CPC C12Q 1/6823 (2013.01) [C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/682 (2013.01); C12N 2310/20 (2017.05); C12N 2310/315 (2013.01); C12N 2310/321 (2013.01); C12N 2310/3231 (2013.01); C12N 2320/10 (2013.01)] | 25 Claims |
1. A composition of matter comprising:
a ribonucleoprotein complex (RNP) comprising a nucleic acid-guided nuclease and a gRNA complementary to synthesized activating molecules;
a plurality of template molecules comprising a sequence having homology to the gRNA and a primer binding domain;
a plurality of blocked primer molecules comprising a sequence complementary to the primer binding domain of the plurality of template molecules and having a Formula IV, wherein Formula IV in the 5′-to-3′ direction comprises:
T-D-M-A-Lp-C;
wherein T is 17-31 nucleotides in length;
D is 0-15 nucleotides in length;
M is 1-25 nucleotides in length;
A is 0-15 nucleotides in length and comprises a sequence complementary to D; and
L is 3-25 nucleotides in length;
p is 0 or 1;
C is 4-15 nucleotides in length and comprises a sequence complementary to T; and
a polymerase and dNTPs;
wherein when blocked, the blocked primer molecules cannot bind to the template molecules and be extended by the polymerase and wherein when unblocked, the blocked primer molecules can bind to the template molecules and be extended by the polymerase to produce synthesized activating molecules.
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