US5389512A
Expired / Lapsed
US
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Method for determining the relative amount of a viral nucleic acid segment in a sample by the polymerase chain reaction
Assignee: Roche Molecular Systems Inc
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Filing: 1994-05-20
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Granted: 1995-02-14
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Expires: 2014-05-20
Abstract
Abstract The present invention provides a method for determining the relative amount of a nucleic acid segment in a sample by the polymerase chain reaction. The method involves the simultaneous amplification of the nucleic acid segment and a second nucleic acid segment present in the sample. The amount of amplified DNA from each segment is determined and compared to standard curves to determine the amount of the nucleic acid segment present in the sample before amplification expressed as a ratio of first segment to second segment. The method is especially preferred for determining the viral load, or copies of virus genome/host cell, in a sample of cells from an individual infected with a virus.
Inventors
- 👤 Shirley Y. Kwok
- 👤 John J. Sninsky
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-01 14:29
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6851; C12Q1/6876; C12Q1/6881; C12Q1/70; C12Q1/701; C12Q1/702; C12Q1/703
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
181 words
A method for determining the amount of a viral nucleic acid sequence relative to the amount of a cellular nucleic acid sequence in a sample comprising virally infected host cells, comprising the steps of: (a) co-amplifying the viral nucleic acid sequence and the cellular nucleic acid sequence in an amplification reaction mixture that comprises a first primer pair for amplifying said viral nucleic acid sequence, a second primer pair for amplifying said cellular nucleic acid sequence and DNA polymerase, wherein the relative concentrations of said first and second primer pairs are adjusted so that the concentration of the primer pair for amplification of the nucleic acid sequence present in the greatest amount is lower than the concentration of the primer pair for amplification of the nucleic acid sequence present in the smallest amount, (b) measuring the amounts of amplified viral and cellular nucleic acid sequences produced in step (a); and (c) determining the amount of said viral nucleic acid sequence relative to the amount of said cellular nucleic acid sequence present in said sample prior to the co-amplification in step (a).
Claim 2
Independent Claim
181 words
A method for determining the amount of a viral nucleic acid sequence relative to the amount of a cellular nucleic acid sequence in a sample comprising virally infected host cells, comprising the steps of: (a) co-amplifying the viral nucleic acid sequence and the cellular nucleic acid sequence in an amplification reaction mixture that comprises a first primer pair for amplifying said viral nucleic acid sequence, a second primer pair for amplifying said cellular nucleic acid sequence and DNA polymerase, wherein the relative concentrations of said first and second primer pairs are adjusted so that the concentration of the primer pair for amplification of the nucleic acid sequence present in the greatest amount is lower than the concentration of the primer pair for amplification of the nucleic acid sequence present in the smallest amount, (b) measuring the amounts of amplified viral and cellular nucleic acid sequences produced in step (a); and (c) determining the amount of said viral nucleic acid sequence relative to the amount of said cellular nucleic acid sequence present in said sample prior to the co-amplification in step (a).
Claim 3
Dependent Claim
12 words
The method of claim 1, wherein said DNA polymerase is Taq polymerase.
Claim 4
Dependent Claim
28 words
The method of claim 1, wherein said first primer pair is present in said amplification reaction mixture at a concentration higher than that of said second primer pair.
Claim 5
Dependent Claim
17 words
The method of claim 3, wherein said viral nucleic acid sequence is indicative of a disease state.
Claim 6
Dependent Claim
18 words
The method of claim 4, wherein said genomic sequence is a segment of human cellular nucleic acid DNA.
Claim 7
Dependent Claim
18 words
The method of claim 5, wherein said amplified viral and cellular nucleic acid sequences are distinguishable by size.
Claim 8
Dependent Claim
14 words
The method of claim 5, wherein said genomic DNA is from an HLA locus.
Claim 9
Dependent Claim
24 words
The method of claim 1, wherein said sample comprises viral nucleic acid in the range of 1 copy per every 100 to 10,000 cells.
Claim 10
Dependent Claim
17 words
The method of claim 1, wherein said viral nucleic acid is a segment from an AIDS virus.
Claim 11
Dependent Claim
12 words
The method of claim 7, wherein said second primer pair is GH26/GH27.
Claim 12
Dependent Claim
12 words
The method of claim 9, wherein said first primer pair is SK38/SK39.
Claim 13
Dependent Claim
12 words
The method of claim 9, wherein said first primer pair is SK101/SK145.
Claim 14
Dependent Claim
46 words
The method of claim 1, wherein the amplification reaction mixture comprises about 50 picomoles of the first primer pair, about 300 femtomoles to about 2 picomoles of the second primer pair, and the cellular nucleic acid sequence is about 1 to 3 μg of cellular DNA.
Claim 15
Dependent Claim
41 words
The method of claim 3 wherein the concentration of the first primer pair is related to the concentration of the second primer pair by a ratio in a range of from about 170-fold higher concentration to about a 25-fold higher concentration.
References Cited (Backward Citations)
US4582788A
Cited
US4683194A
Cited
US4683202A
Cited
US4683202B1
Cited
EP0229701A2
Cited
US4683195A
Cited
US4683195B1
Cited
EP0237362A1
Cited
EP0258017A2
Cited
US4965188A
Cited
EP0269445A2
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1998-02-13 | 1998-08-15 | 1999-02-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2002-02-13 | 2002-08-15 | 2003-02-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2006-02-13 | 2006-08-15 | 2007-02-14 | $7700.00 | Window Closed / Lapsed |