Primers and methods for the detection and discrimination of nucleic acids

• US 10,041,117 B2
• Filed: 07/11/2014
• Issued: 08/07/2018
• Est. Priority Date: 10/23/2001
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Nazarenko, Irina, Rashtchian, Ayoub, Solus, Joseph, Pires, Richard M., Darfler, Marlene, Gebeyehu, Gulilat, Astatke, Mekbib
• Primary Examiner(s): Thomas, David C
• Application Number:
  US14/329,773
• Publication Number:
  US 20150037792A1
• Time in Patent Office: 1,488 Days
• Field of Search: None
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence

Abstract

The present invention provides novel primers and methods for the detection of specific nucleic acid sequences. The primers and methods of the invention are useful in a wide variety of molecular biology applications and are particularly useful in allele specific PCR.

Claims

1. A composition comprising one or more target nucleic acid molecules and at least two different oligonucleotides, wherein said oligonucleotides are extendable by nucleic acid synthesis and wherein said oligonucleotides, when extended by nucleic acid synthesis, are incorporated into a double-stranded nucleic acid product on opposite strands, wherein said first oligonucleotide comprises a fluorescent moiety at or near its 3′

-terminal nucleotide and said second oligonucleotide comprises a quenching moiety at or near its 3′

-terminal nucleotide; and

wherein said first oligonucleotide exhibits a detectable change upon incorporation into said double-stranded nucleic acid product.

2. The composition of claim 1, further comprising at least one component selected from the group consisting of one or more nucleotides, one or more DNA polymerases and one or more reverse transcriptases.

4. The composition of claim 1, wherein said fluorescent moiety and said quencher moiety form a FRET pair.

5. The composition of claim 1, wherein said fluorescent moiety is selected from the group consisting of a fluorescein phosphoramidite (FAM) moiety, a 6-carboxyfluorescein succinimidyl ester (6-FAM, SE) moiety, a fluorescein-5-isothiocyanate (FITC) moiety, a 5-(6-)-carboxytetramethyl rhodamine (TAMRA) moiety, a succinimidyl ester moiety, and a 4,4-difluoro-5, 7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY 530/550) moiety.

6. The composition of claim 1, wherein said target nucleic acid molecule is a cDNA molecule.

10. The composition of claim 1, wherein said target nucleic acid is an RNA target.

11. The composition of claim 1, wherein said first and second oligonucleotides are linear.

12. The composition of claim 1, wherein said first and/or second oligonucleotides further comprise a specificity enhancing group.

13. The composition of claim 12, wherein said specificity enhancing group comprises a 2′

-O-alkyl ribonucleotide.

3. A method for amplifying a double-stranded nucleic acid molecule, comprising:

providing a first and second primer, wherein said first primer is complementary to a portion of the first strand of said nucleic molecule and said second primer is complementary to a portion of the second strand of said nucleic acid molecule;

hybridizing said first primer to said first strand and said second primer to said second strand in the presence of one or more polymerases, under conditions such that a third nucleic acid molecule complementary to all or a portion of said first strand and a fourth nucleic acid molecule complementary to all or a portion said second strand are synthesized;

wherein said first and second primers are incorporated into said third and said fourth nucleic acid molecules, respectively;

denaturing said first and third strand, and said second and fourth strands; and

repeating the above steps one or more times, wherein said first primer comprises a fluorescent moiety at or near its 3′

-terminal nucleotide and said second primer comprises a quencher moiety at or near its 3′

-terminal nucleotide, wherein said fluorescent moiety exhibits a detectable change upon incorporation of said first primer into said third nucleic acid molecules.

7. The method of claim 3, wherein said fluorescent moiety and said quencher moiety form a FRET pair.

8. The method of claim 3, wherein said fluorescent moiety is selected from the group consisting of a fluorescein phosphoramidite (FAM) moiety, a 6-carboxyfluorescein succinimidyl ester (6-FAM, SE) moiety, a fluorescein-5-isothiocyanate (FITC) moiety, a 5-(6-)-carboxytetramethyl rhodamine (TAMRA) moiety, a succinimidyl ester moiety, and a 4,4-difluoro-5, 7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY 530/550) moiety.

9. The method of claim 3, wherein said double-stranded nucleic acid molecule is a DNA/RNA molecule.

14. The composition of claim 3, wherein said first and second primers are linear.

15. The composition of claim 3, wherein said first and/or second primers further comprise a specificity enhancing group.

16. The composition of claim 15, wherein said specificity enhancing group comprises a 2′

-O-alkyl ribonucleotide.