EP0628640A1
Expired / Lapsed
EP
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Simultaneous amplification of multiple targets
Assignee: Becton Dickinson and Co
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Filing: 1994-05-26
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Granted: —
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Expires: 2014-05-26
Abstract
Abstract Methods for multiplex amplification of target nucleic acid sequences using a single pair of primers. Defined sequences are appended to the ends of multiple target sequences as part of the amplification reaction so that no steps in addition to amplification are required. The target sequences with the appended defined sequences need not be isolated prior to amplification. In one embodiment for coamplification of two target sequences, a sequence corresponding to a terminal segment of the first target sequence is appended to one end of the second target sequence and a sequence corresponding to a terminal segment of the second target sequence is appended to one end of the first target sequence. Amplification of the two targets then requires only a single pair of primers. Alternatively, a single defined sequence may be appended to the 5' and 3' ends of any number of selected targets. All such modified target sequences may then be amplified using a single pair of primers which hybridize to the defined end-sequences.
Inventors
- 👤 George Terrance Walker
- 👤 James G. Nadeau
- 👤 Michael C. Little
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-30 20:29
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6853; G; G11; G11B; G11B19/00; G11B19/02; G11B19/04; G11B5/00; G11B5/02; G11B5/09; G11B5/127; G11B5/187; G11B5/1875; G11B5/1877; G11B5/1878; G11B5/31; G11B5/3109; G11B5/3113; G11B5/455; G11B5/465; G11B2005/0002; G11B2005/0005; G11B2005/0008
Claims Analysis
Total Claims: 10
Claim 1
Independent Claim
366 words
A method for simultaneously amplifying two target nucleic acid sequences by Strand Displacement Amplification (SDA) comprising: a) hybridizing a first amplification primer to the 3' end of a first target sequence, the first amplification primer comprising a 3' target binding sequence and a 5' recognition site for a restriction enzyme capable of nicking one strand of a double stranded hemimodified recognition site for the enzyme, extending the first amplification primer with polymerase to form a first extension product complementary to the first target sequence, and displacing the first extension product; b) hybridizing a first adapter primer to the first extension product at the 3' end of the complement of the first target sequence, the 3' end of the first adapter primer comprising a target binding sequence capable of hybridizing to the first extension product and the 5' end of the first adapter primer being substantially identical to a 3' target binding sequence of a second amplification primer capable of hybridizing to a second target sequence; c) extending the first adapter primer with polymerase to form a second extension product and displacing the second extension product; d) hybridizing the second amplification primer to the 3' end of the second target sequence, the second amplification primer comprising the 3' target binding sequence and a 5' recognition site for a restriction enzyme capable of nicking one strand of a double stranded hemimodified recognition site for the enzyme, extending the second amplification primer with polymerase to form a third extension product complementary to the second target sequence, and displacing the third extension product; e) hybridizing a second adapter primer to the third extension product at the 3' end of the complement of the second target sequence, the 3' end of the second adapter primer comprising a target binding sequence capable of hybridizing to the third extension product and the 5' end of the second adapter primer being substantially identical to the target binding sequence of the first amplification primer; f) extending the second adapter primer with polymerase to form a fourth extension product and displacing the fourth extension product, and; g) simultaneously amplifying the second and fourth extension products in an SDA reaction using the first and second amplification primers.
Claim 2
Dependent Claim
22 words
The method according to Claim 1 wherein the first, second, third and fourth extension products are displaced by extension of bumper primers.
Claim 3
Dependent Claim
215 words
The method of Claim 1 further comprising the steps of: a) hybridizing the first adapter primer to the 3' end of a second strand of the first target sequence, extending the first adapter primer to form a fifth extension product complementary to the second strand of the first target sequence, and displacing the fifth extension product; b) hybridizing the first amplification primer to the fifth extension product at the 3' end of the complement of the second strand of the first target sequence, extending the first amplification primer with polymerase to form a seventh extension product, and displacing the seventh extension product; c) hybridizing the second adapter primer to the 3' end of a second strand of the second target sequence, extending the second adapter primer to form a sixth extension product complementary to the second strand of the second target sequence, and displacing the sixth extension product; d) hybridizing the second amplification primer to the sixth extension product at the 3' end of the complement of the second strand of the second target sequence, extending the second amplification primer with polymerase to form an eighth extension product, and displacing the eighth extension product, and; e) simultaneously amplifying the seventh and eighth extension products in the SDA reaction using the first and second amplification primers.
Claim 4
Independent Claim
235 words
A method for simultaneously amplifying multiple target nucleic acid sequences by Strand Displacement Amplification (SDA) comprising: a) hybridizing a first adapter primer to the 3' end of each target sequence, the 3' end of the adapter primer comprising a target binding sequence capable of hybridizing to the target sequence and the 5' end of the adapter primer comprising a first adapter sequence; b) extending the first adapter primer with polymerase to produce a first extension product for each target sequence which is complementary to each target sequence and displacing the first extension products; c) hybridizing a second adapter primer to the first extension product of each target sequence at the 3' end of the complement of each target sequence, the 3' end of the second adapter primer comprising a target binding sequence capable of hybridizing to the first extension product and the 5' end of the second adapter primer comprising a second adapter sequence; d) extending the second adapter primer with polymerase to produce a second extension product for each target sequence, displacing the second extension products, and; e) simultaneously amplifying the second extension products of the target sequences in an SDA reaction using amplification primers comprising 3' target binding sequences which are substantially identical to the first and second adapter sequences and 5' recognition sites for a restriction enzyme capable of nicking one strand of a hemimodified double stranded recognition site for the enzyme.
Claim 5
Dependent Claim
32 words
The method according to Claim 4 wherein the first and second adapter sequences are substantially identical and the second extension product of each target sequence is amplified using a single amplification primer.
Claim 6
Dependent Claim
70 words
The method according to Claim 4 wherein the first and second adapter sequences are not substantially identical and the second extension product of each target sequence is amplified using a pair of amplification primers, one member of the pair having a target binding sequence substantially identical to the first adapter sequence and the other member of the pair having a target binding sequence substantially identical to the second adapter sequence.
Claim 7
Independent Claim
25 words
The method according to any one of Claims 5 or 6 wherein the first and second extension products are displaced by extension of bumper primers.
Claim 8
Dependent Claim
118 words
The method according to Claim 4 further comprising the steps of: a) hybridizing the second adapter primer to the 3' end of a second strand of each target sequence, extending the second adapter primer to form a third extension product complementary to the second strand of each target sequence, and displacing the third extension product; b) hybridizing the first adapter primer to the third extension product of each target sequence at the 3' end of the complement of each target sequence, extending the first adapter primer to form a fourth extension product, and displacing the fourth extension product, and; c) simultaneously amplifying the fourth extension products of the target sequences in the SDA reaction using the amplification primers.
Claim 9
Independent Claim
316 words
A method for simultaneously amplifying two target nucleic acid sequences in a primer extension amplification reaction comprising: a) hybridizing a first amplification primer to the 3' end of a first target sequence, the first amplification primer comprising a 3' target binding sequence, extending the first amplification primer with polymerase to form a first extension product complementary to the first target sequence, and displacing the first extension product; b) hybridizing a first adapter primer to the first extension product at the 3' end of the complement of the first target sequence, the 3' end of the first adapter primer comprising a target binding sequence capable of hybridizing to the first extension product and the 5' end of the first adapter primer being substantially identical to a 3' target binding sequence of a second amplification primer capable of hybridizing to a second target sequence; c) extending the first adapter primer with polymerase to form a second extension product and displacing the second extension product; d) hybridizing the 3' target binding sequence of second amplification primer to the 3' end of the second target sequence, extending the second amplification primer with polymerase to form a third extension product complementary to the second target sequence, and displacing the third extension product; e) hybridizing a second adapter primer to the third extension product at the 3' end of the complement of the second target sequence, the 3' end of the second adapter primer comprising a target binding sequence capable of hybridizing to the third extension product and the 5' end of the second adapter primer being substantially identical to the target binding sequence of the first amplification primer; f) extending the second adapter primer with polymerase to form a fourth extension product and displacing the fourth extension product, and; g) simultaneously amplifying the second and fourth extension products in a primer extension amplification reaction using the first and second amplification primers.
Claim 10
Independent Claim
215 words
A method for simultaneously amplifying multiple target nucleic acid sequences in a primer extension amplification reaction comprising: a) hybridizing a first adapter primer to the 3' end of each target sequence, the 3' end of the adapter primer comprising a target binding sequence capable of hybridising to the target sequence and the 5' end of the adapter primer comprising a first adapter sequence; b) extending the first adapter primer with polymerase to produce a first extension product for each target sequence which is complementary to each target sequence and displacing the first extension products; c) hybridizing a second adapter primer to the first extension product of each target sequence at the 3' end of the complement of each target sequence, the 3' end of the second adapter primer comprising a target binding sequence capable of hybridizing to the first extension product and the 5' end of the second adapter primer comprising a second adapter sequence; d) extending the second adapter primer with polymerase to produce a second extension product for each target sequence, displacing the second extension products, and; e) simultaneously amplifying the second extension products of the target sequences in a primer extension amplification reaction using amplification primers comprising 3' target binding sequences which are substantially identical to the first and second adapter sequences.
References Cited (Backward Citations)
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