ASSAY FOR SIMULTANEOUS GENOMIC AND PROTEOMIC ANALYSIS

• US 20180208975A1
• Filed: 01/17/2018
• Published: 07/26/2018
• Est. Priority Date: 01/20/2017
• Status: Abandoned Application

• Current Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Original Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Inventors: Peterson, Vanessa Marie, Klappenbach, Joel Albert
• Application Number:
  US15/873,331
• Publication Number:
  US 20180208975A1
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The present invention is directed to a biochemical assay for simultaneous genomic and proteomic analysis.

Claims

1. A method for detecting a plurality of target proteins and mRNA in a cell or cell lysate comprising:

a. contacting a cell or cell lysate with

1) a plurality of protein target probes, wherein each target probe in the plurality comprises;

i. a protein-binding molecule;

ii. a target nucleotide sequence comprising a primer for a polymerase chain reaction (PCR), a protein identification sequence that identifies the protein-binding molecule, a linker sequence that hybridizes to a sequence in the detection probe;

iii. a linker between the protein-binding molecule and the target nucleotide sequence; and

2) a plurality of microparticles comprising a plurality of detection probes comprising;

i. a protein detection nucleotide sequence comprising a primer for a polymerase chain reaction (PCR), a cell identification sequence that identifies the cell, a unique molecular identifier (UMI), and a complimentary linker sequence that hybridizes to the linker sequence in the target probe;

ii. an mRNA detection nucleotide sequence comprising a primer for a polymerase chain reaction (PCR), an cell identification sequence that identifies the cell, a unique molecular identifier, and polydT;

b. allowing the target nucleotide sequence to hybridize to the protein detection nucleotide sequence and the mRNA to hybridize to the mRNA detection nucleotide sequence in the microparticle;

c. conducting reverse transcription and/or polymerase extension to generate

i. a first analyte sequence comprising the primer, cell identification sequence, unique molecular identifier, and complimentary linker sequence in the detection probe; and

protein identification sequence and the primer from the protein target probe;

ii. a second analyte sequence comprising the primer, cell identification sequence, unique molecular identifier, and polydT in the detection probe; and

cDNA of the mRNA;

d. enriching the analyte sequences from unbound target and detection nucleotide sequences in the sample;

e. detecting signals from the analyte sequences based on PCR amplification and sequencing, wherein the signals are distinguishable for each protein and mRNA.

3. The method of claim 1, wherein the protein-binding molecule is an antibody or an aptamer.

4. The method of claim 1, wherein the linker is cleavable.

5. The method of claim 1, wherein the linker sequence that hybridizes to the detection probe is universal for the target probe.

6. The method of claim 1, wherein the primer comprises a common sequence to enable PCR amplification.

7. The method of claim 1, wherein the protein identification sequences have a length of about 5-20 nucleotides.

8. The method of claim 1, wherein the protein identification nucleotide sequences have a length of about 10 nucleotides.

9. The method of claim 1, wherein the cell identification sequences have a length of about 5-20 nucleotides.

10. The method of claim 1, wherein the cell identification sequences have a length of about 10-15 nucleotides.

11. The method of claim 1, wherein the UMI sequences have a length of about 4-20 nucleotides.

12. The method of claim 1, wherein the polydT sequences have a length of about 20-40 nucleotides.

13. The method of claim 1, wherein the microparticle comprises a polymer bead, magnetic bead, hydrogel microsphere, or resin that has the first and mRNA detection nucleotide sequence bound to or encapsulated in the microparticle.

2. A method for detecting a plurality of target proteins and mRNA in a single cell:

a. contacting a single cell with a plurality of protein target probes, wherein each target probe in the plurality comprises;

1) a plurality of protein target probes, wherein each target probe in the plurality comprises;

i. a protein-binding molecule;

ii. a target nucleotide sequence comprising a primer for a polymerase chain reaction (PCR), a protein identification sequence that identifies the protein-binding molecule, a linker sequence that hybridizes to a sequence in the detection probe;

iii. a linker between the protein-binding molecule and the target nucleotide sequence; and

b. forming an emulsion droplet or microwell comprising the single cell and a microparticle comprising a plurality of detection probes comprising;

i. a protein detection nucleotide sequence comprising a primer for a polymerase chain reaction (PCR), a cell identification sequence that identifies the cell, a unique molecular identifier, and a complimentary linker sequence that hybridizes to the linker sequence in the target probe;

ii. an mRNA detection nucleotide sequence comprising a primer for a polymerase chain reaction (PCR), a cell identification sequence that identifies the cell, a unique molecular identifier, and polydT;

c. allowing the target nucleotide sequence to hybridize to the protein detection nucleotide sequence and the mRNA to hybridize to the mRNA detection nucleotide sequence in the microparticle;

d. conducting reverse transcription and/or polymerase extension to generate

i. a first analyte sequence comprising the primer, cell identification sequence, unique molecular identifier, and complimentary linker sequence in the detection probe; and

protein identification sequence and the primer from the protein target probe;

ii. a second analyte sequence comprising the primer, cell identification sequence, unique molecular identifier, and polydT in the detection probe; and

cDNA of the mRNA;

e. enriching the analyte sequences from unbound target and detection nucleotide sequences in the sample;

f. detecting signals from the analyte sequences based on PCR amplification and sequencing, wherein the signals are distinguishable for each protein and mRNA

14. A kit for multiplexed detection of a plurality of proteins and mRNA from a sample comprisinga plurality of protein target probes, wherein each target probe in the plurality comprises:

i. a protein-binding molecule;

ii. a target nucleotide sequence comprising a primer for a polymerase chain reaction (PCR), a protein identification sequence that identifies the protein-binding molecule, a linker sequence that hybridizes to a sequence in the detection probe;

iii. a linker between the protein-binding molecule and the target nucleotide sequence; and

2) a plurality of microparticles comprising a plurality of detection probes comprising;

i. a protein detection nucleotide sequence comprising a primer for a polymerase chain reaction (PCR), a cell identification sequence that identifies the cell, a unique molecular identifier, and a complimentary linker sequence that hybridizes to the linker sequence in the target probe; and

ii. an mRNA detection nucleotide sequence comprising a primer for a polymerase chain reaction (PCR), a cell identification sequence that identifies the cell, a unique molecular identifier, and polydT.