Phosphotriester-modified oligonucleotide primers for nucleic acid amplification
Inventors
Zon, Gerald • Lebedev, Alexandre
Assignees
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Publication Number
US-8361753-B2
Publication Date
2013-01-29
Expiration Date
Abstract
The present invention provides methods and compositions for nucleic acid amplification. These methods involve the use of oligonucleotide primers in temperature dependent nucleic acid amplification reactions. In certain aspects, the methods are accomplished by use of certain modified oligonucleotide primers which provide utility in nucleic acid amplification. In preferred embodiments, the oligonucleotide primers are modified with particular chemical groups such as esters.
Core Innovation
A method of amplifying nucleic acids uses a modified oligonucleotide primer that includes a modification group at one or two internucleotide linkages. The modification group is positioned at the n, n-1, n-2, or n-3 positions, where n is the 3′ terminal internucleotide linkage, and the presence of the modification group impairs nucleic acid polymerase extension of the modified oligonucleotide primer.
The modification group is configured to be non-reversibly thermally dissociated, so that the modified primer is rendered inactive during early steps and then converted upon thermal treatment. The modified oligonucleotide primer is described as having structure I, where Q is a modification group comprising one or more thermally cleavable groups.
Structure I includes defined relationships for nucleoside content and linkage components within the primer, where Nuc is a nucleoside within the primer sequence. The definitions specify allowed options for U and Z, each selected independently from O, S, Se, NR9, or CR9R10; define R9 and R10 as hydrogen or optionally substituted straight or branched hydrocarbyl; define Y as O, S or Se; and define W as O, S, S(O), S(O)2, Se, C(O), C(S), C(O)NH, NH or NR9.
Claims Coverage
The document includes one independent claim defining a modified primer-based nucleic acid amplification method, with dependent claims that refine the chemical definition of the thermally cleavable modification group Q and add dissociation kinetics, temperature constraints, and PCR-related implementation. The inventive features center on the primer structure and positioning, polymerase extension impairment, non-reversible thermal dissociation, and the structure I chemical framework containing Q.
Modified primer with thermally dissociable modification group
A method of amplifying nucleic acids using a modified oligonucleotide primer, wherein the modified oligonucleotide primer comprises a modification group at one or two internucleotide linkages; the presence of the modification group impairs nucleic acid polymerase extension; the modification group can be non-reversibly thermally dissociated; and the modified oligonucleotide primer has structure I, wherein Q is a modification group comprising one or more thermally cleavable groups.
Modification group at n, n-1, n-2, or n-3 positions
The modification groups are at the n, n-1, n-2, or n-3 positions, where n is the 3′ terminal internucleotide linkage.
Structure I chemical definition including linkage components and Q
The modified oligonucleotide primer has structure I, where Nuc is a nucleoside within the primer sequence; U and Z are independently O, S, Se, NR9, or CR9R10; R9 and R10 are each independently hydrogen or optionally substituted straight or branched hydrocarbyl; Y is O, S or Se; W is O, S, S(O), S(O)2, Se, C(O), C(S), C(O)NH, NH or NR9; and Q is a modification group comprising one or more thermally cleavable groups.
Overall claim coverage centers on amplifying nucleic acids with a modified oligonucleotide primer that positions a thermally dissociable modification group at one or two defined internucleotide linkages near the 3′ end, impairs polymerase extension, and then non-reversibly thermally dissociates via thermally cleavable modification group Q within a defined structure I chemical framework.
Stated Advantages
Improves primer inactivity prior to thermal dissociation by impairing nucleic acid polymerase extension of the modified oligonucleotide primer.
Reduces primer dimer formation and improves amplicon specificity in PCR and RT-PCR.
Documented Applications
Hot-start nucleic acid amplification formats including PCR, RT-PCR, and multiplex PCR.
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