Methods of detecting analytes and compositions thereof

Abstract

The invention relates to methods of detecting analytes in samples by generating analyte-based DNA libraries amenable for sequencing. The methods include the use of proximity probe pairs, each probe comprising an analyte binding domain and oligonucleotide domain. The methods further provide for integrated DNA and RNA library preparations and methods of making and uses thereof. The invention also provides compositions useful in the methods.

Description

[0001] Background of the invention

[0002] Next-generation sequencing (NGS) technology has been used in nucleic acid analysis, for example in DNA variant detection and RNA transcriptome profiling. Equally important to DNA / RNA are protein biomarkers in translational research. However, most protein analyzes are performed on entirely different platforms. For example, protein analysis can be accomplished by traditional ELISA analysis or mass spectrometry. Being able to analyze nucleic acid and protein biomarkers on the same platform will significantly reduce analysis time and provide more insights.

[0003] Protein detection has been successfully converted to nucleic acid detection through the use of oligonucleotide-conjugated antibodies (Abs). Immuno-PCR is one such technique described decades ago (Sano, T. et al., Science 258:120-2 (1992)). In this case, antigen-specific Abs are conjugated to oligonucleotide sequences and used in a typical ELISA procedure. Although many ELIS...

Examples

Embodiment 1

[0159] The following is an illustrative example showing how the methods described herein can be used for protein analysis. In total, 96 pairs of probes were designed to detect 96 different protein targets. Four of these are controls for data normalization purposes. Control 1 and Control 2 were used for exogenous protein targets that were not in the test sample. The 5' ends of all oligonucleotides were conjugated to their respective antibodies. Control 3 is an extension control in which both oligo A and oligo B are conjugated to the same antibody, so extension is not related to antigen binding. Control 4 is an assay control used to monitor changes in PCR amplification, where the intact full-length oligonucleotide was incorporated directly into the reaction.

Embodiment 2

[0161] sample:

[0162] 1. Human serum samples

[0163] 2. Human serum sample + protein targets #1 and #2 spiked at 5ng / ml

[0164] 3. PBS (negative control)

[0165] Antibody Binding:

[0166]

[0167] Incubate overnight (16 hr) at 4°C.

[0168] extend:

[0169]

[0170] Incubate at 50°C for 20 min → 95°C for 5 min → 17 cycles (95°C for 30 sec → 54°C for 1 min → 60°C for 1 min) → hold at 4°C in a thermal cycler with a heating lid.

[0171] Library amplification:

[0172]

[0173] Incubate in a thermal cycler with a heated lid at 95 °C for 13 min → 98 °C for 2 min → 20 cycles (98 °C for 15 sec → 60 °C for 2 min) → 72 °C for 5 min → 4 °C hold.

[0174] Purification: 75uL of ice-cold water was added to each of the 25uL samples from the previous step for a total of 100uL. One round of 1.2x Ampure XP bead purification (eluted in 20 uL of water) was performed.

[0175] Library quantification using Agilent Bioanalyzer High Sensitivity DNA chip: Dilute the purified lib...

Embodiment 3

[0177] Starting material: purified genomic DNA and total RNA. For example, 50ng gDNA and 50ng total RNA were purified from THP-1 cell line. Ideally, the relative amounts of gDNA and RNA should be representative of the amount in the sample.

[0178] DNA / RNA Fragmentation:

[0179]

[0180] RNA polyadenylation

[0181]

[0182]

[0183] DNA connection:

[0184]

[0185] Purification: Add 50uL of ice-cold water to 50uL of the sample from the previous step, making a total of 100uL. 2 rounds of 1.2×Ampure XP bead purification were performed following the manufacturer's manual with the following exceptions: the first round was eluted in 52 uL of water; and the second round was eluted in 13 uL of water.

[0186] reverse transcription:

[0187]

[0188]

[0189] Purification: 75uL of ice-cold water was added to 25uL of the sample from the previous step for a total of 100uL. Follow the manufacturer's manual for 1 round of 1.2x Ampure XP bead purification and elu...

[0001] Background of the invention

[0002] Next-generation sequencing (NGS) technology has been used in nucleic acid analysis, for example in DNA variant detection and RNA transcriptome profiling. Equally important to DNA / RNA are protein biomarkers in translational research. However, most protein analyzes are performed on entirely different platforms. For example, protein analysis can be accomplished by traditional ELISA analysis or mass spectrometry. Being able to analyze nucleic acid and protein biomarkers on the same platform will significantly reduce analysis time and provide more insights.

[0003] Protein detection has been successfully converted to nucleic acid detection through the use of oligonucleotide-conjugated antibodies (Abs). Immuno-PCR is one such technique described decades ago (Sano, T. et al., Science 258:120-2 (1992)). In this case, antigen-specific Abs are conjugated to oligonucleotide sequences and used in a typical ELISA procedure. Although many ELIS...

Examples

Embodiment 1

[0159] The following is an illustrative example showing how the methods described herein can be used for protein analysis. In total, 96 pairs of probes were designed to detect 96 different protein targets. Four of these are controls for data normalization purposes. Control 1 and Control 2 were used for exogenous protein targets that were not in the test sample. The 5' ends of all oligonucleotides were conjugated to their respective antibodies. Control 3 is an extension control in which both oligo A and oligo B are conjugated to the same antibody, so extension is not related to antigen binding. Control 4 is an assay control used to monitor changes in PCR amplification, where the intact full-length oligonucleotide was incorporated directly into the reaction.

Embodiment 2

[0161] sample:

[0162] 1. Human serum samples

[0163] 2. Human serum sample + protein targets #1 and #2 spiked at 5ng / ml

[0164] 3. PBS (negative control)

[0165] Antibody Binding:

[0166]

[0167] Incubate overnight (16 hr) at 4°C.

[0168] extend:

[0169]

[0170] Incubate at 50°C for 20 min → 95°C for 5 min → 17 cycles (95°C for 30 sec → 54°C for 1 min → 60°C for 1 min) → hold at 4°C in a thermal cycler with a heating lid.

[0171] Library amplification:

[0172]

[0173] Incubate in a thermal cycler with a heated lid at 95 °C for 13 min → 98 °C for 2 min → 20 cycles (98 °C for 15 sec → 60 °C for 2 min) → 72 °C for 5 min → 4 °C hold.

[0174] Purification: 75uL of ice-cold water was added to each of the 25uL samples from the previous step for a total of 100uL. One round of 1.2x Ampure XP bead purification (eluted in 20 uL of water) was performed.

[0175] Library quantification using Agilent Bioanalyzer High Sensitivity DNA chip: Dilute the purified lib...

Embodiment 3

[0177] Starting material: purified genomic DNA and total RNA. For example, 50ng gDNA and 50ng total RNA were purified from THP-1 cell line. Ideally, the relative amounts of gDNA and RNA should be representative of the amount in the sample.

[0178] DNA / RNA Fragmentation:

[0179]

[0180] RNA polyadenylation

[0181]

[0182]

[0183] DNA connection:

[0184]

[0185] Purification: Add 50uL of ice-cold water to 50uL of the sample from the previous step, making a total of 100uL. 2 rounds of 1.2×Ampure XP bead purification were performed following the manufacturer's manual with the following exceptions: the first round was eluted in 52 uL of water; and the second round was eluted in 13 uL of water.

[0186] reverse transcription:

[0187]

[0188]

[0189] Purification: 75uL of ice-cold water was added to 25uL of the sample from the previous step for a total of 100uL. Follow the manufacturer's manual for 1 round of 1.2x Ampure XP bead purification and elu...

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