Detection Of Analytes And Nucleic Acids
Methods of detecting at least one analyte and at least one nucleic acid in a sample are provided. Reagents for carrying out the methods are also provided.
1 . A method of detecting at least one target analyte and at least one target nucleic acid in a cell, comprising:
a) lysing the cell in a multifunctional lysis buffer to produce a cell lysate;
b) detecting at least one target analyte in the cell lysate using a proximity detection assay; and
c) detecting at least one target nucleic acid in the cell lysate using a quantitative nucleic acid detection assay;
wherein (b) and (c) occur in the same vessel.
2 . The method of claim 1 , wherein at least one target analyte is selected from a protein, a peptide, a carbohydrate, and a hormone.
3 . The method of claim 2 , wherein at least one target analyte is a protein.
4 . The method of claim 1 , wherein the proximity detection assay comprises:
a) incubating the cell lysate with a first proximity detection probe and a second proximity detection probe under conditions allowing interaction between the first and second proximity detection probes; and
b) detecting the interaction between the first and second proximity detection probes.
5 . The method of claim 4 , wherein the first proximity detection probe comprises a first oligonucleotide moiety and a first analyte binding moiety, and wherein the second proximity detection probe comprises a second oligonucleotide moiety and a second analyte binding moiety.
6 . The method of claim 5 , wherein the first analyte binding moiety and the second analyte binding moiety are capable of binding to the same target analyte.
7 . The method of claim 5 , wherein the first analyte binding moiety and the second analyte binding moiety are capable of binding to different target analytes.
8 . The method of claim 5 , wherein the interaction between the first and second proximity detection probes comprises at least one method selected from hybridization between the first and second oligonucleotide moieties and ligation of the first and second oligonucleotide moieties.
9 . The method of claim 8 , wherein the interaction between the first and second proximity detection probes comprises ligation of the first and second oligonucleotide moieties.
10 . The method of claim 4 , wherein the incubating comprises at least one splint oligonucleotide.
11 . The method of claim 1 , wherein the at least one target nucleic acid is at least one mRNA.
12 . The method of claim 11 , wherein the quantitative nucleic acid detection assay comprises reverse-transcription and real-time PCR.
13 . The method of claim 1 , wherein at least one of the at least one target nucleic acids encodes at least one of the at least one target analytes.
14 . The method of claim 1 , wherein the multifunctional lysis buffer comprises at least one chemical selected from NDSB-201, LDAO, CHAPS, DEDTAB, Zwittergent 3-10, and CAPSO.
15 . The method of claim 14 , wherein the multifunctional lysis buffer comprises NDSB-201.
16 . The method of claim 1 , wherein the detecting the at least one target analyte comprises a first real-time PCR reaction and the detecting the at least one target nucleic acid comprises a second real-time PCR reaction.
17 . A method of detecting at least one target analyte and at least one target nucleic acid in a cell, comprising:
a) lysing the cell in a multifunctional lysis buffer to produce a cell lysate;
b) incubating the cell lysate with:
i) at least one proximity detection probe set, wherein each proximity detection probe set comprises at least two proximity detection probes, and wherein each proximity detection probe comprises at least one analyte binding moiety and at least one oligonucleotide moiety;
ii) at least one splint oligonucleotide; and
iii) at least one ligase;
such that at least one ligated proximity detection probe set is formed;
c) incubating the cell lysate with at least one protease;
d) detecting the at least one ligated proximity detection probe set; and
e) detecting at least one target nucleic acid.
18 . The method of claim 17 , wherein at least one target analyte is a protein.
19 . The method of claim 17 , wherein (d) and (e) occur in the same vessel.
20 . A method of detecting at least one target analyte and at least one target nucleic acid in a cell, comprising:
a) lysing the cell in a multifunctional lysis buffer to produce a cell lysate;
b) incubating the cell lysate with at least one proximity detection probe set such that at least one hybridized proximity detection probe set is formed, wherein each proximity detection probe set comprises at least two proximity detection probes, and wherein each proximity detection probe comprises at least one analyte binding moiety and at least one oligonucleotide moiety;
c) incubating the cell lysate with at least one protease;
d) detecting the at least one hybridized proximity detection probe set; and
e) detecting at least one target nucleic acid.
21 . The method of claim 20 , wherein at least one target analyte is a protein.
22 . The method of claim 20 , wherein (d) and (e) occur in the same vessel.
23 . The method of claim 20 , wherein the incubating in (b) comprises at least one splint oligonucleotide.
24 . A multifunctional lysis buffer comprising at least one chemical selected from NDSB-201, LDAO, CHAPS, DEDTAB, Zwittergent 3-10, and CAPSO.
25 . The multifunctional lysis buffer of claim 24 , comprising NDSB-201.
26 . The multifunctional lysis buffer of claim 24 , wherein at least one chemical is present at a concentration of 0.05% to 5%.
27 . A kit for detecting at least one target analyte and at least one target nucleic acid, comprising at least one multifunctional lysis buffer comprising at least one chemical selected from NDSB-201, LDAO, CHAPS, DEDTAB, Zwittergent 3-10, and CAPSO.
28 . The kit of claim 27 , wherein the kit comprises at least one ligase.
29 . The kit of claim 27 , wherein the kit comprises at least one proximity detection probe set.
30 . The kit of claim 27 , wherein the kit comprises at least one protease.
31 . The kit of claim 27 , wherein the kit comprises at least one primer, at least one detector probe, and at least one polymerase.
32 . A composition comprising a lysate, wherein the lysate comprises a multifunctional lysis buffer comprising at least one chemical selected from NDSB-201, LDAO, CHAPS, DEDTAB, Zwittergent 3-10, and CAPSO, and at least one proximity detection probe set.