QUANTIFICATION OF NUCLEIC ACIDS AND PROTEINS USING OLIGONUCLEOTIDE MASS TAGS

• US 20090305237A1
• Filed: 05/26/2006
• Published: 12/10/2009
• Est. Priority Date: 05/26/2005
• Status: Abandoned Application

• Current Assignee: Trustees of Boston University
• Original Assignee: Trustees of Boston University
• Inventors: Zhang, Lingang, Kasif, Simon, Cantor, Charles R.
• Application Number:
  US11/914,970
• Publication Number:
  US 20090305237A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

The invention provides a method for detecting and quantifying the amount of target molecules, such as nucleic acids or proteins in a sample. The target molecules are first recognized and bounded by target-specific probes, generally nucleic acids or proteins that bind specifically to the targets, each of which is labeled with a short single-stranded nucleic acid probe, either DNA or RNA, with distinct molecular weight. This label is called an oligonucleotide mass tag. One or several standard oligonucleotide sequences can be designed with similar sequence but distinct molecular weight to those oligonucleotide mass tags. Then the oligonucleotide mass tags associated with bounded probes and the standard sequences are co-amplified using a pair of common primers. The presence and/or amount of each oligonucleotide mass tag, which corresponds to the amount of corresponding target molecule, is determined by a primer extension reaction and quantification of the primer extension product.

Claims

1. A method for detecting a target nucleic acid in a sample comprising the steps of

a) designing two 5′

-3′

probes, probe 1 and probe 2, wherein

i) the 5′

end of the probe 1 comprises a region A that is a nucleic acid sequence wherein a primer 1 anneals, a region B that comprises a massTag sequence that has a known unique mass, and a region C that is the most 3′

-region of the probe 1 and comprises the target recognition sequence that binds the target molecule, and

ii) the probe 2 comprises in its most 5′

-region a region D that is the target recognizing sequence, then a region E that comprises a massTag, and a region F in its most 3′

-region that is a constant region recognized by primer 2;

b) combining the target nucleic acid in a double-stranded form with the probe 1 and the probe 2, wherein the region C and the region D of the probes 1 and 2, respectively, anneal to the target;

c) ligating the probe 1 and the probe 2 together, wherein the ligated product consists of a continuous nucleic acid sequence comprising 5′

-A-B-C-D-E-F-3″

;

d) amplifying the 5′

-A-B-C-D-E-F-3′

using the primer 1 and primer 2;

e) optionally removing the excess dNTPs; and

f) detecting the amplified product.

2. The method of claim 1, further comprising a step of quantifying the amount of the 5′

-A-B-C-D-E-F-3′

.

3. The method of claim 1, further comprising a step of quantifying the amount of the 5′

-A-B-C-D-E-F-3′

, wherein the quantification is performed using a primer extension reaction using a primer extension primer that anneals to either A or F region or both mid wherein the primer extension reaction is performed so that it ends at the beginning of the region C or end of the region D so as to amplify the massTag in its entirety and analyzing the primer extension products.

4. The method of claim 1, further comprising a step of quantifying the amount of the 5′

-A-B-C-D-E-F-3′

wherein wherein the quantification is performed using a primer extension reaction using a primer extension primer that anneals to either A or F region or both and wherein the primer extension reaction is performed so that it ends at the beginning of the region C or end of the region D so as to amplify the massTag in its entirety arid analyzing the primer extension products the detecting in step f) is performed using mass spectrometry.

5. The method of claim 1, further comprising a step of quantifying the amount of the 5′

-A-B-C-D-E-F-3′

wherein the quantification is performed using a primer extension reaction using a primer extension primer that anneals to either A or F region or both and wherein the primer extension reaction is performed so that it ends at the beginning of the region C or end of the region D so as to amplify the massTag in its entirety and analyzing the primer extension products, wherein a known amount of one or more standard sequences is added to the amplification reaction, wherein the standard sequences comprise a sequence otherwise identical to 5′

-A-B-C-D-E-F-3′

except that the massTag region is designed to have a difference in its mass compared to the template 5′

-A-B-C-D-E-F-3′

, and wherein the primer extension reaction of the template and the standard allows determination of the absolute amount of target in the original sample.

6. A method for detecting or quantifying a target protein in a sample comprising the steps of

a) designing a probe comprising at least four regions, a region P in the most 5′

-end of the probe that comprises a protein capture molecule specific to the target protein, a region A that comprises a constant nucleic acid sequence recognized by a primer 1, a region B that comprises a massTag sequence, and a region C that comprises a sequence recognized by a primer 2;

b) combining a sample suspected of containing the target protein with the P-5-A-B-C-3′

-probe;

c) amplifying the 5′

-A-B-C-3′

combination using the primer 1 and the primer 2;

d) optionally removing excess nucleotides;

e) performing a primer extension reaction wherein the massTag of the 5′

-A-B-C-3′

is extended and results in a primer extension product if the target protein is present in the sample; and

f) detecting the primer extension product.

7. The method of claim 6, wherein a known amount of at least one standard probes otherwise identical to 5′

-A-B-C-3′

except for a distinct mass Tag sequence (region B) is added to the amplification reaction, wherein the primer extension reaction of the probes and the standards allows the absolute quantification of the mass Tag sequence.

8. The method of claim 6, wherein the primer extension products is analyzed using mass spectrometry.

9. The method of claim 6, wherein the target protein is further quantified by, in addition to primer 1 and primer 2, adding to the amplification reaction a known amount of at least one standard oligonucleotide with otherwise identical sequence to 5′

-A-B-C-3′

except for a different massTag, wherein after the primer extension reaction, if the target protein is present in the sample, a first primer extension product corresponding to the target protein and a second primer extension product corresponding to the standard oligonucleotide is produced, and wherein the primer extension products are detected using mass spectrometry and the ratio of the surface area that corresponds to the primer extension products with different mass indicates the amount of the target protein in the sample.

10. The method of claim 7, wherein the mass spectrometry is MALDI-TOF.

12. The method of claim 10, wherein the analysis is performed using mass spectrometry.

11. A method for detecting a protein-protein interaction comprising the steps of

a) designing a unique single-stranded oligonucleotide label for each of the proteins or protein fragments that are involved or suspected to be involved in the protein-protein interaction, wherein each label consists of 3 regions, named from 5′

to 3′

a region A1, a region A2, and a region A3 for first protein or protein fragment (A), and a region B1, a region B2, and a region B3 for second protein or fragment (B) and so forth, and wherein the region A1 and the region B1 are 15-20 bases long, and the region A2 and the B2 are 5-30 bases long, and the region A3 and the region B3 are 10 bases long, and so forth;

b) linking the protein or protein fragments that are involved or suspected to be involved in the protein-protein interaction to their corresponding single-stranded oligonucleotide labels;

c) mixing the labeled proteins or protein fragments that are involved or suspected to be involved in tire protein-protein interaction with a connector, together with a ligase, wherein the region A3, the region B3, arid so forth, can be ligated together only if they are in sufficient proximity which occurs if the proteins or protein fragments interact with each other; and

d) amplifying the ligated template using primer A1 that recognizes at least part of the region A1 and primer B1′

that is antisense to the region B1;

e) optionally removing excess dNTPs;

f) performing primer extension reaction, wherein the extension ends at the end of region 2 (A2 or B2′

);

g) detecting the primer extension products wherein presence of a primer extension product is indicative of protein-protein interaction between the proteins or protein fragments, and wherein absence of primer extension product is indicative of no interaction between the protein or protein fragments.

13. The method of claim 11, further comprising adding to the amplification reaction a known amount of at least one standard probe with sequence identical to each protein probe except for the massTag sequence, wherein the primer extension reaction of the template and the standard allows determination of the absolute amount of the protein or protein fragment in the protein complex.

14. A method for analyzing DNA-protein binding specificities comprising the steps of

a) designing probes for each candidate protein-binding sequence, wherein each probe usually comprises four regions, named from 5′

to 3′

region A, comprising a constant nucleic acid sequence recognized by one PCR primer, region B, comprising the OMT sequence, region C, comprising the candidate protein-binding sequence, and region D, a constant sequence recognized by the other PCR primer;

b) annealing each probe with equal molar of its reverse complementary to form double-stranded probe;

c) incubating target protein with a mixture of double-stranded probes, and the antibody to the target protein (first antibody);

d) adding beads coated with a second antibody specific to the first antibody;

e) washing up beads and PCR amplifying probes immobilized on the beads;

f) removing excess dNTPs;

g) performing a primer extension reaction, wherein the extension products comprise 5′

-A-B-3′

; and

h) quantifying the primer extension products.

15. The method of claim 14, wherein the analysis is performed using mass spectroscopy.