Methods and reagents for profiling quantities of nucleic acids
First Claim
1. A method of quantitatively analyzing a set of target nucleic acid sequences, said method comprising:
- (a) hybridizing a set of oligonucleotide probe precursors to said target nucleic acid sequences to produce hybrids, wherein a unique set of oligonucleotide precursors is employed for each set of target nucleic acid sequences, (b) processing said hybrids to alter the mass of each of said oligonucleotide probe precursors in said hybrids in a target sequence-mediated reaction to produce oligonucleotide products, each of which has a unique mass characteristic of its respective target nucleic acid sequence, which unique mass is not a result of the presence of a mass tag in said oligonucleotide product, and (c) analyzing said oligonucleotide products by means of mass spectrometry and relating the results thereof to the amount of said target nucleic acid sequences in said set.
1 Assignment
0 Petitions
Accused Products
Abstract
Methods and reagents are disclosed for quantitatively analyzing a set of target nucleic acid sequences. In the method a unique set of oligonucleotide probe precursors is hybridized to the target nucleic acid sequences to produce hybrids. The hybrids are processed to alter the mass of each of the oligonucleotide probe precursors in the hybrids in a target sequence-mediated reaction to produce oligonucleotide products, each of which has a unique mass that is not a result of the presence of a mass tag in the oligonucleotide product. The processing of the hybrids may involve polymerase extension or ligation. The products are analyzed by means of mass spectrometry and the results are related to the amount of the target nucleic acid sequences in the set. Kits for carrying out the above methods are also disclosed.
8 Citations
45 Claims
-
1. A method of quantitatively analyzing a set of target nucleic acid sequences, said method comprising:
-
(a) hybridizing a set of oligonucleotide probe precursors to said target nucleic acid sequences to produce hybrids, wherein a unique set of oligonucleotide precursors is employed for each set of target nucleic acid sequences, (b) processing said hybrids to alter the mass of each of said oligonucleotide probe precursors in said hybrids in a target sequence-mediated reaction to produce oligonucleotide products, each of which has a unique mass characteristic of its respective target nucleic acid sequence, which unique mass is not a result of the presence of a mass tag in said oligonucleotide product, and (c) analyzing said oligonucleotide products by means of mass spectrometry and relating the results thereof to the amount of said target nucleic acid sequences in said set. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
-
-
18. A method of determining the expression of genes in a set of genes, said method comprising:
-
(a) hybridizing a set of oligonucleotide probe precursors to said set of genes to produce hybrids, wherein (i) the composition of each of said genes is known to the extent necessary to select said set of oligonucleotide probe precursors and (ii) each of said oligonucleotide probe precursors has a unique mass, binds specifically to a respective gene, is substantially incapable of hybridizing to another of said oligonucleotide probe precursors to produce a hybrid capable of enzymatic extension, and is modifiable by enzymatic extension to yield oligonucleotide products each having a unique mass that is not a result of the presence of a mass tag in said oligonucleotide product, (b) extending each of said hybridized oligonucleotide probe precursors by polymerizing at least one nucleotide at the 3′
-end of said hybridized oligonucleotide probe precursors to produce said oligonucleotide products, and(c) analyzing said oligonucleotide products by means of mass spectrometry and relating the results thereof to said expression of said genes in said set. - View Dependent Claims (19, 20, 21, 22)
-
-
23. A method of determining the expression of genes in a set of genes, said method comprising:
-
(a) hybridizing a set of oligonucleotide probe precursors to said genes to produce hybrids, wherein (i) the composition of each of said genes is known to the extent necessary to select said set of oligonucleotide probe precursors and (ii) at least two of said oligonucleotide probe precursors in said set bind specifically and adjacently to a respective gene and are ligatable to yield oligonucleotide products each having a different mass that is'"'"'not a result of the presence of a mass tag in said oligonucleotide product, (b) ligating adjacent oligonucleotide probe precursors to produce oligonucleotide products, each of which has a unique mass and (c) analyzing said oligonucleotide products by means of mass spectrometry and relating the results thereof to the amount of expression of said genes in said set. - View Dependent Claims (24, 25, 26, 27, 28)
-
-
29. A composition comprising a set of oligonucleotide probe precursors characterized as follows:
-
(a) each of said oligonucleotide probe precursors in said set binds specifically to a respective target nucleic acid sequence, (b) said oligonucleotide probe precursors are substantially incapable of hybridizing to one another to produce hybrids capable of enzymatic extension, and (c) said oligonucleotide probe precursors can be mass modified by enzymatic extension to yield oligonucleotide products each having a different mass that is not a result of the presence of a mass tag in said oligonucleotide product. - View Dependent Claims (30, 31, 32, 33)
-
-
34. A composition comprising a set of oligonucleotide probe precursors characterized as follows:
-
(a) each of said oligonucleotide probe precursors has a length of about 5 to about 10 nucleotides and (b) at least three of said oligonucleotide probe precursors in said set bind specifically to a respective target nucleic acid sequence and are ligatable to yield an oligonucleotide product having a different mass that is not a result of the presence of a mass tag in said oligonucleotide product. - View Dependent Claims (35, 36, 37, 38, 39)
-
-
40. A method of determining the expression of genes in a set of genes, said method comprising:
-
(1) hybridizing said set of genes to a multiplicity of nucleic acid probes attached to a surface in an array wherein said multiplicity of nucleic acid sequence probes comprise (i) a cleavable linker attached to said surface and (ii) a nucleic acid sequence having a 3′
-end and a terminal 5′
-phosphate wherein said 3′
-end of said nucleic acid sequence is attached to said cleavable linker;
(2) hybridizing a set of oligonucleotide probe precursors to said genes, wherein a unique set of oligonucleotide precursors is employed for each set of genes, (3) processing said hybrids to alter the mass of each of said oligonucleotide probe precursors in said hybrids in a target sequence-mediated reaction to produce oligonucleotide products, each of which has a unique mass that is not a result of the presence of a mass tag in said oligonucleotide product, (4) cleaving said cleavable linker; and
(5) analyzing said oligonucleotide products by mass spectrometry and relating the results thereof to the amount of said genes in said set. - View Dependent Claims (41, 42, 43, 44, 45)
-
Specification