Amplification of DNA with a control sequence differing in GC content

• US 6,723,507 B1
• Filed: 03/06/2001
• Issued: 04/20/2004
• Est. Priority Date: 09/07/1998
• Status: Expired due to Fees

• Current Assignee: Secretary of State For Defence
• Original Assignee: Her Majesty The Queen In Right of Canada As Represented By The Secretary of State For Defence
• Inventors: Brightwell, Gale, Lee, Martin A
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/786,446
• Time in Patent Office: 1,141 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

A method for amplifying a target DNA sequence, said method comprising amplifying said sequence in the presence of: a) a nucleic acid polymerase; b) at least one primer capable of hybridising to said target polynucleotide; c) an internal control sequence to which said primer is capable of hybridising and which is of similar length to the target DNA sequence but with a different percentage GC content; and d) label means for detecting the hybridisation of nucleic acids in the reaction; and detecting the hybridisation of the target and control sequences at different temperatures.

Claims

1. A method for amplifying a target DNA sequence, said method comprising

(i) amplifying said sequence in the presence of

(a) a nucleic acid polymerase,

(b) at least one primer capable of hybridizing to said target DNA sequence,

(c) a control sequence to which said primer is capable of hybridizing and which is of a similar length to the target DNA sequence but with a different percentage GC content and

(d) label means for detecting hybridization of one or more of:

1—

the primer to either of the target sequence and control sequence

2—

the target sequence to a complement of the target sequence; and

3—

the control sequence to a complement of the control sequence;

said amplifying comprising at least one hybridizing step and at least one denaturation step; and

(ii) detecting hybridization of the target and control sequences at different temperatures;

wherein the conditions used in the amplification are such that amplification of one of either the target sequence or the control sequence is favoured, said conditions favouring amplification of the sequence which is present in smaller amounts wherein the temperature of the denaturation step of the amplification is controlled such that the sequence which is present in smaller amounts is denatured in preference to the other sequence, and the control sequence is designed such that the temperature of denaturation of the control sequence is lower than the temperature of denaturation of the target sequence when the control sequence is present in smaller amounts, and the temperature of denaturation of the control sequence is greater than the temperature of denaturation of the target sequence when the target sequence is present in smaller amounts.

2. A method according to claim 1 wherein at least a forward primer and a reverse primer are used to amplify both the target nucleotide sequence and the control sequence.

3. A method according to claim 1 or claim 2 wherein the label means comprises a fluorescent label.

4. A method according to claim 3 wherein the label means comprises an intercalating dye.

5. A method according to claim 4 wherein the intercalating dye is SYBRGreen™

, SYBRGold™

or ethidium bromide.

6. A method according to claim 3 wherein the fluorescent label utilises fluorescence resonance transfer (FRET) as the basis of the signal.

7. A method according to claim 6 wherein the control DNA sequence is provided with a reporter and a quencher molecule arranged so that formation of a duplex by the control sequence alters the spatial relationship between the quencher and reporter molecules.

8. A method according to claim 7 wherein the control sequence is a single stranded sequence, where the end portions hybridise together, and in which the reporter molecule is attached in the region of either the 5′

or the 3′

end of the sequence and the quencher molecule is attached at the opposite end.

9. A method according to claim 7 wherein the reporter and quencher molecules are located on opposite strands of a double stranded DNA control sequence and are located such that on hybridisation of the strands, they are brought into close proximity to each other so that FRET can occur.

10. A method according to claim 6 wherein the label means comprises an intercalating dye and quencher molecule, provided on the control sequence, which can absorb radiation from the dye when in the form of a double stranded molecule.

11. A method according to claim 6 in which a reporter molecule is provided on a first oligonucleotide strand and a quencher molecule is provided on a second oligonucleotide strand, said first and second strands being off similar length to the target DNA sequence and having a similar GC content to each other, said first and second strands being hybrisable to a third DNA sequence present in the reaction mixture such that on hybridisation, the reporter and quencher molecules are brought into close proximity to each other.