Determining DNA sequences by mass spectrometry

• US 5,003,059 A
• Filed: 06/20/1988
• Issued: 03/26/1991
• Est. Priority Date: 06/20/1988
• Status: Expired due to Term

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Genomyx Incorporated
• Inventors: Brennan, Thomas M.
• Primary Examiner(s): Griffin, Ronald W.
• Assistant Examiner(s): Kunz, Gary L.
• Application Number:
  US07/209,247
• Time in Patent Office: 1,009 Days
• Field of Search: 536/27-29, 435/6
• US Class Current: 536/25.32
• 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 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

This invention relates to the methods, apparatus, reagents and mixtures of reagents for sequencing natural or recombinant DNA and other polynucleotides. In particular, this invention relates to a method for sequencing polynucleotides based on mass spectrometry to determine which of the four bases (adenine, guanine, cytosine or thymine) is a component of the terminal nucleotide. In particular, the present invention relates to identifying the individual nucleotides by the mass of stable nuclide markers contained within either the dideoxynucleotides, the DNA primer, or the deoxynucleotide added to the primer. This invention is particularly useful in identifying specific DNA sequences in very small quantities in biological products produced by fermentation or other genetic engineering techniques. The invention is therefore useful in evaluating safety and other health concerns related to the presence of DNA in products resulting from genetic engineering techniques.

Claims

1. In a process for determining DNA sequence by the dideoxynucleotide chain termination method, the improvement comprising incorporating ³² S, ³³ S, ³⁴ S and ³⁶ S in the formation of the chain terminated sequence so that a unique sulfur isotope is associated with the terminal nucleotide in the chain terminated sequence, separating the chain terminated sequences by capillary gel electrophoresis, combusting the separated chain terminated sequences to convert the incorporated sulfur to SO₂, and determining the terminal nucleotide by measuring ⁶⁴ SO₂, ⁶⁵ SO₂, ⁶⁷ SO₂, and ⁶⁸ SO₂ in a mass spectrometer thereby determining the sequence of the DNA.

2. A process according to claim 1 wherein the isotope is incorporated only into the 2'"'"', 3'"'"'-dideoxyribonucleotides.

3. A process according to claim 1 wherein the isotope is incorporated only into the 2'"'"'-deoxyribonucleotides.

4. A process according to claim 1 wherein the isotope is incorporated into a DNA primer.