide-T⁵ is selected from:

63. The kit according to claim 60 wherein the plurality is at least 3.

64. The kit according to claim 60 wherein the plurality is at least 5.

65. A kit for genotyping, comprising a plurality of amplification primer pairs, wherein at least one of the primers has the formula:

T^ms-L-X

wherein,

T^ms is an organic group detectable by mass spectrometry, comprising carbon, at least one of hydrogen and fluoride, and optional atoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine;

L is an organic group which allows a T^ms-containing moiety to be cleaved from the remainder of the compound, wherein the T^ms-containing moiety comprises a functional group which supports a single ionized charge state when the compound is subjected to mass spectrometry and is selected from tertiary amine, quaternary amine and organic acid; and

X is a nucleic acid fragment wherein L is conjugated to X at a location other than the 3′

end of X; and

each primer pair associates with a different locus;

the compound also having the formula;

wherein

G is (CH₂)_1-6 wherein a hydrogen on one and only one of the CH₂ groups is replaced with —

(CH₂)_c-Amide-T⁴;

T² and T⁴ are organic moieties of the formula C_1-25N_0-9O_0-9H_α

F_β

wherein the sum of α and

β

is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms;

Amide is

R¹ is hydrogen or C_1-10 alkyl;

c is an integer ranging from 0 to 4; and

T⁵ is an organic moiety of the formula C_1-25N_0-9O_0-9H_α

F_β

wherein the sum of α and

β

is sufficient to satisfy the otherwise unsatisfied valencies of the C, N, and O atoms; and

T⁵ includes a tertiary or quaternary amine or an organic acid; and

m is an integer ranging from 0-49 where when m is greater than 1, then G, c, Amide, R¹ and T⁴ are independently selected.

66. The kit according to claim 65 wherein the -Amide-T⁵ is selected from:

67. The kit according to claim 65 wherein -Amide-T⁵ is selected from:

68. The kit according to claim 65 wherein the plurality is at least 3.

69. The kit according to claim 65 wherein the plurality is at least 5.