Method for nucleic acid amplification by transcription using displacement, and reagents and kit therefor

• US 5,849,547 A
• Filed: 04/24/1995
• Issued: 12/15/1998
• Est. Priority Date: 07/26/1993
• Status: Expired due to Fees

• Current Assignee: Bio Merieux
• Original Assignee: Bio Merieux
• Inventors: Cleuziat, Philippe, Mallet, Francois, Levasseur, Pierre, Guillou-Bonnici, Francoise
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/406,849
• Time in Patent Office: 1,331 Days
• Field of Search: 435/91.21, 435/91.2, 536/24.33
• US Class Current: 435/91.21
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid

Abstract

A target nucleic acid sequence is amplified by providing a polynucleotide such as VII including the sequence to be amplified as well as an RNA polymerase promoter sequence, and contacting said polynucleotide, in the presence of a system having the RNA polymerase activity, a RNA-dependent DNA polymerase activity, and a DNA-dependent DNA polymerase activity and capable of strand displacement, with a set of primers. The primers include a primer such as A capable of hybridizing with a segment complementary to a portion of the sequence to be amplified, wherein A includes an upstream RNA polymerase promoter sequence followed by an arbitrary sequence, a primer such as D containing said RNA polymerase promoter sequence, and primer such as C containing said arbitrary sequence. The elongation product of D is displaced by the elongation product of C, and the elongation product of D is thus obtained in the form of single-stranded VIII. Hybridization of A on VIII followed by elongation forms double stranded product X whiuch can undergo transcription, with amplification, to give single-stranded RNA VII bis. An analogous system of 3 primers B, E and F similarly produces single-stranded DNA VII from VII bis. Hence, the amplification method, which can operate isothermally, is cyclic.

Claims

1. Method of amplifying a sequence of a target nucleic acid, said sequence comprising, from its 5'"'"' end, in the 5'"'"'-3'"'"' direction, an upstream sequence having at least 5 nucleotides and from its 3'"'"' end in the 3'"'"'-5'"'"' direction, a downstream sequence having at least 5 nucleotides,

said method comprising the steps of:

obtaining a polynucleotide comprising a first segment corresponding to the sequence to be amplified and at least a second segment comprising the sense sequence of a first RNA polymerase promoter or the antisense sequence of a second RNA polymerase promoter or at least a portion of said sense or antisense sequence, wherein such a second segment comprising said sense sequence of the first promoter or portion thereof is situated upstream of the 5'"'"' end of said first segment, and such a second segment comprising said antisense sequence of the second promoter or portion thereof is situated downstream of the 3'"'"' end of said first segment, and

bringing said polynucleotide into contact with an excess amount of a set of primers, in the presence of a system having RNA polymerase activity, RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity and strand displacement activity, under conditions allowing the function of the activities, and in the presence of an excess amount of deoxyribonucleoside triphosphates and ribonucleoside triphosphates, such that said sequence of the target nucleic acid is amplified, said set of primers comprising;

a) at least one primer selected from the group consisting of;

1) a first primer comprising successively from its 5'"'"' end towards its 3'"'"' end;

a first optional polynucleotide segment of an arbitrary sequence comprising at least 5 nucleotides,

a second segment comprising at least a portion of the sense sequence of said first RNA polymerase promoter including its 3'"'"'-terminal portion,

and a third segment having the same length as said upstream sequence and being either homologous to said upstream sequence, or capable of hybridizing with said upstream sequence, and

2) a second primer comprising successively, from its 5'"'"' end towards its 3'"'"' end;

a first optional segment of an arbitrary sequence comprising at least 5 nucleotides,

a second segment comprising at least a portion of the sense sequence of said second RNA polymerase promoter including its 3'"'"'-terminal portion,

and a third segment having the same length as said downstream sequence and being either homologous to said downstream sequence, or capable of hybridizing with said downstream sequence,

wherein, when said first and second primers are both present, the third segment of one of the first and second primers is homologous to one of the upstream or downstream sequences of said sequence to be amplified and the third segment of the other primer is capable of hybridizing with the other downstream or upstream sequence, and

b) at least one primer selected from the group consisting of;

1) a third primer comprising at its 3'"'"'-end;

either a sequence containing a segment that is homologous to all or part of the second segment of the first primer and containing the 5'"'"'-end of said sense sequence of said first RNA polymerase promoter,

or a sequence homologous to a portion of the first segment of the first primer but not comprising its 5'"'"' end,

2) a fourth primer comprising at its 3'"'"'-end;

either a sequence containing a segment that is homologous to all or part of the second segment of the second primer and containing the 5'"'"'-end of said sense sequence of said second RNA polymerase promoter,

or a sequence homologous to a portion of the first segment of the second primer but not comprising its 5'"'"'-end,

3) a fifth primer comprising at its 3'"'"'-end;

either a sequence homologous to a portion of the third primer, said portion not comprising the 3'"'"'-terminal nucleotide of said third primer, or a sequence homologous to at least a portion of said first segment of the first primer,

wherein, when said third and fifth primers are both present, the 3'"'"'-end of the third primer hybridizes downstream of the 3'"'"'-end of the fifth primer, and

4) a sixth primer comprising at its 3'"'"'-end;

either a sequence homologous to a portion of the fourth primer, said portion not comprising the 3'"'"'-terminal nucleotide of the fourth primer,

or a sequence homologous to at least a portion of said first segment of the second primer,

wherein, when said fourth and sixth primers are both present, the 3'"'"'-end of the fourth primer hybridizes downstream of the 3'"'"'-end of the sixth primer.

2. Method according to claim 1, wherein said polynucleotide further comprises at least one third segment selected from the group consisting of:

a) upstream of the 5'"'"' end of a said second segment comprising the sense sequence of said first promoter or portion thereof, a segment homologous to the first segment of either the first primer or the second primer, and

b) downstream of the 3'"'"' end of a said second segment comprising the antisense sequence of said second promoter or portion thereof, a segment capable of hybridizing with the first segment of either the first primer or the second primer.

3. Method according to claim 1, characterized by the fact that when the starting nucleic acid is in the form of a double strand, it is subjected to a preliminary denaturing operation.

5. Amplification method according to claim 1, wherein said set of primers comprises said first primer and said second primer.

9. Method according to claim 1, wherein said first RNA polymerase promoter is the same as said second RNA polymerase promoter.

4. Set of primers for amplifying a sequence of a target nucleic acid, comprising:

a) at least one primer selected from the group consisting of;

1) a primer A1 comprising successively, from its 5'"'"' end towards its 3'"'"' end;

a first optional polynucleotide segment of an arbitrary sequence comprising at least 5 nucleotides,

a second segment comprising at least a portion of the sense sequence of a first RNA polymerase promoter including its 3'"'"'-terminal end,

and a third segment capable of hybridizing with a target sequence, and

2) a primer B1 comprising successively, from its 5'"'"' end towards its 3'"'"' end;

a first optional polynucleotide segment of an arbitrary sequence comprising at least 5 nucleotides,

a second segment comprising at least a portion of the sense sequence of a second RNA polymerase promoter including its 3'"'"'-terminal end,

and a third segment capable of hybridizing with a nucleotide sequence complementary to a sequence of the target situated, on the target, upstream of the sequence with which primer Al is capable of hybridizing; and

b) at least one primer selected from the group consisting of;

1) a primer A2 comprising at its 3'"'"'-end;

either a sequence homologous to all or part of the second segment of the primer A1, and comprising the 5'"'"' end of the sense sequence of said RNA polymerase promoter,

or a sequence homologous to a portion of the first segment of said primer A1 or B1, but not comprising its 5'"'"' portion,

2) a primer A3 comprising at its 3'"'"'-end;

either a sequence homologous to a portion of the primer A2, not comprising the 3'"'"'-terminal nucleotide of A2,

or a sequence homologous to at least a portion of the first segment of primer A1,

wherein, when said primers A1 and A2 are both present, the 3'"'"'-end of the primer Al hybridizes downstream of the 3'"'"'-end of the primer A2,

3) a primer B2 comprising at its 3'"'"'-end;

either a sequence homologous to all or part of the second segment of the primer B1, and comprising the 5'"'"' end of the sense sequence of said RNA polymerase promoter,

or a sequence homologous to a portion of the first segment of said primer A1 or B1, but not comprising its 5'"'"' portion, and

4) a primer B3 comprising at its 3'"'"'-end;

either a sequence homologous to a portion of the primer B2, not comprising the 3'"'"'-terminal nucleotide of B2,

or a sequence homologous to at least a portion of the first segment of primer B1,

wherein, when said primers B1 and B2 are both present, the 3'"'"'-end of the primer B1 hybridizes downstream of the 3'"'"'-end of the primer B2.

6. The set of primer of claim 4, further comprising at least one of a seventh primer capable of hybridizing with a region of the target nucleic acid downstream of the sequence to be amplified and an eighth primer capable of hybridizing with a sequence complementary to a region of the target nucleic acid upstream of the sequence to be amplified.

10. Set of primers according to claim 4, wherein said first RNA polymerase promoter is the same as said second RNA polymerase promoter.

7. Method of amplifying a sequence of a target nucleic acid, said sequence comprising, from its 5'"'"' end, in the 5'"'"'-3'"'"' direction, an upstream sequence having at least 5 nucleotides and from its 3'"'"' end in the 3'"'"'-5'"'"' direction, a downstream sequence having at least 5 nucleotides, and said target nucleic acid comprising the sequence to be amplified and extending, beyond the 3'"'"' end of said sequence to be amplified, via a downstream region, and beyond the 5'"'"' end of said sequence to be amplified, via an upstream region,

said method comprising bringing said target nucleic acid into contact with an excess amount of a set of primers, in the presence of a system having RNA polymerase activity, RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity and strand displacement activity, under conditions allowing the function of the activities, and in the presence of an excess amount of deoxyribonucleoside triphosphates and ribonucleoside triphosphates, such that said sequence of the target nucleic acid is amplified, said set of primers comprising:

a) at least one primer selected from the group consisting of;

1) a first primer comprising successively from its 5'"'"' end towards its 3'"'"' end;

a first optional polynucleotide segment of an arbitrary sequence comprising at least 5 nucleotides,

a second segment comprising at least a portion of the sense sequence of a first RNA polymerase promoter including its 3'"'"'-terminal portion,

and a third segment having the same length as said upstream sequence and being either homologous to said upstream sequence, or capable of hybridizing with said upstream sequence, and

2) a second primer comprising successively, from its 5'"'"' end towards its 3'"'"' end;

a first optional segment of an arbitrary sequence comprising at least 5 nucleotides,

a second segment comprising at least a portion of the sense sequence of a second RNA polymerase promoter including its 3'"'"'-terminal portion,

and a third segment having the same length as said downstream sequence and being either homologous to said downstream sequence, or capable of hybridizing with said downstream sequence,

wherein, when said first and second primers are both present, the third segment of one of the first and second primers is homologous to one of the upstream or downstream sequences of said sequence to be amplified and the third segment of the other primer is capable of hybridizing with the other downstream or upstream sequence,

b) at least one primer selected from the group consisting of;

1) a third primer comprising at its 3'"'"'-end;

either a sequence containing a segment that is homologous to all or part of the second segment of the first primer and containing the 5'"'"'-end of said sense sequence of said first RNA polymerase promoter,

or a sequence homologous to a portion of the first segment of the first primer but not comprising its 5'"'"' end,

2) a fourth primer comprising at its 3'"'"'-end;

either a sequence containing a segment that is homologous to all or part of the second segment of the second primer and containing the 5'"'"'-end of said sense sequence of said second RNA polymerase promoter,

or a sequence homologous to a portion of the first segment of the second primer but not comprising its 5'"'"' end,

3) a fifth primer comprising at its 3'"'"'-end;

either a sequence homologous to a portion of the third primer, said portion not comprising the 3'"'"'-terminal nucleotide of said third primer,

or a sequence homologous to at least a portion of said first segment of the first primer,

wherein, when said third and fifth primers are both present, the 3'"'"'-end of the third primer hybridizes downstream of the 3'"'"'-end of the fifth primer, and

4) a sixth primer comprising at its 3'"'"'-end;

either a sequence homologous to a portion of the fourth primer, said portion not comprising the 3'"'"'-terminal nucleotide of the fourth primer,

or a sequence homologous to at least a portion of said first segment of the second primer,

wherein, when said fourth and sixth primers are both present, the 3'"'"'-end of the fourth primer hybridizes downstream of the 3'"'"'-end of the sixth primer, and

c) at least one primer selected from the group consisting of;

1) a seventh primer capable of hybridizing with said downstream region of the target nucleic acid, and

2) an eighth primer capable of hybridizing with a sequence complementary to said upstream region of the target nucleic acid.

8. Amplification method according to claim 7, wherein said set of primers comprises said first primer and said second primer.

11. Method according to claim 7, wherein said first RNA polymerase promoter is the same as said second RNA polymerase promoter.