Methods and compositions for determining the sequence of nucleic acid molecules

• US 6,623,928 B2
• Filed: 05/14/2001
• Issued: 09/23/2003
• Est. Priority Date: 01/23/1996
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Qiagen Genomics, Inc.
• Inventors: Van Ness, Jeffrey, Mulligan, John T., Tabone, John C., Howbert, J. Jeffry
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/855,999
• Publication Number:
  US 20020119456A1
• Time in Patent Office: 862 Days
• Field of Search: 435/6, 436/173, 536/26.6
• US Class Current: 435/6.12
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07D 211/78 Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
• 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: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

Methods and compounds, including compositions therefrom, are provided for determining the sequence of nucleic acid molecules. The methods permit the determination of multiple nucleic acid sequences simultaneously. The compounds are used as tags to generate tagged nucleic acid fragments which are complementary to a selected target nucleic acid molecule. Each tag is correlative with a particular nucleotide and, in a preferred embodiment, is detectable by mass spectrometry. Following separation of the tagged fragments by sequential length, the tags are cleaved from the tagged fragments. In a preferred embodiment, the tags are detected by mass spectrometry and the sequence of the nucleic acid molecule is determined therefrom. The individual steps of the methods can be used in automated format, e.g., by the incorporation into systems.

Claims

1. A composition comprising first and second compounds of the formula T^ms—

L^hv—

X wherein,

L^hv is a chemical group which, upon exposure to light of selected wavelength, allows cleavage of T^ms from X;

T^ms is an organic group which is detectable by mass spectrometry and comprises a variable mass component; and

X is nucleic acid;

with the proviso that the variable mass component in the first compound has a mass that is not identical to the mass of the variable mass component of the second compound, but the T^ms groups in the first and second compounds are otherwise identical.

3. A composition according to claim 1, wherein L^hv has the formula L¹-L²-L³, wherein L² is a molecular fragment that absorbs actinic radiation to promote the cleavage of T^ms from X, and L¹ and L³ are independently a direct bond or an organic moiety, wherein L¹ separates L² from T^ms and L³ separates L² from X, and neither L¹ nor L³ undergo bond cleavage when L² absorbs the actinic radiation.

5. The composition according to claim 3 wherein L^hυ

has the formula L¹-L²-L³ and -L²-L³ has the formula;

with one carbon atom at positions a, b, c, d or e being substituted with -L³-X and optionally one or more of positions b, c, d or e being substituted with alkyl, alkoxy, fluoride, chloride, hydroxyl, carboxylate or amide; and

R¹ is hydrogen or hydrocarbyl.

6. The composition according to claim 5 wherein -L³-X is located at position a.

2. A composition comprising first and second compounds of the formula T^ms—

L—

X, wherein

T^ms has the formula

 

wherein

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

(CH₂)_c-Amide-T⁴;

Amide is

R¹ is hydrogen or C_1-10 alkyl;

c is an integer ranging from 0 to 4; and

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

T⁵ is an organic moiety of the formula C_1-25N_0-9O_0-9S_0-3P_0-3H_α

F_β

I_δ

wherein the sum of α

, β and

δ

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

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

m is an integer ranging from 0-49;

L is a chemical group which, upon exposure to light of selected wavelength, allows cleavage of T^ms from X; and

X is nucleic acid;

with the proviso that m in the first compound is different from m in the second compound, but the T^ms groups in the first and second compounds are otherwise identical.

4. A composition according to claim 2, wherein L^hv has the formula L¹-L²-L³, wherein L² is a molecular fragment that absorbs actinic radiation to promote the cleavage of T^ms from X, and L¹ and L³ are independently a direct bond or an organic moiety, wherein L¹ separates L² from T^ms and L³ separates L² from X, and neither L¹ nor L³ undergo bond cleavage when L² absorbs the actinic radiation.

7. The composition according to claim 4 wherein L^hυ

has the formula L¹-L²-L³ and -L²-L³ has the formula;

with one carbon atom at positions a, b, c, d or e being substituted with -L³-X and optionally one or more of positions b, c, d or e being substituted with alkyl, alkoxy, fluoride, chloride, hydroxyl, carboxylate or amide; and

R¹ is hydrogen or hydrocarbyl.

8. The composition according to claim 7 wherein -L³-X is located at position a.