UNIVERSAL TAGS WITH NON-NATURAL NUCLEOBASES

摘要

The present invention relates to amplification primers with a universal tag and sequencing primers comprising at least one non-natural nucleobase capable of hybridizing to a complementary non-natural nucleobase. The present invention further relates to amplification methods of nucleic acid amplification and sequencing using an amplification primer with a universal tag and sequencing primers, as well as kits and solid supports comprising such primers and tags.

主权项

1. A kit for sequencing a polynucleotide target, comprising:
(a) an amplification primer comprising:
(i) a 3′ target-specific nucleotide sequence; and(ii) a 5′ non-target-specific universal sequencing tag comprising at least one natural nucleobase that is not target specific and at least three contiguous non-natural nucleobases, each capable of complementary binding to a corresponding non-natural nucleobase, wherein the at least three contiguous non-natural nucleobases is are capable of being replicated during amplification, wherein the amplification primer is utilized with a target sequence and an amplification polymerase to produce an amplified polynucleotide sequencing template, and wherein when the amplified polynucleotide sequencing template is exposed to a sequencing polymerase, the at least three contiguous non-natural nucleobases present in the amplified polynucleotide sequencing template are not capable of being sequenced and cause termination of the primer extension by the sequencing polymerase, whereby the portion of (ii) that is 5′ to the at least three contiguous non-natural nucleobases cannot be sequenced; and (b) a universal sequencing primer comprising an oligonucleotide of the sequence comprising the 5′ non-target-specific universal sequencing tag.