Oligonucleotide which can be used as primer in a method of amplification based on a replication accompanied by strand displacement

Claim 2

Oligonucleotide comprising successively, from its 5' end to its 3' end, a first portion capable of self-pairing in order to form a stem-loop structure comprising an upstream segment and a downstream segment which are paired, and a second portion not capable of self-pairing, wherein said first portion comprises at least one blocking agent capable of blocking the replication, by a polymerase, of said oligonucleotide, such that at least a portion of the downstream segment is replicated and such that the upstream segment is not replicated totally or partially.

Claim 3

Oligonucleotide according to claim 1, wherein the blocking agent is such that the portion of the downstream segment capable of being replicated has a length of at least 2 nucleotides.

Claim 4

Oligonucleotide according to claim 1, wherein the downstream end of the upstream segment and the upstream end of the downstream segment are linked by a loop.

Claim 5

Oligonucleotide according to claim 1, wherein said upstream and downstream segments each contain from 2 to 30 nucleotides.

Claim 6

Oligonucleotide according to claim 1, wherein said first portion comprises, as the blocking agent, either a hydrocarbon arm or a modified nucleotide which, when present in an oligonucleotide template for a polymerase, stops replication and transcription by said polymerase at the level of said modified nucleotide.

Claim 7

Oligonucleotide according to claim 1, wherein the oligonucleotide contains, between said first and second portions, a third portion containing a sense sequence of a promoter for an RNA polymerase.

Claim 8

Oligonucleotide according to claim 1, wherein said second portion contains from 5 to 40 nucleotides.

Claim 9

Oligonucleotide primer that can be used in a process for the amplification of a target sequence of a nucleic acid, said target sequence comprising a downstream region of at least 5 nucleotides, said oligonucleotide primer comprising an oligonucleotide as defined in claim 1, wherein said second portion contains, at its 3' end, a sequence capable of hybridizing with said downstream region of the target.

Claim 10

Process for cyclic amplification of a target sequence of a nucleic acid, said target sequence comprising, at its 5' ends an upstream region and, at its 3'end, a downstream region, said process comprising the steps of: (1) obtaining a single-stranded polynucleotide comprising a first segment corresponding to the target sequence to be amplified, a second segment situated upstream of the 5' end of the first segment and a third segment situated downstream of the 3' end of the first segment, wherein said second segment of said single-stranded polynucleotide is capable of self-pairing in order to form a stem-loop structure comprising an upstream segment and a downstream segment which are paired, and said third segment of said single-stranded polynucleotide has any sequence, (2) exposing said polynucleotide to: (a) a first primer as defined in claim 8, capable of hybridizing with the downstream region of the target sequence, wherein at least a downstream portion of the downstream segment of the first portion of the first primer is complementary to the third segment of the single-stranded polynucleotide, (b) optionally a second primer as defined in claim 8, capable of hybridizing with a downstream region of a sequence complementary to the target sequence, said downstream region being complementary to said upstream region of the target sequence, wherein the first portion of the second primer is a homologue of the second segment of said single-stranded polynucleotide, (c) a third primer whose sequence is homologous to at least a downstream portion of a sequence of the downstream segment of said first portion of the first primer, (d) a fourth primer whose sequence is homologous to at least a portion of a sequence of the downstream segment of said second segment of said single-stranded polynucleotide, and (e) an enzymatic system having at least an activity for replicating said nucleic acid and a strand displacement activity, and (3) incubating a mixture obtained in step (2) under conditions allowing hybridization to occur and the enzymatic activities to function.

Claim 11

Process according to claim 9, wherein said first primer is an oligonucleotide comprising successively, from its 5' end to its 3' end, a first portion capable of self-pairing in order to form a stem-loop structure comprising an upstream segment and a downstream segment which are paired, a second portion not capable of self-pairing, and between said first and second portions, a third portion containing a sense sequence of a promoter for an RNA polymerase, wherein said first portion comprises at least one blocking agent capable of blocking the replication, by a polymerase, of said oligonucleotide, such that at least a portion of the downstream segment is replicated and such that the upstream segment is not replicated totally or partially, wherein said single-stranded polynucleotide contains, between said first segment and said third segment, a fourth segment complementary to said sense sequence contained in the first primer, and wherein said enzymatic system further contains an RNA polymerase activity under the control of the promoter corresponding to the sense sequence.

Claim 12

Process according to claim 9, wherein said polynucleotide is exposed to the second primer and said second primer is an oligonucleotide comprising successively, from its 5' end to its 3' end, a first portion capable of self-pairing in order to form a stem-loop structure comprising an upstream segment and a downstream segment which are paired, a second portion not capable of self-pairing, and between said first and second portions, a third portion containing a sense sequence of a promoter for an RNA polymerase, wherein said first portion comprises at least one blocking agent capable of blocking the replication, by a polymerase, of said oligonucleotide, such that at least a portion of the downstream segment is replicated and such that the upstream segment is not replicated totally or partially, wherein said single-stranded polynucleotide contains, between said first segment and said second segment, an additional segment whose sequence is the same as the sense sequence contained in the second primer, and wherein said enzymatic system further contains, an RNA polymerase activity under the control of the promoter corresponding to the sense sequence contained in the second primer.

Claim 13

Oligonucleotide according to claim 2, wherein the downstream segment capable of being replicated has a length of at least 3 nucleotides.

Claim 14

Oligonucleotide according to claim 4, wherein said upstream and downstream segments each contain from 3 to 15 nucleotides.

Claim 15

A process according to claim 10, wherein the blocking agent is such that the portion of the downstream segment capable of being replicated has a length of at least two nucleotides.

Claim 16

A process according to claim 9, wherein the downstream end of the upstream segment and the upstream end of the downstream segment are linked by a loop.

Claim 17

A process according to claim 9, wherein said upstream and downstream segments each contain from 2 to 30 nucleotides.

Claim 18

A process according to claim 10, wherein said first portion comprises, as the blocking agent, either a hydrocarbon arm or a modified nucleotide which, when present in an oligonucleotide template for a polymerase, stops replication and transcription by said polymerase at the level of said modified nucleotide.

Claim 19

A process according to claim 9, wherein the downstream segment capable of being replicated has a length of at least 3 nucleotides.

Claim 20

A process according to claim 9, wherein said upstream and downstream segments each contain from 3 to 15 nucleotides.