Chemically modified nucleoside 5′-triphosphates for thermally initiated amplification of nucleic acid

Inventors

Lebedev, Alexandre • Koukhareva, Inna

Assignees

Trilink Biotechnologies LLC

Abstract

Provided herein are methods and compositions for nucleic acid replication. These methods involve the use of 3′-substituted nucleoside 5′-triphosphates or 3′-substituted terminated primers in nucleic acid replication reactions. In certain aspects, the methods are accomplished by use of 3′-substituted NTPs and/or 3′-substituted terminated primers which provide utility in nucleic acid replication. In preferred embodiments, the NTPs and/or primers are substituted at the 3′-position with particular heat labile chemical groups such as ethers, esters or carbonate esters.

Core Innovation

The invention relates to replicating nucleic acids using at least one NTP that comprises a thermally labile 3′-substitution group. The replication reaction mixture does not contain chemical reagents for removal of the thermally labile 3′-substitution group, enabling the 3′-substituted NTPs to remain in the mixture during replication.

The invention relates to hot-start nucleic acid amplification and replication using 3′-substituted nucleoside 5′-triphosphates (NTPs) or 3′-substituted terminating primers comprising thermally labile 3′-protecting groups. The 3′-substitution renders the NTPs non-substrate or terminating prior to initial denaturation, thereby preventing extension and reducing mis-priming and primer dimer artifacts.

Upon heating, the thermally labile 3′-protecting group converts and/or dissociates to provide an open 3′-OH, providing an active form for replication after the initial denaturation step without the need for chemical reagents for removal of the thermally labile 3′-substitution group. The disclosure includes broad chemical formulas and structural embodiments for thermally labile 3′-substituted NTP analogs and terminated primer constructs.

Claims Coverage

The independent claims cover nucleic-acid replication or amplification methods that use at least one NTP bearing a thermally labile 3′-substitution group in a replication reaction mixture that does not contain chemical reagents for removal of that group. The claim coverage includes four inventive features centered on the thermally labile 3′-substituted NTPs, exclusion of removal reagents, and stated improvements in specificity or efficiency.

The claim set centers on performing nucleic-acid replication or amplification with NTPs bearing thermally labile 3′-substitution groups while excluding chemical reagents that remove those groups. The claims further frame the same constraint pattern as improving specificity or improving efficiency, and the broader disclosure also mentions conversion to an open 3′-hydroxyl, limited fractions of 3′-substituted NTPs, multiple substitution groups, and specified PCR, amplification, sequencing, and reverse-transcription workflows.

Stated Advantages

Documented Applications