WO1996036986B1 - Methods and apparatus for sequencing polymers with a statistical certainty using mass spectrometry - Google Patents
Methods and apparatus for sequencing polymers with a statistical certainty using mass spectrometryInfo
- Publication number
- WO1996036986B1 WO1996036986B1 PCT/US1996/007146 US9607146W WO9636986B1 WO 1996036986 B1 WO1996036986 B1 WO 1996036986B1 US 9607146 W US9607146 W US 9607146W WO 9636986 B1 WO9636986 B1 WO 9636986B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polymer
- fragments
- mass
- charge ratio
- exonuclease
- Prior art date
Links
- 229920000642 polymer Polymers 0.000 title claims abstract 36
- 238000004949 mass spectrometry Methods 0.000 title abstract 3
- 230000003301 hydrolyzing Effects 0.000 claims abstract 23
- 238000006243 chemical reaction Methods 0.000 claims abstract 8
- 238000004458 analytical method Methods 0.000 claims abstract 6
- 239000003795 chemical substances by application Substances 0.000 claims 20
- 101700008821 EXO Proteins 0.000 claims 7
- 101700083023 EXRN Proteins 0.000 claims 7
- 229940088598 Enzyme Drugs 0.000 claims 6
- 102000004190 Enzymes Human genes 0.000 claims 6
- 108090000790 Enzymes Proteins 0.000 claims 6
- 229920001222 biopolymer Polymers 0.000 claims 6
- 102000018389 Exopeptidases Human genes 0.000 claims 5
- 108010091443 Exopeptidases Proteins 0.000 claims 5
- 238000006460 hydrolysis reaction Methods 0.000 claims 5
- 238000005259 measurement Methods 0.000 claims 5
- 229920000160 (ribonucleotides)n+m Polymers 0.000 claims 3
- 108010029607 4-nitrophenyl-alpha-glucosidase Proteins 0.000 claims 3
- 101710028797 AFUA_4G13800 Proteins 0.000 claims 3
- 101710007399 DDS Proteins 0.000 claims 3
- 108060002716 Exonucleases Proteins 0.000 claims 3
- 102000013165 Exonucleases Human genes 0.000 claims 3
- 101700028499 LECG Proteins 0.000 claims 3
- 102000005840 alpha-Galactosidase Human genes 0.000 claims 3
- 108010030291 alpha-Galactosidase Proteins 0.000 claims 3
- 102000012086 alpha-L-Fucosidase Human genes 0.000 claims 3
- 108010061314 alpha-L-Fucosidase Proteins 0.000 claims 3
- 150000001720 carbohydrates Chemical class 0.000 claims 3
- 235000014633 carbohydrates Nutrition 0.000 claims 3
- 229920003013 deoxyribonucleic acid Polymers 0.000 claims 3
- -1 exoglycosidases Proteins 0.000 claims 3
- 108010050669 glucosidase I Proteins 0.000 claims 3
- 101700053671 manC Proteins 0.000 claims 3
- 102000004196 processed proteins & peptides Human genes 0.000 claims 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims 3
- 102000004169 proteins and genes Human genes 0.000 claims 3
- 108090000623 proteins and genes Proteins 0.000 claims 3
- 102000003670 Carboxypeptidase B Human genes 0.000 claims 2
- 108090000087 Carboxypeptidase B Proteins 0.000 claims 2
- 108010080937 Carboxypeptidases A Proteins 0.000 claims 2
- 102000000496 Carboxypeptidases A Human genes 0.000 claims 2
- 108010059081 Cathepsin A Proteins 0.000 claims 2
- 102000005572 Cathepsin A Human genes 0.000 claims 2
- 102000003902 Cathepsin C Human genes 0.000 claims 2
- 108090000267 Cathepsin C Proteins 0.000 claims 2
- 102000010567 DNA Polymerase II Human genes 0.000 claims 2
- 108010063113 DNA Polymerase II Proteins 0.000 claims 2
- 102000002704 EC 3.4.11.1 Human genes 0.000 claims 2
- 108010004098 EC 3.4.11.1 Proteins 0.000 claims 2
- 102100001542 EXO1 Human genes 0.000 claims 2
- 108010046914 Exodeoxyribonuclease V Proteins 0.000 claims 2
- 102000019236 Exonuclease V Human genes 0.000 claims 2
- 102100016213 HEXD Human genes 0.000 claims 2
- 101700022374 HEXD Proteins 0.000 claims 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims 2
- 101700068378 XRN1 Proteins 0.000 claims 2
- 108010012864 alpha-Mannosidase Proteins 0.000 claims 2
- 102000019199 alpha-Mannosidase Human genes 0.000 claims 2
- 101710043144 apeI Proteins 0.000 claims 2
- 102000005936 beta-Galactosidase Human genes 0.000 claims 2
- 108010005774 beta-Galactosidase Proteins 0.000 claims 2
- 108010054847 carboxypeptidase P Proteins 0.000 claims 2
- 230000000875 corresponding Effects 0.000 claims 2
- 108010007577 exodeoxyribonuclease I Proteins 0.000 claims 2
- 108010052305 exodeoxyribonuclease III Proteins 0.000 claims 2
- 239000007788 liquid Substances 0.000 claims 2
- 101710028320 ltah-1.1 Proteins 0.000 claims 2
- 239000011159 matrix material Substances 0.000 claims 2
- PTFCDOFLOPIGGS-UHFFFAOYSA-N zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 claims 2
- 108090000317 Chymotrypsin Proteins 0.000 claims 1
- 108010059378 Endopeptidases Proteins 0.000 claims 1
- 102000005593 Endopeptidases Human genes 0.000 claims 1
- 102000002464 Galactosidases Human genes 0.000 claims 1
- 108010093031 Galactosidases Proteins 0.000 claims 1
- 102100013287 MOGS Human genes 0.000 claims 1
- 238000000692 Student's t-test Methods 0.000 claims 1
- 108090001109 Thermolysin Proteins 0.000 claims 1
- 108090000631 Trypsin Proteins 0.000 claims 1
- 102000004142 Trypsin Human genes 0.000 claims 1
- 230000002745 absorbent Effects 0.000 claims 1
- 239000002250 absorbent Substances 0.000 claims 1
- 229960002376 chymotrypsin Drugs 0.000 claims 1
- 230000001419 dependent Effects 0.000 claims 1
- 230000003100 immobilizing Effects 0.000 claims 1
- 108010009689 mannosyl-oligosaccharide 1,2-alpha-mannosidase Proteins 0.000 claims 1
- 239000000178 monomer Substances 0.000 claims 1
- 108010030544 peptidyl-Lys metalloendopeptidase Proteins 0.000 claims 1
- 229960001322 trypsin Drugs 0.000 claims 1
- 239000012588 trypsin Substances 0.000 claims 1
Abstract
The method and apparatus disclosed herein are useful for sequencing polymers using mass spectrometry. The methods involve differing ratios of hydrolyzing agent to polymer disposed upon a reaction surface adapted for use with a mass spectrometer. The methods further involve integrating data obtained from mass spectrometry analysis of a plurality of series of hydrolyzed polymer fragments, and provide statistical interpretation paradigms and computer software therefor. The apparatus involves a mass spectrometer sample holder, having hydrolyzing agent disposed thereon, which is useful for adapting any mass spectrometer for polymer sequencing.
Claims
6 7
AMENDED CLAIMS
[received by the International Bureau on 3 December 1996 (03.12.96); original claims 1, 3-7, 18, 19, 43, 45, 46 and 61 amended; new claims 108 and 109 added; remaining claims unchanged (14 pages)]
1. A method of obtaining sequence information about a polymer comprising a plurality of monomers of known mass, said method comprising the steps of:
a) providing a set of polymer fragments, each differing by one or more monomers;
b) measuring a difference x between the mass-to-charge ratio of at least one pair of fragments;
c) asserting a mean difference μ between the mass-to-charge ratio of the pair of fragments measured in step b, wherein μ corresponds to a known mass-to-charge ratio of one or more differing monomers;
d) selecting a desired confidence level for μ;
e) analyzing x to determine if it is statistically different from μ at the selected confidence level; and
f) determining if the asserted mean μ is assignable to the mass difference x with the selected confidence level based upon the analysis in step e).
2. The method of claim 1 wherein a statistical difference determined in the analysis of step e) indicates that the asserted mean μ is not assignable to the mass difference x with the selected confidence level.
3. The method of claim 1 comprising repeating steps c) through f) for a plurality of desired values of μs.
6 8
4. The method of claim 1 wherein the analysis of step e) comprises a two-tailed t-test for an experimental mean.
5. The method of claim 1 wherein the analyzing in step e) comprises:
g) repeating step b) a number of times, n, to determine a measured mean mass-to-charge ratio difference x between at least one pair of fragments;
h) determining a standard deviation s ofthe mean mass-to-charge ratio difference x determined in step g);
i) comparing x to the asserted mean difference μ;
j) repeating steps c) through i) for a plurality of desired values of μs.
6 9
6. The method of claim 5 comprising repeating steps b) through j) for additional pairs of fragments.
7. The method of claim 5 wherein the comparing in step i) comprises taking the absolute value ofthe asserted mean difference.
8. The method of claim 5 further comprising the step of determining the number of measurements, n, based upon the analysis in step e).
9. The method of claim 1 wherein the polymer is a biopolymer.
10. The method of claim 9 wherein the biopolymer is selected from the group consisting of DNAs, RNAs, PNAs, proteins, peptides, carbohydrates and modified forms thereof.
11. The method of 1 further comprising the step of hydrolyzing the polymer to obtain the polymer fragments in step a).
12. The method of claim 1 further comprising hydrolyzing, on a reaction surface, the polymer with a hydrolyzing agent.
13. The method of claim 12 wherein the polymer is hydrolyzed on a reaction surface, said surface providing differing amounts of a hydrolyzing agent which hydrolyzes said polymer thereby to break inter-monomer bonds.
14. The method of claim 11, 12 or 13 wherein the hydrolyzing agent is an exohydrolase or an endohydrolase.
15. The method of claim 14 wherein hydrolyzing with said exohydrolase produces a series of fragments comprising a sequence-defining ladder of said polymer.
16. The method of claim 15 wherein the exohydrolase is selected from the group consisting of: exonucleases, exoglycosidases, and exopeptidases.
7 0
17. The method of claim 16 wherein the exopeptidase is selected from the group consisting of carboxypeptidase Y, carboxypeptidase A, carboxypeptidase B, carboxypeptidase P, aminopeptidase 1, leucine aminopeptidase, proline aminodipeptidase and cathepsin C.
7 ]
18. The method of claim 16 wherein the exoglycosidase is selected from the group consisting of
a) α - ■ Mannosidase I b) α - - Mannosidase c) β - ■ Hexosaminodase d) β - ■ Galactosidase e) α - - Fucosidase I and II f) α - - Galactosidase g) α - - Neuraminidase and h) a - - Glucosidase I and II
19. The method of claim 16 wherein the exonuclease is selected from the group consisting of
a) λ- exonuclease b) t7 Gene 1 exonuclease c) exonuclease III d) Exonuclease I e) Exonuclease V f) Exonuclease II and g) DNA Polymerase II.
20. The method of claim 14 wherein hydrolyzing with said endohydrolase produces a series of fragments defining a map of said polymer.
21. The method of claim 20 wherein said endohydrolase is an endopeptidase selected from the group consisting of: trypsin, chymotrypsin, endo-proteinase Lys-C, endoproteinase Arg-C and thermolysin.
7
22. The method of claim 12 wherein the agent is a hydrolyzing agent other than an enzyme.
23. The method of claim 12 wherein said agent capable of hydrolyzing said polymer comprises a combination of at least one enzyme and at least one agent other than an enzyme.
24. The method of claim 13 wherein the reaction surface comprises an array of discrete separable zones, each zone comprising a differing amount of said hydrolyzing agent.
38. The method of claim 12 wherein hydrolysis is accomplished by immobilizing said agent on said reaction surface.
39. The method of claim 12 wherein hydrolysis is accomplished using a hydrolyzing agent in liquid or gel form, said liquid or gel form being resistant to physical dislocation.
40. The method of claim 1 comprising the additional step of combining a light- absorbent matrix with said fragments prior to step b).
41. The method of claim 1 comprising the additional step of combining said polymer fragments with moieties for selectively shifting the mass of hydrolyzed sequences prior to step b).
42. The method of claim 1 comprising the additional step of combining said polymer fragments with moieties for improving ionization prior to step b).
43. A method for obtaining sequence information about a polymer comprising a series of different monomers of known mass, said method comprising the steps of:
a) providing a set of polymer fragments, each differing by one or more monomers;
b) measuring the mass-to-charge ratio difference x between a pair of fragments;
c) asserting a mean difference μ, which is related to a known mass-to-charge ratio of one or more monomers;
d) selecting a desired confidence level for μ;
7 4
e) repeating step b) to obtain a number of measurements n, thereby to determine the measured mean mass-to-charge ratio difference JC between the pair of fragments;
f) determining the standard deviation s ofthe measured mean mass-to-charge ratio difference x determined in st~p e;
g) calculating a test statistic tcaicuiated with the following algorithm:
x — μ n calculated — '
h) comparing the test statistic tcaicuiated calculated in step g to a t-distribution corresponding to the number of measurements and the desired confidence level; and
i) determining if the asserted mean μ is assignable to the mass difference x with the selected confidence level based upon the comparison in step h.
7 5
44. The method of claim 43 further comprising a comparison ofthe calculated test statistic tcaicuiated in step g) to a t-distribution corresponding to the number of measurements and the desired confidence level.
45. The method of claim 43 further comprising repeating steps b) - i) for additional pairs of fragments thereby to obtain sequence information.
46. The method of claim 43 further comprising the step of determining the number of measurements, n, based upon the comparison in step h).
47. The method of claim 43 wherein the polymer is a biopolymer.
48. The method of claim 47 wherein the biopolymer is selected from the group consisting of DNAs, RNAs, PNAs, proteins, peptides, carbohydrates and modified forms thereof.
49. The method of claim 43 further comprising the step of hydrolyzing the polymer with a hydrolyzing agent to create the fragments in step a).
50. The method of claim 49 wherein the hydrolyzing agent is an exohydrolase which produces a series of fragments comprising a sequence-defining ladder of said polymer.
51. The method of claim 50 wherein the exohydrolase is selected from the group consisting of: exonucleases, exoglycosidases, exopeptidases.
52. The method of claim 51 wherein the exopeptidase is selected from the group consisting of carboxypeptidase Y, carboxypeptidase A, carboxypeptidase B, carboxypeptidase P, aminopeptidase 1, leucine aminopeptidase, proline aminodipeptidase and cathepsin C.
7 6
53. The method of claim 51 wherein the exoglycosidase is selected from the group consisting of
a) α - Mannosidase I b) α - Mannosidase c) β - Hexosaminidase d) β - Galactosidase e) α - Fucosidase I and II f) α - Galactosidase
7 7
g) α - Neuraminidase and h) α - Glucosidase I and II.
54. The method of claim 51 wherein the exonuclease is selected from the group consisting of
a) Exonuclease b) λ- exonuclease c) t7 Gene 1 exonuclease d) exonuclease III e) Exonuclease I f) Exonuclease V g) Exonuclease II h) DNA Polymerase II.
55. The method of claim 49 wherein the hydrolyzing agent is other than an enzyme.
56. The method of claim 49 wherein the agent comprises a combination of at least one enzyme and at least one agent other than an enzyme.
57. The method of claim 49 wherein hydrolysis is performed on a reaction surface, said surface providing differing amounts of a hydrolyzing agent.
58. The method of claim 57 wherein the reaction surface comprises an array of discrete separable zones, each zone comprising a differing amount of said hydrolyzing agent.
59. The method of claim 49 wherein the reaction surface comprises a continuous concentration gradient ofa hydrolyzing agent.
60. The method of claim 43 further comprising adding a matrix to the polymer fragments before measuring the mass-to-charge ratio in step b).
7 8
61. A method for obtaining sequence information about a polymer having a plurality of monomers of known mass, said method comprising: a) providing a set of polymer fragments, each differing by one or more monomers; b) measuring a difference x between the mass-to-charge ratio ofa pair of fragments; c) asserting a mean difference μ between the mass-to-charge ratio ofthe pair of fragments measured in step b, wherein μ corresponds to a known mass- to-charge ratio of one or more monomers; d) selecting the desired confidence level for μ; e) analyzing x to determine if it is statistically different from μ at the selected confidence level; f) repeating steps b)-e) a number of times n, until a plurality of desired values of μs have been asserted; g) determining if the asserted mean μ is assignable to the mass difference x with the selected confidence level based upon the analysis in step e; and h) repeating steps b) -g) for additional pairs of fragments.
62. The method of claim 61 wherein the polymer is a biopolymer.
63. The method of claim 62 wherein the biopolymer is selected from the group consisting of DNAs, RNAs, PNAs, proteins, peptides, carbohydrates and modified forms thereof.
64. The method of claim 61 wherein the polymer fragments in step a) are created by concentration dependent hydrolysis ofthe polymer.
65. The method of claim 61 further comprising the step of hydrolyzing said polymer with a hydrolyzing agent to produce the polymer fragments in step a).
7 9
66. The method of claim 65 wherein the hydrolyzing agent is an exohydrolase.
67. The method of claim 66 wherein the hydrolysis caused by said exohydrolase produces a series of fragments defining a ladder of said polymer.
68. The method of claim 66 wherein the exohydrolase is selected from the group consisting of: exonucleases, exoglycosidases, and exopeptidases.
69. The method of claim 68 wherein the exoglycosidase is selected from the group consisting of
a) α - Mannosidase I b) α - Mannosidase c) β - Hexosaminidase d) β - Galactosidase e) α - Fucosidase I and II f) α - Galactosidase g) α - Neuraminidase and h) α - Glucosidase I and II.
8 0
107. The sample plate of any one of claims 78, 85, 99, 100 or 101 wherein said plate is disposable.
108. A method of obtaining information about the identity of a polymer comprising a plurality of monomers of known mass, said method comprising the steps of: a) providing a set of polymer fragments created by the endohydrolysis of said polymer; b) measuring the mass-to-charge ratio of a fragment; c) asserting a hypothetical identity for the fragment, wherein the hypothetical identity corresponds to a known identity of a fragment ofa reference polymer, said fragment having a known mass-to-charge ratio; d) selecting a desired confidence level for the hypothetical identity; and e) determining whether the measured mass-to-charge ratio is statistically different from the mass-to-charge ratio ofthe asserted hypothetical fragment; f) determimng if the asserted hypothetical identity is assignable to the measured mass-to-charge ratio ofthe fragment with the selected confidence level based upon the determination in step e; and g) repeating steps b)- e).
109. The method of claim 108 wherein the hypothetical identity in step c) corresponds to a known identity derived from a computer database of known sequences.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP08535084A JP2001500606A (en) | 1995-05-19 | 1996-05-17 | Method and apparatus for statistically certain polymer sequencing using mass spectrometry |
| EP96916490A EP0827628A1 (en) | 1995-05-19 | 1996-05-17 | Methods and apparatus for sequencing polymers with a statistical certainty using mass spectrometry |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US44605595A | 1995-05-19 | 1995-05-19 | |
| US08/447,175 | 1995-05-19 | ||
| US08/447,175 US5869240A (en) | 1995-05-19 | 1995-05-19 | Methods and apparatus for sequencing polymers with a statistical certainty using mass spectrometry |
| US08/446,055 | 1995-05-19 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1996036986A1 WO1996036986A1 (en) | 1996-11-21 |
| WO1996036986B1 true WO1996036986B1 (en) | 1997-01-09 |
Family
ID=27034484
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1996/007146 WO1996036986A1 (en) | 1995-05-19 | 1996-05-17 | Methods and apparatus for sequencing polymers with a statistical certainty using mass spectrometry |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0827628A1 (en) |
| JP (2) | JP2001500606A (en) |
| WO (1) | WO1996036986A1 (en) |
Families Citing this family (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69433811T2 (en) | 1993-01-07 | 2005-06-23 | Sequenom, Inc., San Diego | DNA SEQUENCING BY MASS SPECTROMONY |
| US5605798A (en) | 1993-01-07 | 1997-02-25 | Sequenom, Inc. | DNA diagnostic based on mass spectrometry |
| US6146854A (en) * | 1995-08-31 | 2000-11-14 | Sequenom, Inc. | Filtration processes, kits and devices for isolating plasmids |
| US5777324A (en) | 1996-09-19 | 1998-07-07 | Sequenom, Inc. | Method and apparatus for maldi analysis |
| DE19714558A1 (en) * | 1997-04-09 | 1998-10-15 | Joachim W Prof Dr Engels | A new method for sequencing biopolymers using mass spectrometry |
| WO1998054571A1 (en) * | 1997-05-28 | 1998-12-03 | The Walter And Eliza Hall Institute Of Medical Research | Nucleic acid diagnostics based on mass spectrometry or mass separation and base specific cleavage |
| NZ516848A (en) | 1997-06-20 | 2004-03-26 | Ciphergen Biosystems Inc | Retentate chromatography apparatus with applications in biology and medicine |
| US6207370B1 (en) | 1997-09-02 | 2001-03-27 | Sequenom, Inc. | Diagnostics based on mass spectrometric detection of translated target polypeptides |
| GB9821393D0 (en) | 1998-10-01 | 1998-11-25 | Brax Genomics Ltd | Protein profiling 2 |
| US6994969B1 (en) | 1999-04-30 | 2006-02-07 | Methexis Genomics, N.V. | Diagnostic sequencing by a combination of specific cleavage and mass spectrometry |
| US20040198950A1 (en) | 2001-04-30 | 2004-10-07 | George Jackowski | Biopolymer marker indicative of disease state having a molecular weight of 1518 daltons |
| DE10123711A1 (en) * | 2001-05-15 | 2003-02-27 | Wolfgang Altmeyer | Procedure for determining the origin of biological materials |
| AU2004235331B2 (en) * | 2003-04-25 | 2008-12-18 | Sequenom, Inc. | Fragmentation-based methods and systems for De Novo sequencing |
| WO2005024068A2 (en) | 2003-09-05 | 2005-03-17 | Sequenom, Inc. | Allele-specific sequence variation analysis |
| EP2395098B1 (en) | 2004-03-26 | 2015-07-15 | Agena Bioscience, Inc. | Base specific cleavage of methylation-specific amplification products in combination with mass analysis |
| WO2009138207A2 (en) * | 2008-04-28 | 2009-11-19 | Kuehn Andreas | Method and arrangement for the control of measuring systems, corresponding computer programme and corresponding computer-readable storage medium |
| EP4032538A3 (en) | 2009-03-02 | 2022-10-26 | Massachusetts Institute of Technology | Methods and products for in vivo enzyme profiling |
| WO2012116131A1 (en) * | 2011-02-23 | 2012-08-30 | Leco Corporation | Correcting time-of-flight drifts in time-of-flight mass spectrometers |
| US10006916B2 (en) * | 2011-03-15 | 2018-06-26 | Massachusetts Institute Of Technology | Multiplexed detection with isotope-coded reporters |
| EP3440013A4 (en) | 2016-04-08 | 2021-03-17 | Massachusetts Institute of Technology | Methods to specifically profile protease activity at lymph nodes |
| CA3022928A1 (en) | 2016-05-05 | 2017-11-09 | Massachusetts Institute Of Technology | Methods and uses for remotely triggered protease activity measurements |
| CA3059358A1 (en) | 2017-04-07 | 2018-10-11 | Massachusetts Institute Of Technology | Methods to spatially profile protease activity in tissue and sections |
| US11054428B2 (en) | 2018-03-05 | 2021-07-06 | Massachusetts Institute Of Technology | Inhalable nanosensors with volatile reporters and uses thereof |
| US11835522B2 (en) | 2019-01-17 | 2023-12-05 | Massachusetts Institute Of Technology | Sensors for detecting and imaging of cancer metastasis |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1514782A1 (en) * | 1987-07-16 | 1989-10-15 | Tikhookeanskij I Bioorg Khim D | Method of detecting bacterial endotoxin |
| US5288644A (en) * | 1990-04-04 | 1994-02-22 | The Rockefeller University | Instrument and method for the sequencing of genome |
-
1996
- 1996-05-17 WO PCT/US1996/007146 patent/WO1996036986A1/en active Application Filing
- 1996-05-17 EP EP96916490A patent/EP0827628A1/en not_active Withdrawn
- 1996-05-17 JP JP08535084A patent/JP2001500606A/en not_active Ceased
-
2006
- 2006-10-05 JP JP2006274536A patent/JP2007206054A/en not_active Ceased
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