主权项 |
1. A multiplex amplification reaction mixture comprising:
a) a nucleic acid sample having a plurality of different target nucleic acids; and b) a mixture of two or more different loopable primers, each loopable primer including a single-stranded nucleic acid molecule having in a 3′ to 5′ direction
(i) a single-stranded target-specific 3′ region;(ii) a first loop-forming region which forms a first strand of a double-stranded stem region;(iii) a non-complementary region and a universal priming region which forms a single-stranded loop;(iv) a second loop-forming region which forms a second strand of the double-stranded stem region; and(v) a non-complementary single-stranded 5′ tail,wherein the single-stranded target specific 3′ region includes at least one inosine,wherein the first and the second loop-forming regions hybridize to each other to form a double-stranded stem region,wherein the single-stranded loop is located between the first and second loop-forming regions, and the non-complementary region of (iii) contains only adenine residues,wherein the two or more different loopable primers have non-complementary single-stranded 5′ tails containing different identification sequences, andwherein the stem and the loop, when formed, are configured to allow the single-stranded target specific 3′ region to hybridize to a target nucleic acid without the target nucleic acid hybridizing to the double-stranded stem, the non-complementary region of (iii) or the non-complementary 5′ tail. |