Patent US5837891A successfully refreshed from Google Patents.
US5629178A
Expired / Lapsed
US
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Method for enhancing amplification in the polymerase chain reaction employing peptide nucleic acid (PNA)
Assignee: Applied Biosystems LLC
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Filing: 1994-10-28
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Granted: 1997-05-13
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Expires: 2014-10-28
Abstract
Abstract A process for producing a particular nucleic acid sequence from a given sequence of DNA or RNA in amounts which are large compared to the amount initially present, using PNAs in conjunction with the polymerase chain reaction (PCR) is disclosed.
Inventors
- 👤 Daniel B. Demers
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 16:34
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/686
Claims Analysis
Total Claims: 125
Claim 1
Independent Claim
65 words
A process for amplifying a nucleic acid sequence of interest which is in a sample containing at least one nucleic acid molecule, comprising the steps of: treating the sample by including at least one PNA oligomer which binds within the nucleic acid sequence to be amplified and a polymerase; and amplifying a nucleic acid sequence which is complementary to the nucleic acid sequence of interest.
Claim 2
Dependent Claim
65 words
A process for amplifying a nucleic acid sequence of interest which is in a sample containing at least one nucleic acid molecule, comprising the steps of: treating the sample by including at least one PNA oligomer which binds within the nucleic acid sequence to be amplified and a polymerase; and amplifying a nucleic acid sequence which is complementary to the nucleic acid sequence of interest.
Claim 3
Dependent Claim
69 words
The process according to claim 1 wherein said step of amplifying comprises: annealing at least one oligonucleotide primer to said nucleic acid sequence of interest and extending the at least one oligonucleotide primer so as to synthesize at least one extension product employing said nucleic acid sequence as a template wherein the at least one PNA oligomer is displaced from the template during the synthesis of the extension product.
Claim 4
Dependent Claim
31 words
The process according to claim 2, wherein said nucleic acid sequence of interest includes a sequence portion which is substantially repeated at least once within the nucleic acid sequence of interest.
Claim 5
Dependent Claim
36 words
The process according to claim 3, wherein the at least one PNA oligomer comprises a mixture of several PNA oligomers, each of which is substantially complementary to a portion of said nucleic acid sequence of interest.
Claim 6
Dependent Claim
30 words
The process according to claim 2, wherein said nucleic acid sequence of interest includes a sequence portion which is repeated at least once within the nucleic acid sequence of interest.
Claim 7
Dependent Claim
47 words
The process according to claim 2, wherein during the PNA oligomer binding step, said at least one PNA oligomer forms a duplex with a portion of the nucleic acid sequence of interest, said duplex having a melting temperature within the range of 70° C. to 80° C.
Claim 8
Dependent Claim
21 words
The process according to claim 6, wherein said duplex has a melting temperature within the range of 74° to 78° C.
Claim 9
Dependent Claim
36 words
The process according to claim 2, wherein the at least one PNA oligomer comprises a pair of PNA oligomers at least a portion of the sequence of each of said pair being complementary to the other.
Claim 10
Dependent Claim
20 words
The process according to claim 2, wherein said at least one PNA oligomer each comprises 5 to 20 PNA monomers.
Claim 11
Dependent Claim
20 words
The process according to claim 9, wherein said at least one PNA oligomer each comprises 8 to 16 PNA monomers.
Claim 12
Independent Claim
251 words
A method of amplifying a nucleic acid sequence of interest in a sample containing at least one nucleic acid molecule, comprising: (a) treating the sample with (i) a polymerase, (ii) at least one oligonucleotide primer which binds to the nucleic acid sequence of interest, (iii) at least one PNA oligomer which is substantially complementary to a portion of and which binds within the nucleic acid sequence of interest at a position different from the position at which the oligonucleotide primer binds, and (iv) four different nucleoside triphospates, forming a first mixture; (b) heating the first mixture to within a first temperature range so as to denature the nucleic acid and to separate any of the at least one oligonucleotide primer and any of the at least one PNA oligomer which are bound to the nucleic acid, forming a second mixture; (c) cooling the second mixture to within a second temperature range at which the at least one PNA oligomer binds to the nucleic acid sequence of interest, forming a third mixture; (d) bringing the third mixture to within a third temperature range at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest and at which an extension product is synthesized from the oligonucleotide primer on each strand, provided the nucleic acid sequence of interest is present, said synthesis employing the nucleic acid as a template, and whereby the at least one PNA oligomer is displaced from the nucleic acid sequence of interest during said synthesis.
Claim 13
Independent Claim
251 words
A method of amplifying a nucleic acid sequence of interest in a sample containing at least one nucleic acid molecule, comprising: (a) treating the sample with (i) a polymerase, (ii) at least one oligonucleotide primer which binds to the nucleic acid sequence of interest, (iii) at least one PNA oligomer which is substantially complementary to a portion of and which binds within the nucleic acid sequence of interest at a position different from the position at which the oligonucleotide primer binds, and (iv) four different nucleoside triphospates, forming a first mixture; (b) heating the first mixture to within a first temperature range so as to denature the nucleic acid and to separate any of the at least one oligonucleotide primer and any of the at least one PNA oligomer which are bound to the nucleic acid, forming a second mixture; (c) cooling the second mixture to within a second temperature range at which the at least one PNA oligomer binds to the nucleic acid sequence of interest, forming a third mixture; (d) bringing the third mixture to within a third temperature range at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest and at which an extension product is synthesized from the oligonucleotide primer on each strand, provided the nucleic acid sequence of interest is present, said synthesis employing the nucleic acid as a template, and whereby the at least one PNA oligomer is displaced from the nucleic acid sequence of interest during said synthesis.
Claim 14
Dependent Claim
17 words
The method according to claim 11, comprising repeating steps (b), (c), and (d) at least one time.
Claim 15
Dependent Claim
29 words
The method according to claim 11, further comprising the step of (e) treating the product of step (d) under denaturing conditions to separate the extension product from said template.
Claim 16
Dependent Claim
24 words
The method according to claim 13, further comprising the step of (f) detecting the presence of any of said nucleic acid sequence of interest.
Claim 17
Dependent Claim
31 words
The method according to claim 11, wherein the nucleic acid sequence of interest includes a sequence portion which is substantially repeated at least once within the nucleic acid sequence of interest.
Claim 18
Dependent Claim
30 words
The method according to claim 15, wherein the at least one PNA oligomer comprises a mixture of several PNA oligomers, each of which is substantially complementary to said sequence portion.
Claim 19
Dependent Claim
30 words
The method according to claim 11, wherein said nucleic acid sequence of interest includes a sequence portion which is repeated at least once within the nucleic acid sequence of interest.
Claim 20
Dependent Claim
43 words
The method according to claim 11, wherein during step (c) the at least one PNA oligomer forms a duplex with a portion of the nucleic acid sequence of interest, said duplex having a melting temperature within the range of 70° to 80° C.
Claim 21
Dependent Claim
21 words
The method according to claim 18, wherein said duplex has a melting temperature within the range of 74° to 78° C.
Claim 22
Dependent Claim
36 words
The method according to claim 11, wherein the at least one PNA oligomer comprises a pair of PNA oligomers, at least a portion of the sequence of each of said pair being complementary to the other.
Claim 23
Dependent Claim
19 words
The method according to claim 11, wherein the first temperature range is a range of 80° to 100° C.
Claim 24
Dependent Claim
19 words
The method according to claim 11, wherein the second temperature range is a range of 70° to 80° C.
Claim 25
Dependent Claim
19 words
The method according to claim 22, wherein the second temperature range is a range of 72° to 78° C.
Claim 26
Dependent Claim
19 words
The method according to claim 11, wherein the third temperature range is a range of 40° to 80° C.
Claim 27
Dependent Claim
21 words
The method according to claim 11, wherein each of said at least one PNA oligomer comprises 5 to 20 PNA monomers.
Claim 28
Dependent Claim
21 words
The method according to claim 25, wherein each of said at least one PNA oligomer comprises 8 to 16 PNA monomers.
Claim 29
Dependent Claim
18 words
The method according to claim 11, wherein, while performing steps (c) and (d), the temperature is continuously varied.
Claim 30
Dependent Claim
28 words
The method according to claim 11, wherein, during each of steps (c) and (d), the temperature is maintained at a substantially constant value for an interval of time.
Claim 31
Dependent Claim
105 words
The method according to claim 11, wherein step (d) comprises the steps of: (d1) bringing the third mixture to within a fourth temperature range which is within said third temperature range and at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest, forming a fourth mixture; (d2) heating the fourth mixture to within a fifth temperature range which is within said third temperature range and at which said extension product is synthesized from the oligonucleotide primer on each strand, whereby the at least one PNA oligomer is displaced from the nucleic acid sequence of interest, forming a fifth mixture.
Claim 32
Dependent Claim
17 words
The method according to claim 29, comprising repeating steps (b), (c), and (d) at least one time.
Claim 33
Dependent Claim
29 words
The method according to claim 29, further comprising the step of (e) treating the product of step (d) under denaturing conditions to separate the extension product from said template.
Claim 34
Dependent Claim
24 words
The method according to claim 31, further comprising the step of (f) detecting the presence of any of said nucleic acid sequence of interest.
Claim 35
Dependent Claim
19 words
The method according to claim 29, wherein the first temperature range is a range of 80° to 100° C.
Claim 36
Dependent Claim
19 words
The method according to claim 29, wherein the second temperature range is a range of 70° to 80° C.
Claim 37
Dependent Claim
19 words
The method according to claim 34, wherein the second temperature range is a range of 72° to 78° C.
Claim 38
Dependent Claim
19 words
The method according to claim 29, wherein the fourth temperature range is a range of 40° to 72° C.
Claim 39
Dependent Claim
19 words
The method according to claim 29, wherein the fifth temperature range is a range of 60° to 80° C.
Claim 40
Dependent Claim
107 words
The process according to claim 1 using a kit wherein said kit comprises: a) a container containing at least one oligonucleotide primer for each strand of each nucleic acid sequence of interest, each of said at least one oligonucleotide primer being substantially complementary to a strand of each nucleic acid sequence of interest; b) a container containing at least one PNA oligomer which is substantially complementary to a portion of and which binds within each nucleic acid sequence of interest at a position different form the position at which the at least one oligonucleotide primer binds; and c) a container containing each of four different nucleoside triphosphates.
Claim 41
Dependent Claim
19 words
The process according to claim 38, wherein said kit further comprises: (d) a container containing an agent for polymerization.
Claim 42
Dependent Claim
30 words
The process according to claim 38 wherein said nucleic acid of interest includes a sequence portion which is substantially repeated at least once within the nucleic acid sequence of interest.
Claim 43
Dependent Claim
30 words
The process according to claim 40, wherein the at least one PNA oligomer comprises a mixture of several PNA oligomers, each of which is substantially complementary to said sequence portion.
Claim 44
Dependent Claim
30 words
The process according to claim 38 wherein said nucleic acid sequence of interest includes a sequence portion which is repeated at least once within the nucleic acid sequence of interest.
Claim 45
Dependent Claim
40 words
The process according to claim 38, wherein the at least one PNA oligomer forms a duplex with a portion of the nucleic acid sequence of interest, said duplex having a melting temperature within the range of 70° to 80° C.
Claim 46
Dependent Claim
21 words
The process according to claim 43, wherein said duplex has a melting temperature within the range of 74° to 78° C.
Claim 47
Dependent Claim
36 words
The process according to claim 38, wherein the at least one PNA oligomer comprises a pair of PNA oligomers, at least a portion of the sequence of each of said pair being complementary to the other.
Claim 48
Dependent Claim
21 words
The process according to claim 38, wherein each of said at least one PNA oligomer comprises 5 to 20 PNA monomers.
Claim 49
Dependent Claim
21 words
The process according to claim 46, wherein each of said at least one PNA oligomer comprises 8 to 16 PNA monomers.
Claim 50
Dependent Claim
302 words
A process for cloning into a vector a nucleic acid sequence of interest in a sample containing at least one nucleic acid, comprising: (a) treating the sample with i) a polymerase, ii) at least one oligonucleotide primer containing a restriction site at its 5' end and which is substantially complementary to and binds to a strand of each different nucleic acid sequence of interest, iii) at least one PNA oligomer which is substantially complementary to a portion of and which binds within the nucleic acid sequence of interest at a position different from the position at which the oligonucleotide primer binds, and iv) four different nucleoside triphosphates, forming a first mixture; (b) heating the first mixture to within a first temperature range so as to denature the nucleic acid and to separate any of the at least one oligonucleotide primer and any of the at least one PNA oligomer which is bound to the nucleic acid, forming a second mixture; (c) cooling the second mixture to within a second temperature range at which the at least one PNA oligomer binds to the nucleic acid, forming a third mixture; (d) bringing the third mixture to within a third temperature range at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest and at which an extension product is synthesized from the oligonucleotide primer on each strand, provided the nucleic acid sequence of interest is present, said synthesis employing the nucleic acid as a template, and whereby the at least one PNA oligomer is displaced from the nucleic acid sequence of interest during said synthesis (e) adding to the product of step (d) a restriction enzyme which cleaves at said restriction site to thereby obtain cleaved products; and (f) ligating the cleaved products into at least one cloning vector.
Claim 51
Dependent Claim
302 words
A process for cloning into a vector a nucleic acid sequence of interest in a sample containing at least one nucleic acid, comprising: (a) treating the sample with i) a polymerase, ii) at least one oligonucleotide primer containing a restriction site at its 5' end and which is substantially complementary to and binds to a strand of each different nucleic acid sequence of interest, iii) at least one PNA oligomer which is substantially complementary to a portion of and which binds within the nucleic acid sequence of interest at a position different from the position at which the oligonucleotide primer binds, and iv) four different nucleoside triphosphates, forming a first mixture; (b) heating the first mixture to within a first temperature range so as to denature the nucleic acid and to separate any of the at least one oligonucleotide primer and any of the at least one PNA oligomer which is bound to the nucleic acid, forming a second mixture; (c) cooling the second mixture to within a second temperature range at which the at least one PNA oligomer binds to the nucleic acid, forming a third mixture; (d) bringing the third mixture to within a third temperature range at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest and at which an extension product is synthesized from the oligonucleotide primer on each strand, provided the nucleic acid sequence of interest is present, said synthesis employing the nucleic acid as a template, and whereby the at least one PNA oligomer is displaced from the nucleic acid sequence of interest during said synthesis (e) adding to the product of step (d) a restriction enzyme which cleaves at said restriction site to thereby obtain cleaved products; and (f) ligating the cleaved products into at least one cloning vector.
Claim 52
Dependent Claim
17 words
The process according to claim 48, comprising repeating steps (b), (c), and (d) at least one time.
Claim 53
Dependent Claim
31 words
The process according to claim 48, wherein said nucleic acid sequence of interest includes a sequence portion which is substantially repeated at least once within the nucleic acid sequence of interest.
Claim 54
Dependent Claim
30 words
The process according to claim 50, wherein the at least one PNA oligomer comprises a mixture of several PNA oligomers, each of which is substantially complementary to said sequence portion.
Claim 55
Dependent Claim
30 words
The process according to claim 48, wherein said nucleic acid sequence of interest includes a sequence portion which is repeated at least once within the nucleic acid sequence of interest.
Claim 56
Dependent Claim
43 words
The process according to claim 48, wherein during step (c) the at least one PNA oligomer forms a duplex with a portion of the nucleic acid sequence of interest, said duplex having a melting temperature within the range of 70° to 80° C.
Claim 57
Dependent Claim
21 words
The process according to claim 53, wherein said duplex has a melting temperature within the range of 74° to 78° C.
Claim 58
Dependent Claim
36 words
The process according to claim 48, wherein the at least one PNA oligomer comprises a pair of PNA oligomers, at least a portion of the sequence of each of said pair being complementary to the other.
Claim 59
Dependent Claim
19 words
The process according to claim 48, wherein the first temperature range is a range of 80° to 100° C.
Claim 60
Dependent Claim
19 words
The process according to claim 48, wherein the second temperature range is a range of 70° to 80° C.
Claim 61
Dependent Claim
19 words
The process according to claim 57, wherein the second temperature range is a range of 72° to 78° C.
Claim 62
Dependent Claim
19 words
The process according to claim 48, wherein the third temperature range is a range of 40° to 80° C.
Claim 63
Dependent Claim
21 words
The process according to claim 48, wherein each of said at least one PNA oligomer comprises 5 to 20 PNA monomers.
Claim 64
Dependent Claim
21 words
The process according to claim 60, wherein each of said at least one PNA oligomer comprises 8 to 16 PNA monomers.
Claim 65
Dependent Claim
18 words
The process according to claim 48, wherein, while performing steps (c) and (d), the temperature is continuously varied.
Claim 66
Dependent Claim
28 words
The process according to claim 48, wherein, during each of steps (c) and (d), the temperature is maintained at a substantially constant value for an interval of time.
Claim 67
Dependent Claim
104 words
The process according to claim 48, wherein step (d) comprises the steps of: (di) bringing the third mixture to within a fourth temperature range which is within said third temperature range and at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest, forming a fourth mixture; (dii) heating the fourth mixture to within a fifth temperature range which is within said third temperature range and at which said extension product is synthesized from the oligonucleotide primer on each strand, whereby at least one PNA oligomer is displaced from the nucleic acid sequence of interest, forming a fifth mixture.
Claim 68
Dependent Claim
19 words
The process according to claim 64, wherein the first temperature range is a range of 80° to 100° C.
Claim 69
Dependent Claim
19 words
The process according to claim 64, wherein the second temperature range is a range of 70° to 80° C.
Claim 70
Dependent Claim
19 words
The process according to claim 66, wherein the second temperature range is a range of 72° to 78° C.
Claim 71
Dependent Claim
19 words
The process according to claim 64, wherein the fourth temperature range is a range of 40° to 72° C.
Claim 72
Dependent Claim
19 words
The process according to claim 64, wherein the fifth temperature range is a range of 60° to 80° C.
Claim 73
Dependent Claim
268 words
A solution containing amplified nucleic acid sequences including an amplified nucleic acid sequence of interest wherein said amplified nucleic acid sequences including said amplified nucleic acid sequence of interest have been amplified by an amplification process comprising the steps of: a) treating a sample containing nucleic acids with i) a polymerase, ii) at least one oligonucleotide primer which binds to a strand of said nucleic acid sequence of interest, iii) at least one PNA oligomer which is substantially complementary to and binds within the nucleic acid sequence of interest at a position different from the position at which the at least one oligonucleotide primer binds, and iv) four different nucleoside triphosphates, forming a first mixture; b) heating the first mixture to within a first temperature range so as to denature the at least one PNA oligomer which is bound to the nucleic acid, forming a second mixture; c) cooling the second mixture to within a second temperature range at which the at least one PNA oligomer binds to the nucleic acid sequence of interest forming a third mixture; d) bringing the third mixture to within a third temperature range at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest and at which an extension product is synthesized from the oligonucleotide primer on each strand , said synthesis employing the nucleic acid as a template, and whereby the at least one PNA oligomer is displaced from the nucleic acid sequence of interest during said synthesis to form said solution of said amplified nucleic acid sequences including an amplified nucleic acid sequence of interest.
Claim 74
Dependent Claim
268 words
A solution containing amplified nucleic acid sequences including an amplified nucleic acid sequence of interest wherein said amplified nucleic acid sequences including said amplified nucleic acid sequence of interest have been amplified by an amplification process comprising the steps of: a) treating a sample containing nucleic acids with i) a polymerase, ii) at least one oligonucleotide primer which binds to a strand of said nucleic acid sequence of interest, iii) at least one PNA oligomer which is substantially complementary to and binds within the nucleic acid sequence of interest at a position different from the position at which the at least one oligonucleotide primer binds, and iv) four different nucleoside triphosphates, forming a first mixture; b) heating the first mixture to within a first temperature range so as to denature the at least one PNA oligomer which is bound to the nucleic acid, forming a second mixture; c) cooling the second mixture to within a second temperature range at which the at least one PNA oligomer binds to the nucleic acid sequence of interest forming a third mixture; d) bringing the third mixture to within a third temperature range at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest and at which an extension product is synthesized from the oligonucleotide primer on each strand , said synthesis employing the nucleic acid as a template, and whereby the at least one PNA oligomer is displaced from the nucleic acid sequence of interest during said synthesis to form said solution of said amplified nucleic acid sequences including an amplified nucleic acid sequence of interest.
Claim 75
Dependent Claim
27 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein said amplification process further comprises repeating steps (b), (c), and (d) at least one time.
Claim 76
Dependent Claim
38 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein the amplification process further comprises the step of (e) treating the product of step (d) under denaturing conditions to separate the extension product from said template.
Claim 77
Dependent Claim
28 words
The solution containing amplified nucleic acid sequences according to claim 72, wherein the amplification process further comprises the step of (f) detecting the nucleic acid sequence of interest.
Claim 78
Dependent Claim
40 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein in the amplification process said nucleic acid sequence of interest includes a sequence portion which is substantially repeated at least once within the nucleic acid sequence of interest.
Claim 79
Dependent Claim
36 words
The solution containing an amplified nucleic acid sequence according to claim 74, wherein the at least one PNA oligomer comprises a mixture of several PNA oligomers, each of which is substantially complementary to said sequence portion.
Claim 80
Dependent Claim
39 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein in the amplification process said nucleic acid sequence of interest includes a sequence portion which is repeated at least once within the nucleic acid sequence of interest.
Claim 81
Dependent Claim
52 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein during step (c) of the amplification process the at least one PNA oligomer forms a duplex with a portion of the nucleic acid sequence of interest, said duplex having a melting temperature within the range of 70° to 80° C.
Claim 82
Dependent Claim
30 words
The solution containing amplified nucleic acid sequences according to claim 77, wherein in the amplification process said duplex has a melting temperature within the range of 74° to 78° C.
Claim 83
Dependent Claim
45 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein in the amplification process the at least one PNA oligomer comprises a pair of PNA oligomers, at least a portion of the sequence of each of said pair being complementary to the other.
Claim 84
Dependent Claim
28 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein in the amplification process the first temperature range is a range of 80° to 100° C.
Claim 85
Dependent Claim
28 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein in the amplification process the second temperature range is a range of 70° to 80° C.
Claim 86
Dependent Claim
28 words
The solution containing amplified nucleic acid sequences according to claim 81, wherein in the amplification process the second temperature range is a range of 72° to 78° C.
Claim 87
Dependent Claim
28 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein in the amplification process the third temperature range is a range of 40° to 80° C.
Claim 88
Dependent Claim
32 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein in the amplification process said at least one PNA oligomer comprises a PNA polymer containing 5 to 20 PNA monomers.
Claim 89
Dependent Claim
32 words
The solution containing amplified nucleic acid sequences according to claim 84, wherein in the amplification process said at least one PNA oligomer comprises a PNA polymer containing 8 to 16 PNA monomers.
Claim 90
Dependent Claim
27 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein, while performing steps (c) and (d) of the amplification process, the temperature is continuously varied.
Claim 91
Dependent Claim
37 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein, during each of steps (c) and (d) of the amplification process, the temperature is maintained at a substantially constant value for an interval of time.
Claim 92
Dependent Claim
114 words
The solution containing amplified nucleic acid sequences according to claim 70, wherein step (d) of the amplification process comprises the steps of: (di) bringing the third mixture to within a fourth temperature range which is within said third temperature range and at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest, forming a fourth mixture; (dii) heating the fourth mixture to within a fifth temperature range which is within said third temperature range and at which said extension product is synthesized from the oligonucleotide primer on each strand, whereby the at least one PNA oligomer is displaced from the nucleic acid sequence of interest, forming a fifth mixture.
Claim 93
Dependent Claim
28 words
The solution containing an amplified nucleic acid sequences according to claim 88, wherein the defined process further comprises repeating steps (b), (c), and (d) at least one time.
Claim 94
Dependent Claim
38 words
The solution containing amplified nucleic acid sequences according to claim 88, wherein the defined process further comprises the step of; (e) treating the product of step (d) under denaturing conditions to separate the extension product from said template.
Claim 95
Dependent Claim
34 words
The solution containing an amplified nucleic acid sequences according to claim 89, wherein the defined process further comprises the step of: (f) detecting the presence of any of said nucleic acid sequence of interest.
Claim 96
Dependent Claim
28 words
The solution containing amplified nucleic acid sequences according to claim 88, wherein in the amplification process the first temperature range is a range of 80° to 100° C.
Claim 97
Dependent Claim
28 words
The solution containing amplified nucleic acid sequences according to claim 88, wherein in the amplification process the second temperature range is a range of 70° to 80° C.
Claim 98
Dependent Claim
28 words
The solution containing amplified nucleic acid sequences according to claim 93, wherein in the amplification process the second temperature range is a range of 72° to 78° C.
Claim 99
Dependent Claim
22 words
The amplified nucleic acid sequence according to claim 88, wherein the fourth temperature range is a range of 40° to 72° C.
Claim 100
Dependent Claim
28 words
The solution containing amplified nucleic acid sequences according to claim 88, wherein in the amplification process the fifth temperature range is a range of 60° to 80° C.
Claim 101
Independent Claim
91 words
A method for replicating a target nucleic acid strand with a polymerase to produce a complementary nucleic acid strand of the target nucleic acid strand comprising: (a) providing a peptide nucleic acid (PNA) which, (i) is substantially complementary in sequence to nucleotides in at least a portion of the target strand, (ii) anneals with said portion of the target strand and (b) annealing the PNA to the target strand and replicating the target nucleic acid strand with the polymerase to thereby produce the complementary strand of the target nucleic acid strand.
Claim 102
Independent Claim
91 words
A method for replicating a target nucleic acid strand with a polymerase to produce a complementary nucleic acid strand of the target nucleic acid strand comprising: (a) providing a peptide nucleic acid (PNA) which, (i) is substantially complementary in sequence to nucleotides in at least a portion of the target strand, (ii) anneals with said portion of the target strand and (b) annealing the PNA to the target strand and replicating the target nucleic acid strand with the polymerase to thereby produce the complementary strand of the target nucleic acid strand.
Claim 103
Dependent Claim
42 words
The method according to claim 97, wherein step (a) comprises providing a mixture of PNA oligomers, wherein the nucleobase sequence of at least one of the PNA oligomers in the mixture is complementary in sequence to said portion of the target strand.
Claim 104
Dependent Claim
29 words
The method according to claim 97, wherein the PNA oligomer provided in step (a) comprises 5 to 20 nucleotides complementary in sequence to said portion of the target strand.
Claim 105
Dependent Claim
29 words
The method according to claim 99, wherein the PNA oligomer provided in step (a) comprises 8 to 16 nucleotides complementary in sequence to said portion of the target strand.
Claim 106
Dependent Claim
19 words
The method according to claim 97, wherein the target strand comprises, a multiplicity of perfect or imperfect sequence repeats.
Claim 107
Dependent Claim
13 words
The method according to claim 97, wherein the polymerase is a DNA polymerase.
Claim 108
Dependent Claim
15 words
The method according to claim 102, wherein the DNA polymerase is a thermostable DNA polymerase.
Claim 109
Dependent Claim
33 words
The method according to claim 102, further comprising the step of annealing an oligonucleotide primer to a different portion of the target strand thereby to produce a replication initiation site for the polymerase.
Claim 110
Dependent Claim
33 words
The method according to claim 103, further comprising the step of annealing an oligonucleotide primer to a different portion of the target strand thereby to produce a replication initiation site for the polymerase.
Claim 111
Independent Claim
123 words
A method for replicating a target nucleic acid strand with a thermostable DNA polymerase to produce a complementary nucleic acid strand of the target nucleic acid strand comprising: (a) providing a peptide nucleic acid (PNA) which, (i) is substantially complementary in sequence to nucleotides in at least a portion of the target nucleic acid strand, (ii) anneals with said portion of the target nucleic acid strand and (iii) when annealed with said portion of the target nucleic acid strand, does not serve as a replication initiation site for the polymerase; and (b) annealing the PNA to the target nucleic acid strand and replicating the target nucleic acid strand with the polymerase to thereby produce the complementary strand of the target nucleic acid strand.
Claim 112
Independent Claim
123 words
A method for replicating a target nucleic acid strand with a thermostable DNA polymerase to produce a complementary nucleic acid strand of the target nucleic acid strand comprising: (a) providing a peptide nucleic acid (PNA) which, (i) is substantially complementary in sequence to nucleotides in at least a portion of the target nucleic acid strand, (ii) anneals with said portion of the target nucleic acid strand and (iii) when annealed with said portion of the target nucleic acid strand, does not serve as a replication initiation site for the polymerase; and (b) annealing the PNA to the target nucleic acid strand and replicating the target nucleic acid strand with the polymerase to thereby produce the complementary strand of the target nucleic acid strand.
Claim 113
Dependent Claim
42 words
The method according to claim 106, wherein step (a) comprises providing a mixture of PNA oligomers, wherein the nucleobase sequence of at least one of the PNA oligomers in the mixture is complementary in sequence to said portion of the target strand.
Claim 114
Dependent Claim
29 words
The method according to claim 106, wherein the PNA oligomer provided in step (a) comprises 5 to 20 nucleotides complementary in sequence to said portion of the target strand.
Claim 115
Dependent Claim
28 words
The method according to claim 108, wherein the PNA provided in step (a) comprises 8 to 16 nucleotides complementary in sequence to said portion of the target strand.
Claim 116
Dependent Claim
17 words
The method according to claim 106, wherein the target strand comprises a multiplicity of nucleotide sequence repeats.
Claim 117
Dependent Claim
35 words
The method according to claim 106, further comprising the step of: (C) annealing an oligonucleotide primer to a second nucleobase sequence of the target strand thereby to produce a replication initiation site for the polymerase.
Claim 118
Independent Claim
130 words
A method for replicating a target nucleic acid strand with a polymerase comprising: (a) providing a target nucleic acid strand having a multiplicity of nucleotide sequence repeats; (b) forming a target nucleic acid strand-PNA oligomer complex by providing a PNA oligomer and annealing said PNA oligomer to the target nucleic acid strand wherein the PNA oligomer is: (i) substantially complementary in sequence to at least a portion of one of the repeats; (ii) anneals with the at least a portion of one of the repeats thereby to form a target nucleic acid strand-PNA oligomer complex; and (iii) when annealed with the at least a portion of the repeats, does not serve as a replication initiation site for the polymerase; and (c) incubating the complex in the presence of the polymerase.
Claim 119
Independent Claim
130 words
A method for replicating a target nucleic acid strand with a polymerase comprising: (a) providing a target nucleic acid strand having a multiplicity of nucleotide sequence repeats; (b) forming a target nucleic acid strand-PNA oligomer complex by providing a PNA oligomer and annealing said PNA oligomer to the target nucleic acid strand wherein the PNA oligomer is: (i) substantially complementary in sequence to at least a portion of one of the repeats; (ii) anneals with the at least a portion of one of the repeats thereby to form a target nucleic acid strand-PNA oligomer complex; and (iii) when annealed with the at least a portion of the repeats, does not serve as a replication initiation site for the polymerase; and (c) incubating the complex in the presence of the polymerase.
Claim 120
Dependent Claim
46 words
The method according to claim 112, wherein step (b) comprises providing a mixture of PNA oligomers, wherein the nucleotide sequence of at least one of the PNA oligomers in the mixture is complementary in sequence to said at least a portion of one of the repeats.
Claim 121
Dependent Claim
33 words
The method according to claim 112, wherein the PNA oligomer provided in step (b) comprises 5 to 20 nucleotides complementary in sequence to said at least a portion of one of the repeats.
Claim 122
Dependent Claim
33 words
The method according to claim 114, wherein the PNA oligomer provided in step (b) comprises 8 to 16 nucleotides complementary in sequence to said at least a portion of one of the repeats.
Claim 123
Dependent Claim
13 words
The method according to claim 112, wherein the polymerase is a DNA polymerase.
Claim 124
Dependent Claim
15 words
The method according to claim 116, wherein the DNA polymerase is a thermostable DNA polymerase.
Claim 125
Dependent Claim
33 words
The method according to claim 112 further comprising the step of: (d) annealing a primer to a different portion of the target strand thereby to produce a replication initiation site for the polymerase.
References Cited (Backward Citations)
WO1992020702A1
Cited
WO1992020703A1
Cited
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