SOLID PHASE SEQUENCING OF BIOPOLYMERS

• US 20110172111A1
• Filed: 08/06/2010
• Published: 07/14/2011
• Est. Priority Date: 04/11/1995
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings), Trustees of Boston University
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Köster, Hubert, Cantor, Charles
• Granted Patent: US 8,758,995 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2523/301 Sonication
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2531/149 Replicase based amplification, e.g. Q beta replicase
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include DNA or RNA in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.

Claims

1. A method for sequencing a target nucleic acid, comprising the steps of:

providing a set of nucleic acid fragments each containing a sequence that corresponds to a sequence of the target nucleic acid;

hybridizing the set to an array of nucleic acid probes to form a target array of nucleic acids, wherein each probe comprises a single-stranded portion comprising a variable region; and

determining molecular weights for nucleic acids of the target array;

whereby the sequence of the target nucleic acid is determined.

2. The method of claim 1, wherein the molecular weights are determined by methods selected from the group consisting of gel electrophoresis, capillary electrophoresis, chromatography, and nuclear magnetic resonance.

3. The method of claim 1, wherein the molecular weights are determined by mass spectrometry.

4. The method of claim 3, wherein the mass spectrometry comprises a step selected from the group consisting of laser heating, droplet release, electrical release, photochemical release, fast atom bombardment, plasma desorption, matrix-assisted laser desorption/ionization, electrospray, and resonance ionization, or a combination thereof.

5. The method of claim 3, wherein the mass spectrometry comprises a step selected from the group consisting of Fourier Transform, ion cyclotron resonance, time of flight analysis with reflection, time of flight analysis without reflection, and quadrupole analysis, or a combination thereof.

6. The method of claim 3, wherein the mass spectrometry comprises matrix-assisted desorption ionization and time of flight analysis.

7. The method of claim 3, wherein the mass spectrometry comprises electrospray ionization and quadrupole analysis.

8. The method of claim 3, wherein two or more molecular weights are determined simultaneously.

9. The method of claim 1, further comprising the step of enzymatically extending the nucleic acid probes of the target array using the hybridized target nucleic acid as a template to form extended strands.

10. The method of claim 2, wherein the extended strands comprise DNA, RNA, PNA or combinations thereof.

11. The method of claim 9, wherein the step of extending is performed in the presence of chain elongating nucleotides and chain terminating nucleotides.

12. The method of claim 1, wherein the array comprises nucleic acid probes having at least one mass-modifying functionality.

13. The method of claim 12, wherein the mass-modifying functionality is coupled to a heterocyclic base, a sugar moiety or a phosphate group.

14. The method of claim 12, wherein the mass-modifying functionality is a chemical moiety that does not interfere with hydrogen bonding for base-pair formation.

15. The method of claim 12, wherein the mass-modifying functionality is coupled to a purine at position C2, N3, N7, or C8.

16. The method of claim 12, wherein the mass-modifying functionality is coupled to a deazapurine at position N7 or N9.

17. The method of claim 12, wherein the mass-modifying functionality is coupled to a pyrimidine at position C5 or C6.

18. The method of claim 12, wherein the mass-modifying functionality is selected from the group consisting of F, Cl, Br, I, SiR3, Si(CH₃)₃, Si(CH₃)₂(C₂H₅), Si(CH₃)(C₂H₅)₂, Si(C₂H₅)₃, (CH₂)_nCH₃, (CH₂)_nNR₂, CH₂CONR₂, (CH₂)_nOH, CH₂F, CHF₂, and CF₃;

wherein n is an integer; and

wherein R is selected from the group consisting of —

H, deuterium and alkyls, alkoxys and aryls of 1-6 carbon atoms, polyoxymethylene, monoalkylated polyoxymethylene, polyethylene imine, polyamide, polyester, alkylated silyl, heterooligo/polyaminoacid and polyethylene glycol.

19. The method of claim 12, wherein the mass-modifying functionality is —

N₃ or —

XR,

wherein X is selected from the group consisting of —

O—

, —

NH—

, —

NR—

, —

S—

, —

OCO(CH₂)_nCOO—

, —

NHCO(CH₂)_nCOO—

, —

OSO₂O—

, —

OCO(CH₂)_n—

, —

NHC(O)—

, and —

C(O)NH—

, and n is an integer from 1 to 20; and

wherein R is selected from the group consisting of —

H, deuterium and alkyls, alkoxys and aryls of 1-6 carbon atoms, polyoxymethylene, monoalkylated polyoxymethylene, polyethylene imine, polyamide, polyester, alkylated silyl, heterooligo/polyaminoacid and polyethylene glycol.

20. The method of claim 19, wherein X is —

NHC(S)—

.