US5578467A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Use of deoxyinosine containing primers to balance primer efficiency in the amplification of nucleic acid molecules
Assignee: Life Technologies Corp
•
Filing: 1994-05-20
•
Granted: 1996-11-26
•
Expires: 2014-05-20
Abstract
Abstract The present invention provides improved methods for amplifying a nucleic acid molecule. Specifically, the invention involves replacing at least one nucleotide of an oligonucleotide with a deoxyinosine residue, so as to more equalize the efficiency with which that oligonucloetide and a second oligonucleotide hybridize to a target molecule, and then amplifying the target molecule using, for example, the polymerase chain reaction. Improved amplification results from the improvement in the relative hybridization efficiencies.
Inventors
- 👤 David M. Schuster
- 👤 Ayoub Rashtchian
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 00:50
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6813; C12Q1/6832; C12Q1/6853; C12Q1/686; C12Q1/6862; C12Q2600/00; C12Q2600/156
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
261 words
An improved method for amplifying the concentration of a target nucleic acid molecule by using a first and second primer to form a double-stranded nucleic acid molecule, wherein said first primer hybridizes to a first strand of said double-stranded nucleic acid molecule, and said second primer hybridizes to a second strand of said double-stranded nucleic acid molecule, and wherein said primers exhibit different T m s with respect to one another, when hybridized to said double-stranded nucleic acid molecule, said improved method comprising the steps: (a) replacing at least one residue in at least one of said primers with at least one deoxyinosine, wherein said replacement equalizes the T m s of the first and second primers with respect to one another, when hybridized to said double-stranded nucleic acid molecule; (b) performing the template-dependent extension of said first primer, said first primer being hybridized to a first strand of said double-stranded molecule, wherein said extension forms a second strand of nucleic acid complementary to said first strand of said double-stranded nucleic acid molecule; (c) performing the template-dependent extension of said second strand, by extending said second primer, said second primer being hybridized to said second strand of said double-stranded molecule, wherein said extension forms a copy of said first strand of said double-stranded nucleic acid molecule; (d) performing the template-dependent extension of said copy of said first strand, to thereby form a copy of said second strand of said double-stranded nucleic acid molecule; (e) repeating steps (b), (c) and (d), to thereby achieve said amplification of said target nucleic acid molecule.
Claim 2
Dependent Claim
261 words
An improved method for amplifying the concentration of a target nucleic acid molecule by using a first and second primer to form a double-stranded nucleic acid molecule, wherein said first primer hybridizes to a first strand of said double-stranded nucleic acid molecule, and said second primer hybridizes to a second strand of said double-stranded nucleic acid molecule, and wherein said primers exhibit different T m s with respect to one another, when hybridized to said double-stranded nucleic acid molecule, said improved method comprising the steps: (a) replacing at least one residue in at least one of said primers with at least one deoxyinosine, wherein said replacement equalizes the T m s of the first and second primers with respect to one another, when hybridized to said double-stranded nucleic acid molecule; (b) performing the template-dependent extension of said first primer, said first primer being hybridized to a first strand of said double-stranded molecule, wherein said extension forms a second strand of nucleic acid complementary to said first strand of said double-stranded nucleic acid molecule; (c) performing the template-dependent extension of said second strand, by extending said second primer, said second primer being hybridized to said second strand of said double-stranded molecule, wherein said extension forms a copy of said first strand of said double-stranded nucleic acid molecule; (d) performing the template-dependent extension of said copy of said first strand, to thereby form a copy of said second strand of said double-stranded nucleic acid molecule; (e) repeating steps (b), (c) and (d), to thereby achieve said amplification of said target nucleic acid molecule.
Claim 3
Dependent Claim
14 words
The method of claim 1, wherein said nucleic acid molecule is an RNA molecule.
Claim 4
Dependent Claim
11 words
The method of claim 2, wherein said RNA molecule is single-stranded.
Claim 5
Dependent Claim
11 words
The method of claim 2, wherein said RNA molecule is double-stranded.
Claim 6
Dependent Claim
14 words
The method of claim 1, wherein said nucleic acid molecule is a DNA molecule.
Claim 7
Dependent Claim
11 words
The method of claim 5, wherein said DNA molecule is single-stranded.
Claim 8
Dependent Claim
11 words
The method of claim 5, wherein said DNA molecule is double-stranded.
Claim 9
Dependent Claim
16 words
The method of claim 1, wherein both of said primers contain at least one deoxyinosine residue.
Claim 10
Dependent Claim
40 words
The method of claim 1, wherein said nucleic acid molecule being amplified is polyadenylated at its 3' end, and wherein one of said primers contains a poly-T sequence, and the other of said primers contains at least one deoxyinosine residue.
Claim 11
Dependent Claim
48 words
The method of claim 1, wherein said nucleic acid molecule being amplified, copy thereof or complementary copy thereof, has been extended to contain a 3' sequence, and wherein one of said primers is capable of hybridizing to said 3' sequence, said primer containing at least one deoxyinosine residue.
Claim 12
Dependent Claim
19 words
The method of claim 1, wherein at least one of said primers is extended using a thermostable DNA polymerase.
Claim 13
Dependent Claim
13 words
The method of claim 11, wherein said thermostable DNA polymerase is Tag polymerase.
Claim 14
Dependent Claim
150 words
An improved method for amplifying the concentration of a target nucleic acid molecule by using a first and a second oligonucleotide primers, wherein said first and second primers hybridize to a nucleic acid molecule, such that they may be ligated to one another and thereby form said target molecule, and exhibit different T m s with respect to one another, when hybridized to said nucleic acid molecule; said improved method comprising the steps: (a) replacing at least one residue in at least one of said primers with at least one deoxyinosine, wherein said replacement equalizes the T m s of the first and second primers with respect to one another, when hybridized to said nucleic acid molecule; (b) hybridizing said first and second primers to said nucleic acid molecule; and (c) ligating said hybridized first and second primers to one another, wherein said ligation forms said target nucleic acid molecule.
Claim 15
Dependent Claim
150 words
An improved method for amplifying the concentration of a target nucleic acid molecule by using a first and a second oligonucleotide primers, wherein said first and second primers hybridize to a nucleic acid molecule, such that they may be ligated to one another and thereby form said target molecule, and exhibit different T m s with respect to one another, when hybridized to said nucleic acid molecule; said improved method comprising the steps: (a) replacing at least one residue in at least one of said primers with at least one deoxyinosine, wherein said replacement equalizes the T m s of the first and second primers with respect to one another, when hybridized to said nucleic acid molecule; (b) hybridizing said first and second primers to said nucleic acid molecule; and (c) ligating said hybridized first and second primers to one another, wherein said ligation forms said target nucleic acid molecule.
Claim 16
Dependent Claim
14 words
The method of claim 13, wherein said target nucleic acid is a cDNA molecule.
Claim 17
Dependent Claim
29 words
The method of claim 13, wherein said oligonucleotides when ligated together form a primer, and wherein said primer is isothermally extended to produce a complement to said target molecule.
Claim 18
Dependent Claim
24 words
The method of claim 13 wherein the capacity of said oligonucleotides to ligate to one another is probative of the presence of a mutation.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-11-26 | 2000-05-27 | 2000-11-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-11-26 | 2004-05-27 | 2004-11-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-11-26 | 2008-05-27 | 2008-11-26 | $7700.00 | Window Closed / Lapsed |