US5580733A
Expired / Lapsed
US
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Vaporization and sequencing of nucleic acids
Assignee: Wayne State University
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Filing: 1994-09-06
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Granted: 1996-12-03
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Expires: 2014-09-06
Abstract
Abstract Method for analyzing a nucleic acid molecule, without fragmenting the molecule, by vaporizing a mixture of the molecule and a matrix by illuminating the mixture with visible laser light absorbed by the matrix and not by the nucleic acid molecule. The method is useful for determining the nucleotide sequence of a polynucleotide by using mass spectrometry to determine the molecular weights of individual single-stranded nucleic acid molecules in a population including a plurality of single-stranded nucleic acid molecules generated from the polynucleotide, each molecule having a different molecular weight, and one defined terminus and one variable terminus which terminates at a specific nucleotide.
Inventors
- 👤 Robert J. Levis
- 👤 Louis J. Romano
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 00:01
CPC Classifications
H; H01; H01J; H01J49/00; H01J49/02; H01J49/04; H01J49/0459; H01J49/0463; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; C12Q1/6872; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/58; G01N33/582; H01J49/10; H01J49/16; H01J49/161; H01J49/162; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333; Y10T436/24
Claims Analysis
Total Claims: 47
Claim 1
Independent Claim
153 words
A method for determining, by mass spectrometry, the nucleotide sequence of a polynucleotide comprising the steps of: (a) generating from said polynucleotide four populations of nucleic acid molecules having from 65 to 1000 nucleotides, wherein the nucleic acid molecules of each population terminate in one of the nucleotides A, T, G or C; (b) for each population of nucleic acid molecules forming a mixture of the nucleic acid molecules with a matrix that absorbs laser light in the visible range; (c) vaporizing said mixture of matrix and nucleic acid molecules by illuminating with visible laser light, wherein said vaporizing occurs without fragmenting the nucleic acid molecules; (d) ionizing the vaporized nucleic acid molecules; (e) determining, by mass spectrometry, the molecular weight of the ionized nucleic acid molecules of each population; and (f) comparing the molecular weights of the nucleic acid molecules generated from the polynucleotide to provide the nucleotide sequence of the polynucleotide.
Claim 2
Independent Claim
153 words
A method for determining, by mass spectrometry, the nucleotide sequence of a polynucleotide comprising the steps of: (a) generating from said polynucleotide four populations of nucleic acid molecules having from 65 to 1000 nucleotides, wherein the nucleic acid molecules of each population terminate in one of the nucleotides A, T, G or C; (b) for each population of nucleic acid molecules forming a mixture of the nucleic acid molecules with a matrix that absorbs laser light in the visible range; (c) vaporizing said mixture of matrix and nucleic acid molecules by illuminating with visible laser light, wherein said vaporizing occurs without fragmenting the nucleic acid molecules; (d) ionizing the vaporized nucleic acid molecules; (e) determining, by mass spectrometry, the molecular weight of the ionized nucleic acid molecules of each population; and (f) comparing the molecular weights of the nucleic acid molecules generated from the polynucleotide to provide the nucleotide sequence of the polynucleotide.
Claim 3
Dependent Claim
18 words
The method of claim 1, wherein said nucleic acid molecules are generated by chemical degradation of said polynucleotide.
Claim 4
Dependent Claim
36 words
The method of claim 1, wherein said nucleic acid molecules are generated by extension of a primer, complementary to a portion of said polynucleotide, by a DNA polymerase in the presence of a chain terminating agent.
Claim 5
Dependent Claim
14 words
The method of claim 3, wherein said chain terminating agent is a dideoxynucleotide triphosphate.
Claim 6
Dependent Claim
13 words
The method of claim 3, wherein said DNA polymerase is T7 DNA polymerase.
Claim 7
Dependent Claim
15 words
The method of claim 3, wherein said extension is performed in the presence of manganese.
Claim 8
Dependent Claim
12 words
The method of claim 1, wherein said polynucleotide is a DNA molecule.
Claim 9
Dependent Claim
12 words
The method of claim 1, wherein said polynucleotide is an RNA molecule.
Claim 10
Dependent Claim
14 words
The method of claim 1, wherein said comparing step is performed using a computer.
Claim 11
Dependent Claim
21 words
The method of claim 1, wherein said vaporizing step is performed using a laser adapted to emit a pulse of light.
Claim 12
Dependent Claim
25 words
The method of claim 10 wherein said vaporizing laser is adapted to emit a pulse of light having a duration of less than 10 nanoseconds.
Claim 13
Dependent Claim
22 words
The method of claim 11, wherein said vaporizing laser is adapted to emit a pulse of light of duration about 5 nanoseconds.
Claim 14
Dependent Claim
24 words
The method of claim 10, wherein the power of said laser light ranges from about 80 mJ/cm 2 to about 500 mJ/cm 2 .
Claim 15
Dependent Claim
21 words
The method of claim 13, wherein the power of said vaporizing laser light is greater than about 120 mJ/cm 2 .
Claim 16
Dependent Claim
18 words
The method of claim 13, wherein the power of said laser light is about 320 mJ/cm 2 .
Claim 17
Dependent Claim
16 words
The method of claim 10, wherein said vaporizing laser is a neodium yttrium aluminum garnet laser.
Claim 18
Dependent Claim
20 words
The method of claim 1, wherein said illuminating is at a wavelength of from about 400 to about 1100 nm.
Claim 19
Dependent Claim
16 words
The method of claim 17, wherein said wavelength is from about 500 to about 550 nm.
Claim 20
Dependent Claim
12 words
The method of claim 18, wherein said wavelength is about 532 nm.
Claim 21
Dependent Claim
12 words
The method of claim 1, wherein said matrix is an organic dye.
Claim 22
Dependent Claim
12 words
The method of claim 20 wherein said organic dye is Rhodamine 6G.
Claim 23
Dependent Claim
29 words
The method of claim 1, wherein said matrix is selected from the group consisting of Rhodamine 6G, Rhodamine 700 or 800, DTTCI, LC8800, DNTTCI, HDITCI, DDCI-4, and dibenzocyanine 45.
Claim 24
Dependent Claim
26 words
The method of claim 1, wherein said matrix has the ability to absorb light at a wavelength of from about 400 nm to about 1100 nm.
Claim 25
Dependent Claim
26 words
The method of claim 23, wherein said matrix has the ability to absorb light at a wavelength of from about 500 nm to about 550 nm.
Claim 26
Dependent Claim
20 words
The method of claim 24, wherein said matrix has the ability to absorb light at a wavelength of 532 nm.
Claim 27
Dependent Claim
20 words
The method of claim 1, wherein said matrix absorbs light at a wavelength at which DNA does not absorb light.
Claim 28
Dependent Claim
20 words
The method of claim 1, wherein said matrix absorbs light at a wavelength at which RNA does not absorb light.
Claim 29
Dependent Claim
22 words
The method of claim 1, wherein the molar ratio of said matrix to said polynucleotide is from about 1:1 to about 100,000:1.
Claim 30
Dependent Claim
15 words
The method of claim 28, wherein said ratio is from about 1000:1 to about 25,000:1.
Claim 31
Dependent Claim
11 words
The method of claim 29, wherein said ratio is about 17,000:1.
Claim 32
Dependent Claim
15 words
The method of claim 1, wherein said nucleic acid molecule has a single positive charge.
Claim 33
Dependent Claim
21 words
The method of claim 31, wherein said single positive charge is placed on said nucleic acid molecule by resonance-enhanced multiphoton ionization.
Claim 34
Dependent Claim
16 words
The method of claim 1, wherein said nucleic acid molecule is bound to an ionizable chromophore.
Claim 35
Dependent Claim
20 words
The method of claim 33, wherein said nucleic acid molecule is chemically bound to said chromophore by a linker arm.
Claim 36
Dependent Claim
16 words
The method of claim 33, wherein said nucleic acid molecule is covalently bound to said chromophore.
Claim 37
Dependent Claim
17 words
The method of claim 33, wherein said chromophobe absorbs light having a wavelength longer than 280 nm.
Claim 38
Dependent Claim
19 words
The method of claim 34, wherein said chromophore is positioned at least 1 atom from said nucleic acid molecule.
Claim 39
Dependent Claim
19 words
The method of claim 34, wherein said chromophore is positioned less than 50 atoms from said nucleic acid molecule.
Claim 40
Dependent Claim
12 words
The method of claim 33, wherein said ionizable chromophore is a dye.
Claim 41
Dependent Claim
10 words
The method of claim 39, wherein said dye is fluorescent.
Claim 42
Dependent Claim
28 words
The method of claim 33, wherein said chromophore is selected from a group consisting of fluorescein, rhodamine, tetramethylrhodamine, sulforhodamine, nitrobenzyl-2-oxa-1-diazole, anthracene, pyrene, coumarin, acridone, N-5-dimethylaminonaphthene, and derivatives thereof.
Claim 43
Dependent Claim
21 words
The method of claim 41, wherein said derivatives are selected from the group consisting of iodoacetamide, maleimide, isothiocyanate and succinimidyl carboxylate.
Claim 44
Dependent Claim
21 words
A methods according to claim 1, wherein said ionizing is performed using a laser adapted to emitting a pulse of light.
Claim 45
Dependent Claim
21 words
The method of claim 43, wherein said laser is adapted to emit a pulse of light of less than 20 nanoseconds.
Claim 46
Dependent Claim
21 words
The method of claim 44, wherein said laser is adapted to emit a pulse of light of less than 10 nanoseconds.
Claim 47
Dependent Claim
14 words
The method of claim 1, wherein said mass spectrometer is a time-of-flight mass spectrometer.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-12-03 | 2000-06-03 | 2000-12-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-12-03 | 2004-06-03 | 2004-12-03 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-12-03 | 2008-06-03 | 2008-12-03 | $7700.00 | Window Closed / Lapsed |