DNA diagnostics based on mass spectrometry

• US 7,501,251 B2
• Filed: 10/02/2006
• Issued: 03/10/2009
• Est. Priority Date: 11/06/1996
• Status: Expired due to Fees

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Hillenkamp, Franz, Koster, Hubert, Ruppert, Andreas, Braun, Andreas, van den Boom, Dirk, Jurinke, Christian
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US11/541,871
• Publication Number:
  US 20070202514A1
• Time in Patent Office: 890 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/6.16
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00504 Pins
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/0052 in the shape of elongated tubes
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 : separated from the reactor apparatus
• CPC Class Codes: B01J 2219/0072 : Organic compounds
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01L 3/0268 : using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07F 9/2408 : of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/2429 : of arylalkanols
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 : Allele-specific amplification
• CPC Class Codes: C12Q 1/6865 : Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6872 : involving mass spectrometry
• CPC Class Codes: C12Q 2523/10 : Characterised by chemical treatment
• CPC Class Codes: C12Q 2525/197 : incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2531/113 : PCR
• CPC Class Codes: C12Q 2535/101 : Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/507 : characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/518 : characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/627 : being a mass spectrometer
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 2035/00237 : Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/1037 : Using surface tension, e.g. pins or wires
• CPC Class Codes: G01N 2035/1069 : by adjusting the spacing between multiple probes of a single transferring head
• CPC Class Codes: G01N 35/1067 : for transfer to or from containers having different spacing
• CPC Class Codes: G01N 35/1074 : arranged in a two-dimensional array
• CPC Class Codes: H01J 49/00 : Particle spectrometers or separator tubes

Abstract

Provided are methods for detecting a target nucleic acid in a biological sample using RNA amplification. In an embodiment, provided is a method that comprises (a) amplifying a target nucleic acid, or portion thereof, using a primer comprising a sequence that is complementary to a polynucleotide sequence in the target nucleic acid, or a complement thereof, and a sequence that encodes an RNA polymerase promoter; (b) synthesizing RNA using an RNA polymerase that recognizes the promoter; and (c) detecting the presence or absence of the resulting RNA using mass spectrometry thereby detecting the presence or absence of the target nucleic acid in the biological sample.

Claims

1. A method for detecting the presence or absence of a target nucleic acid in a biological sample using RNA amplification, comprising:

a. amplifying a target nucleic acid, or portion thereof, using a primer comprising a sequence that is complementary to a polynucleotide sequence in the target nucleic acid, or a complement thereof, and a sequence that encodes an RNA polymerase promoter;

b. synthesizing RNA using an RNA polymerase that recognizes the promoter when the target nucleic acid is present; and

c. detecting the presence or absence of the resulting RNA using mass spectrometry thereby detecting the presence or absence of the target nucleic acid in the biological sample.

8. The process of any one of claims 1-6, wherein the RNA is immobilized to a solid support via a selectively cleavable linker.

9. The process of claim 1, wherein the primer is a peptide nucleic acid.

2. A process of detecting the presence or absence of a target nucleic acid, comprising the steps of:

a. incubating in a reaction mixture

i. an RNA polymerase

ii. nucleoside triphosphates, and

iii. a nucleic acid molecule comprising a sequence that is complementary to a polynucleotide sequence in the target nucleic acid, or a complement thereof, and a promoter for the RNA polymerase,

thereby producing an RNA comprising the polynucleotide sequence of the target nucleic acid, or a complement thereof when the target nucleic acid is present; and

b. detecting the presence or absence of the RNA by mass spectrometry, thereby detecting the presence or absence of the target nucleic acid.

3. The process of claim 2, wherein the target nucleic acid is DNA and the RNA polymerase is DNA dependent RNA polymerase.

4. The process of claim 3, further comprising, following step a) and prior to step b)

a. inactivating the RNA polymerase;

and

b. digesting the DNA using RNAse-free DNAse I.

5. The process of claim 2, further comprising:

a. hybridizing a detector oligonucleotide to the RNA, wherein the detector oligonucleotide is complementary to a portion of the polynucleotide sequence in the target nucleic acid, or complement thereof; and

b. removing unhybridized detector oligonucleotide;

wherein, in step b), the presence or absence of the RNA is detected by detecting the presence or absence of hybridized detector oligonucleotide.

7. The process of claim 5, wherein the detector oligonucleotide is a peptide nucleic acid.

6. A method for detecting the presence or absence of a target nucleic acid, comprising:

a. amplifying the target nucleic acid, or portion thereof, using a primer comprising a sequence that is complementary to at least a portion of the polynucleotide sequence of the target nucleic acid, or a complement thereof, and a sequence that encodes an RNA polymerase promoter, thereby producing an amplified nucleic acid molecule comprising the polynucleotide sequence of the target nucleic acid, or complement thereof, and the RNA polymerase promoter;

b. incubating the amplified nucleic acid molecule with nucleoside triphosphates and an RNA polymerase that recognizes the promoter, thereby producing an RNA corresponding to the target nucleic acid when target nucleic acid is present; and

c. detecting the presence or absence of the RNA using mass spectrometry, thereby detecting the presence or absence of the target nucleic acid.

10. The process of claim 6, wherein the primer is a peptide nucleic acid.