SEQUENCE AMPLIFICATION WITH LINEAR PRIMERS

摘要

The present disclosure relates to the amplification of target nucleic acid sequences for various sequencing and/or identification techniques. The use of these primers, as described herein, allows for the reduction in the amplification of nonspecific hybridization events (such as primer dimerization) while allowing for the amplification of the target nucleic acid sequences.

主权项

1. A method of amplifying a nucleic acid sequence in parallel sequencing technique, said method comprising:
hybridizing a 3′ target specific region of a first linear primer to a first part of a target nucleic acid sequence, wherein the first linear primer comprises a universal priming region that is distinct from the 3′ target specific region, and wherein the 3′ target specific region is a random region that is less than 6 nucleotides in length; extending the first linear primer that is hybridized to the target nucleic acid sequence to form an extended linear primer; hybridizing a second linear primer to a complementary part of the target nucleic acid sequence of the extended linear primer, wherein the second linear primer comprises a same universal priming region as the first linear primer; extending the second linear primer to form a double-extended linear primer; amplifying the double-extended linear primer using an amplification primer, wherein the amplification primer comprises a sequence that is the same as a sequence of the universal priming region, wherein a double-extended linear primer comprising a first insert section is selectively amplified over a double-extended linear primer comprising a second insert section; adding a third primer that comprises a sequence that is complementary to a sequence within the first insert section; performing an amplification reaction using the third primer to amplify the target nucleic acid sequence within the first insert section of the double-extended linear primer, wherein the double-extended linear primer comprising the first insert has its insert section selectively amplified compared to the double-extended linear primer comprising the second insert, wherein the first insert section is larger than the second insert section; and performing a parallel sequencing reaction on an amplified product from the amplification reaction using the third primer.