Patent US4060056A successfully refreshed from Google Patents.
Patent US5527675A successfully refreshed from Google Patents.
US5527675A
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Method for degradation and sequencing of polymers which sequentially eliminate terminal residues
Assignee: Applied Biosystems LLC
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Filing: 1993-08-20
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Granted: 1996-06-18
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Expires: 2013-08-20
Abstract
Abstract A method and apparatus for sequentially degrading at least a portion of a polymer of backbone repeating units, the polymer having a terminal repeating unit comprised of a nucleophile and a backbone carbonyl carbon distant from the nucleophile, comprising the steps of first initiating attack of said nucleophile upon said backbone carbonyl carbon by raising the energy level to activate said nucleophile for said attack. Secondly, forming a ring comprising the terminal repeating unit, thereby simultaneously releasing the ring and generating a shortened polymer having a terminal repeating unit capable of nucleophile attack upon the backbone carbonyl carbon and, lastly, maintaining the reaction conditions necessary for repeating steps a and b until the portion of the polymer desired is degraded. In a related embodiment, polyamide nucleic acid (PNA) sequences can be determined by generating a nested set of polymer fragments, each fragment having N-x repeating units where N is the total number of repeating units in the parent polymer and x is the number of degradation cycles the fragment has been subjected to, and then analyzing the nested set of polymer fragments to determine polymer sequence. An apparatus embodying the method of sequential degradation is also described. Analysis may be by MALD-TOF.
Inventors
- 👤 James M. Coull
- 👤 Leif Christensen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 02:09
CPC Classifications
C; C12; C12P; C12P1/00; C07; C07K; C07K14/00; C07K14/001; C07K14/003; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 35
Claim 1
Independent Claim
124 words
A method for sequentially degrading at least a portion of a polymer of backbone repeating units, said polymer having a terminal repeating unit comprised of a nucleophile and a backbone carbonyl carbon distant from said nucleophile, comprising: (a) attacking the backbone carbonyl carbon with the nucleophile, in the absence of an external reagent by raising the temperature or adjusting the pH of the environment surrounding the polymer; (b) to thereby form a ring comprising said terminal repeating unit, and simultaneously releasing said ring and generating a shortened polymer having a terminal repeating unit capable of nucleophile attack upon said backbone carbonyl carbon; and (c) repeating steps (a) and (b) by maintaining the reaction conditions until at least a portion of the polymer is degraded.
Claim 2
Independent Claim
124 words
A method for sequentially degrading at least a portion of a polymer of backbone repeating units, said polymer having a terminal repeating unit comprised of a nucleophile and a backbone carbonyl carbon distant from said nucleophile, comprising: (a) attacking the backbone carbonyl carbon with the nucleophile, in the absence of an external reagent by raising the temperature or adjusting the pH of the environment surrounding the polymer; (b) to thereby form a ring comprising said terminal repeating unit, and simultaneously releasing said ring and generating a shortened polymer having a terminal repeating unit capable of nucleophile attack upon said backbone carbonyl carbon; and (c) repeating steps (a) and (b) by maintaining the reaction conditions until at least a portion of the polymer is degraded.
Claim 3
Dependent Claim
13 words
The method of claim 1 wherein said polymer is a peptide nucleic acid.
Claim 4
Dependent Claim
20 words
The method of claim 1 wherein said polymer is selected from the group consisting of a polyamide, polyester or polythioester.
Claim 5
Dependent Claim
34 words
The method of claim 1 wherein said polymer is comprised of polymers having a heterogeneous backbone wherein the functional linkages of the backbone are selected from the group consisting of amides, esters, and thioesters.
Claim 6
Dependent Claim
12 words
The method of claim 1 wherein said backbone repeating unit is 2-aminoethylglycine.
Claim 7
Dependent Claim
12 words
The method of claim 1 wherein said backbone repeating unit is 2-hydroxyethylglycine.
Claim 8
Dependent Claim
12 words
The method of claim 1 wherein said backbone repeating unit is 2-thioethylglycine.
Claim 9
Dependent Claim
12 words
The method of claim 1 wherein said nucleophile is an amino group.
Claim 10
Dependent Claim
12 words
The method of claim 1 wherein said nucleophile is a hydroxyl group.
Claim 11
Dependent Claim
12 words
The method of claim 1 wherein said nucleophile is a thiol group.
Claim 12
Dependent Claim
19 words
The method of claim 1 wherein said carbonyl carbon atom is at least 4 atoms distant from said nucleophile.
Claim 13
Dependent Claim
257 words
The method of claim 1 wherein said polymer is of the general formula: ##STR3## wherein, each V is independently selected from the group consisting of OH, SH, NH 2 and NHR, each W is the same or different and is selected from the group consisting of H, R, OR, SR, NHR, NR 2 , F, Cl, Br and I, each Y 1 --Y q-1 is the same or different and is independently selected from the group consisting of O, S, NH and NR, each Z is independently selected from the group consisting of H, OH, OR, NH 2 , NHR, NR 2 , SH, SR, any of the 20 naturally occurring amino acids, polypeptides, proteins, DNA oligomers and RNA oligomers, biotin and fluorescein, each R is the same or different and is an alkyl group having one to five carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group each of A 1 --A q is selected from the group consisting of a single bond, a group of the formula, --(CW.sub.2).sub.s -- and a group of the formula, ##STR4## wherein W is defined above and each s is an integer from one to five, each of L 1 --L q is the same or different and is independently selected from the group consisting of W, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties, biotin, and fluorescein, n=1-2, m=1-2 provided that m does not equal n, p=0-1,000,000.
Claim 14
Dependent Claim
19 words
The method of claim 1 wherein the attack comprises first deblocking the terminal nucleophile of a terminally-blocked parent polymer.
Claim 15
Dependent Claim
20 words
The method of claim 1 wherein the temperature of said polymer's immediate environment is raised until substantial nucleophilic attack begins.
Claim 16
Dependent Claim
20 words
The method of claim 1 wherein the pH of said polymer's immediate environment is adjusted until substantial nucleophilic attack begins.
Claim 17
Independent Claim
150 words
A method for sequencing at least a portion of a polymer of backbone repeating units, said polymer having a terminal repeating unit comprised of a nucleophile and a backbone carbonyl distant from said nucleophile, comprising the steps of: (a) attacking the backbone carbonyl carbon with the nucleophile in the absence of an external reagent by raising the temperature or adjusting the pH of the environment surrounding the polymer thereby simultaneously forming and releasing a ring and generating a shortened polymer having a terminal repeating unit capable of nucleophilic attack upon said backbone carbonyl carbon; (b) repeating step (a) to generate a nested set of polymer fragments, each fragment having N-x repeating units where N is the total number of repeating units in the parent polymer and x is the number of times step (a) is performed; and (c) analyzing said nested set of polymer fragments to determine the polymer sequence.
Claim 18
Independent Claim
150 words
A method for sequencing at least a portion of a polymer of backbone repeating units, said polymer having a terminal repeating unit comprised of a nucleophile and a backbone carbonyl distant from said nucleophile, comprising the steps of: (a) attacking the backbone carbonyl carbon with the nucleophile in the absence of an external reagent by raising the temperature or adjusting the pH of the environment surrounding the polymer thereby simultaneously forming and releasing a ring and generating a shortened polymer having a terminal repeating unit capable of nucleophilic attack upon said backbone carbonyl carbon; (b) repeating step (a) to generate a nested set of polymer fragments, each fragment having N-x repeating units where N is the total number of repeating units in the parent polymer and x is the number of times step (a) is performed; and (c) analyzing said nested set of polymer fragments to determine the polymer sequence.
Claim 19
Dependent Claim
13 words
The method of claim 16 wherein said polymer is a peptide nucleic acid.
Claim 20
Dependent Claim
20 words
The method of claim 16 wherein said polymer is selected from the group consisting of a polyamide, polyester or polythioester.
Claim 21
Dependent Claim
34 words
The method of claim 16 wherein said polymer is comprised of polymers having a heterogeneous backbone wherein the functional linkages of the backbone are selected from the group consisting of amides, esters and thioesters.
Claim 22
Dependent Claim
12 words
The method of claim 16 wherein said backbone repeating unit is 2-aminoethylglycine.
Claim 23
Dependent Claim
12 words
The method of claim 16 wherein said backbone repeating unit is 2-hydroxyethylglycine.
Claim 24
Dependent Claim
12 words
The method of claim 16 wherein said backbone repeating unit is 2-thioethylglycine.
Claim 25
Dependent Claim
12 words
The method of claim 16 wherein said nucleophile is an amino group.
Claim 26
Dependent Claim
12 words
The method of claim 16 wherein said nucleophile is a hydroxyl group.
Claim 27
Dependent Claim
12 words
The method of claim 16 wherein said nucleophile is a thiol group.
Claim 28
Dependent Claim
19 words
The method of claim 16 wherein said carbonyl carbon atom is at least 4 atoms distant from said nucleophile.
Claim 29
Dependent Claim
257 words
The method of claim 16 wherein said polymer is of the general formula: ##STR5## wherein, each V is independently selected from the group consisting of OH, SH, NH 2 and NHR, each W is the same or different and is selected from the group consisting of H, R, OR, SR, NHR, NR 2 , F, Cl, Br and I, each Y 1 --Y q-1 is the same or different and is independently selected from the group consisting of O, S, NH and NR, each Z is independently selected from the group consisting of H, OH, OR, NH 2 , NHR, NR 2 , SH, SR, any of the 20 naturally occurring amino acids, polypeptides, proteins, DNA oligomers and RNA oligomers, each R is the same or different and is an alkyl group having one to six carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group; each of A 1 --A q is selected from the group consisting of a single bond, a group of the formula: --(CW.sub.2).sub.s -- and a group of the formula, ##STR6## wherein W is defined above and each s is an integer from one to five, each of L 1 --L q is the same or different and is independently selected from the group consisting of W, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties, biotin, fluorescein and other reporter ligands, n=1-2, m=1-2 provided that m does not equal n, p=0-1,000,000.
Claim 30
Dependent Claim
19 words
The method of claim 16 wherein initiating attack comprises first deblocking the terminal nucleophile of a terminally-blocked parent polymer.
Claim 31
Dependent Claim
20 words
The method of claim 16 wherein the temperature of said polymer's immediate environment is raised until substantial nucleophilic attack begins.
Claim 32
Dependent Claim
20 words
The method of claim 16 wherein the pH of said polymer's immediate environment is adjusted until substantial nucleophilic attack begins.
Claim 33
Dependent Claim
18 words
The method of claim 16 wherein analysis of the nested set of polymer fragments is by mass spectrometry.
Claim 34
Dependent Claim
18 words
The method of claim 31 wherein the mass spectrometer is a matrix-assisted Laser Desorption Time-of-Flight (MALD-TOF) mass spectrometer.
Claim 35
Dependent Claim
58 words
The method of claim 16 wherein determining the polymer sequence comprises the steps of: (a) calculating the mass difference between two N-x polymer fragments; (b) comparing said mass difference with a table of known masses for terminal repeating units thereby identifying the released terminal repeat unit; and (c) repeating steps a and b until the sequence is determined.
References Cited (Backward Citations)
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-06-18 | 1999-12-18 | 2000-06-18 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-06-18 | 2003-12-18 | 2004-06-18 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-06-18 | 2007-12-18 | 2008-06-18 | $7700.00 | Window Closed / Lapsed |