SAMPLE PREPARATION METHODS, SYSTEMS AND COMPOSITIONS
The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.
1 - 114 . (canceled)
115 . A reaction mixture comprising:
(i) a DNA-dependent polymerase;
(ii) an RNA-selective polymerase
(iii) a cell-free DNA, and
(iv) a free RNA.
116 . The reaction mixture of claim 115 , wherein the DNA-dependent polymerase is selected from a member of the group consisting of: an A-family DNA polymerase, a B-family DNA polymerase, a KOD XL, KOD (exo-), a Bst 2.0, a (New) The rminator, a Deep Vent (exo-), a Pfu DNA polymerase, a Taq, and any combination thereof
117 . The reaction mixture of claim 115 , wherein the RNA-selective polymerase comprises a reverse transcriptase enzyme (RT), an RNA-dependent RNA polymerase, or a combination thereof
118 . The reaction mixture of claim 115 , wherein the DNA-dependent polymerase has non-templated activity.
119 . The reaction mixture of claim 115 , wherein the DNA-dependent polymerase is capable of an addition of a non-templated base to an end of a nascent growing strand of a duplex.
120 . The reaction mixture of claim 119 , wherein the addition of the non-templated base to the end of the nascent growing strand of the duplex occurs at a 3′ end.
121 . The reaction mixture of claim 119 , wherein the non-templated base is N1, wherein N1 is a different nucleic acid base that identifies a first nucleic acid form.
122 . The reaction mixture of claim 121 , wherein the first nucleic acid form comprises DNA, wherein the DNA is single-stranded, double-stranded, or a combination thereof
123 . The reaction mixture of claim 115 , wherein the RNA-selective polymerase has non-templated activity.
124 . The reaction mixture of claim 115 , wherein the RNA-selective polymerase is capable of an addition of a non-templated base to an end of a nascent growing strand of a duplex.
125 . The reaction mixture of claim 124 , wherein the addition of the non-templated base to the end of the nascent growing strand of the duplex occurs at a 3′ end.
126 . The reaction mixture of claim 124 , wherein the non-templated base is N2, wherein N2 is a different nucleic acid base that identifies a second nucleic acid form.
127 . The reaction mixture of claim 125 , wherein the second nucleic acid form comprises RNA, wherein the RNA is single-stranded, double-stranded, or a combination thereof.
128 . The reaction mixture of claim 115 , wherein the cfDNA and the free RNA are single-stranded, double-stranded, or a combination thereof
129 . The reaction mixture of claim 115 , wherein the cell-free DNA (cfDNA) and the free RNA are derived from a sample from a subject.
130 . The reaction mixture of claim 128 , wherein the cfDNA and the free RNA are derived from a same sample from a same subject.
131 . The reaction mixture of claim 129 , wherein the same sample from the same subject comprises a low-quality sample.
132 . The reaction mixture of claim 115 , wherein the cfDNA and free RNA comprise cfDNA, free RNA, or a combination thereof, are derived from a pathogen.
133 . The reaction mixture of claim 132 , wherein the pathogen comprises a virus, a bacterium, a fungus, a parasite, or any combination thereof
134 . The reaction mixture of claim 115 , wherein the reaction mixture is comprised in a container or a tube.
135 . The reaction mixture of claim 134 , wherein the container or the tube comprise a single PCR tube or a single reaction tube.
136 . The reaction mixture of claim 115 , further comprising a ligase, wherein the ligase comprises a DNA ligase, an RNA ligase, or a combination thereof.
137 . A kit comprising:
i) a DNA-dependent polymerase;
ii) an RNA-selective polymerase;
iii) a cell-free DNA; and
iv) a free RNA.