PRIMERS AND METHODS FOR THE DETECTION AND DISCRIMINATION OF NUCLEIC ACIDS

• US 20100233699A1
• Filed: 12/01/2009
• Published: 09/16/2010
• 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: Solus, Joseph, Darfler, Marlene, Pires, Richard M., Nazarenko, Irina, Gebeyehu, Gulilat, Rashtchian, Ayoub, Astatke, Mekbib
• Granted Patent: US 8,394,609 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

1-74. -74. (canceled)

75. A method of distinguishing a single nucleotide polymorphism (SNP) in a target nucleic acid, comprising:

combining said target nucleic acid with a first internally labeled universal primer, a first allele specific primer, a second internally labeled universal primer, a second allele specific primer, and a common reverse or forward primer which hybridizes to said target nucleic acid, wherein said first and second labels are different, and wherein said first allele specific primer has a 5′

tail which hybridizes to said first universal primer and a terminal 3′

nucleotide that is specific to a first allele, and said second allele specific primer has a 5′

tail which hybridizes to said second universal primer and a terminal 3′

nucleotide that is specific to a second allele, under conditions which allow extension of said allele specific primers; and

detecting said first and said second labels, wherein the presence of said first label indicates the presence of said first allele, the presence of said second label indicates the presence of said second allele, and the presence of both labels indicates the presence of both alleles.

76. The method of claim 75, wherein said labels are fluorescent.

77. The method of claim 76, wherein one of said universal primers is labeled with 5-carboxyfluorescein (FAM), and one of said universal primers is labeled with 2′

7′

-dimethoxy-4′

5′

-dichloro-6-carboxyfluorescein (JOE).

78. A method of detecting an allele in a target nucleic acid, comprising:

contacting a target nucleic acid with an internally labeled universal primer, an allele specific primer, and a common reverse or forward primer which hybridizes to said target nucleic acid, wherein said allele specific primer has a 5′

tail which hybridizes to said universal primer and a terminal 3′

nucleotide that is specific to said allele, under conditions which allow extension of said allele specific primer; and

detecting said label, wherein the presence of said label indicates the presence of said allele.

79. The method of claim 78, wherein said label is fluorescent.

80. The method of claim 79, wherein said label is 5-carboxyfluorescein (FAM).

81. The method of claim 79, wherein said label is 2′

7′

-dimethoxy-4′

5′

-dichloro-6-carboxyfluorescein (JOE).

82. The method of claim 79, wherein said label is 6-carboxy-X-rhodamine (ROX).