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 method for replicating a polynucleotide target, comprising the steps of:
hybridizing to a polynucleotide target an amplification primer comprising a 3′ target-specific nucleotide sequence and 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 of which is capable of being replicated during amplification, and extending the amplification primer under PCR conditions, thereby producing a polynucleotide product that is complementary to the polynucleotide target and that further comprises 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, wherein the at least three contiguous non-natural nucleobases present in the polynucleotide product are not capable of being sequenced and cause termination of primer extension by a sequencing polymerase, whereby the portion of the 5′ non-target-specific universal sequencing tag that is 5′ to the at least three contiguous non-natural nucleobases cannot be sequenced.