| CPC C12Q 1/6823 (2013.01) [C12N 9/22 (2013.01); C12N 15/111 (2013.01); C12Q 1/44 (2013.01); C12Q 1/6806 (2013.01); G01N 21/6486 (2013.01); C12N 2310/20 (2017.05)] | 30 Claims |
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1. A method for identifying one or more target nucleic acids of interest from two or more source organisms in a sample comprising the steps of:
obtaining a sample;
lysing the source organisms and fractionating nucleic acids obtained from the lysed source organisms in the sample, wherein the nucleic acids are fractionated into lengths of 50 bp to 100,000 bp;
designing a plurality of first guide nucleic acids (gRNA1s) complementary to a plurality of loci in each genome of the two or more source organisms;
forming first ribonucleoprotein complexes (RNP1s) comprising a first nucleic acid-guided nuclease and the gRNA1s; wherein the first nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity and wherein the RNP1s are formed in partitions where different partitions comprise different gRNA1 sequences;
providing a reaction mixture comprising:
second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acids of interest; wherein the second nucleic acid-guided nuclease exhibits both cis- and trans-cleavage activity; and
a plurality of blocked nucleic acid molecules comprising a sequence corresponding to the second gRNA, wherein a Kd of the blocked nucleic acid molecules binding to the RNP2s is from 100 nM to 100 μM;
contacting the RNP1s in each partition with the reaction mixture and the fractionated nucleic acids in the sample under conditions that allow the target nucleic acids of interest in the sample to bind to RNP1, wherein upon binding of target nucleic acids of interest the RNP1s become active initiating trans-cleavage of at least one of the plurality of blocked nucleic acid molecules thereby producing at least one unblocked nucleic acid molecule, wherein a Kd of the at least one unblocked nucleic acid molecule binding to the RNP2 is from 100 fM to 1 aM and wherein the at least one unblocked nucleic acid molecule binds to RNP2 initiating trans-cleavage of at least one further blocked nucleic acid molecule; and
detecting cleavage products from each partition, thereby detecting the target nucleic acids of interest in the sample.
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