DNA diagnostics based on mass spectrometry

• US 20070202514A1
• Filed: 10/02/2006
• Published: 08/30/2007
• Est. Priority Date: 11/06/1996
• Status: Active Grant

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Braun, Andreas, Hillenkamp, Franz, Koster, Hubert, Van Den Boom, Dirk, Jurinke, Christian, Ruppert, Andreas
• Granted Patent: US 7,501,251 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

Fast and highly accurate mass spectrometry-based processes for detecting a particular nucleic acid sequence in a biological sample are provided. Depending on the sequence to be detected, the processes can be used, for example, to diagnose a genetic disease or chromosomal abnormality; a predisposition to a disease or condition, infection by a pathogenic organism, or for determining identity or heredity.

Claims

1-103. -103. (canceled)

104. A method for detecting a target nucleic acid in a biological sample using RNA amplification, comprising:

a. amplifying a target nucleic acid using a primer comprising a sequence that is complementary to the target sequence, 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 resulting RNA using mass spectrometry thereby detecting the presence of the target nucleic acid sequence in the biological sample.

105. A process of detecting the presence of a target nucleic acid sequence, comprising the steps of:

a. incubating in a reaction mixture

i. an RNA polymerase

ii. nucleoside triphosphates, and

iii. a nucleic acid molecule comprising th target nucleic acid sequence, or a complement thereof, and a promoter for the RNA polymerase,

thereby producing an RNA molecule comprising the target nucleic acid sequence, or a complement thereof; and

b. detecting the RNA molecule by mass spectrometry, thereby detecting the presence of the target nucleic acid sequence.

106. The process of claim 105, wherein the nucleic acid molecule comprising the target nucleic acid sequence is DNA and the RNA polymerase is DNA dependent RNA polymerase.

107. The process of claim 106, 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.

108. The process of claim 105, further comprising:

a. hybridizing a detector oligonucleotide to the RNA molecule comprising the target nucleic acid sequence, wherein the detector oligonucleotide is complementary to a portion of the target nucleic acid sequence; and

b. removing unhybridized detector oligonucleotide;

wherein, in step b), the RNA molecule comprising the target nucleic acid sequence is detected by detecting hybridized detector oligonucleotide.

110. The process of any of claims 105-109, wherein the detector oligonucleotide is a peptide nucleic acid.

111. The process of any of claims 105-109, wherein a nucleic acid is immobilized to a solid support via a selectively cleavable linker.

112. The process of any of claims 105-109, wherein a primer is a peptide nucleic acid.

109. A method for detecting the presence of a target nucleic acid sequence, comprising:

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

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