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, comprising:
a) generating a plurality of cDNA target sequences by a performing reverse transcription reaction on a plurality of RNA; b) hybridizing the plurality of cDNA target sequences with a plurality of first linear primers which contain (i) a terminal 3′ region having a first random sequence and (ii) a 5′ region having a first universal sequence,
wherein the first random sequences of the terminal 3′ region include inosine, adenosine, guanosine, and cytidine, and the first random sequences exclude thymidine,wherein the plurality of the first linear primers include different random sequences,wherein the random sequences hybridize to a sequence of the plurality of cDNA target sequences, andwherein the plurality of the first linear primers include the same universal sequence which does not hybridize to the plurality of cDNA target sequences; and c) forming a plurality of extended linear primer products by extending the first linear primers that are hybridized to the plurality of cDNA target sequences; d) hybridizing the plurality of the extended linear primer products to a plurality of second linear primers which contain (i) a terminal 3′ region having a second random sequence and (ii) a 5′ region having a second universal sequence,
wherein the second random sequences of the terminal 3′ region include inosine, adenosine, guanosine, and cytidine, and the second random sequences exclude thymidine,wherein the plurality of the second linear primers include different random sequences, andwherein the random sequences hybridize to a portion of the extended linear primer products,wherein the plurality of the second linear primers include the same universal sequence as the universal sequence in the first linear primers; and e) forming a plurality of double-extended linear primer products by extending the plurality of second linear primers that are hybridized to the plurality of the extended linear primer products.