High Speed Parallel Molecular Nucleic Acid Sequencing HIGH SPEED PARALLEL MOLECULAR NUCLEIC ACID SEQUENCING US Patent Number 6,982,146 issued on 2006 Jan 3. US Patent Number 8,535,881 issued on 2013 Sep 17. PDF, thanks to www.freepatentsonline.com

Image by Paul Theissan

Fig 2: To sequence a single DNA molecule (16), a primer (18) is annealed and a DNA polymerase (10) is bound. Laser (26) beam (28) hits a Green Fluorescent Protein or other flurorophore (22) attached to the polymerase. Energy is transferred by FRET (30) to base (20) which has a fluorescent compound attached (24). The signal coming out (32) can be seen in the microscope (Fig 3). Fig 3: As the base (20) and the fluorescent dye (24) change, the output signal (32) changes. The signal is picked up by a microscope objective (40) and sent on to a computer (50) for processing into the final DNA sequence (52).

Thomas D. Schneider, Denise Rubens (NCI)
Serial No. 60/151,580 filed 30 Aug 1999

Informal description of the Invention:
Using this technique the sequence of single molecules of DNA or RNA can be read. The method uses a microscope to observe changes in the fluorescence of individual nucleic acid molecules being read by a DNA or RNA polymerase. The data are collected in a computer in parallel, allowing many sequences to be determined from a tiny volume. The technique could be used to read the sequences of mRNA directly from cells, bypassing the current microarray technology. It may also be fast enough to allow individual laboratories to read entire genomes with a single device. This nanotechnology would have diagnostic uses by allowing rapid identification of mutations that cause cancer and other genetic diseases.

Formal description of the Invention: The present application describes a new method and apparatus for DNA sequencing called Two Dye Sequencing (TDS). This method employs engineered DNA polymerases which are labeled with a fluorophore such as Green Fluorescent Protein (GFP) and are combined with an annealed oligonucleotide primer in a chamber of a microscope field of view capable of detecting individual molecules. Four nucleotide triphosphates, each labeled on the base with a different fluorescent dye are introduced to the reaction. Light of a specific wavelength is used to excite the fluorophore on the polymerase, which in turn excites the neighboring fluorophore on the nucleotide by Forster Resonance Energy Transfer (FRET). As nucleotides are added to the primer, their spectral emissions provide sequence information of the DNA molecule.

See also Medusa Sequencing.

• PCT Application Cover page information (html)
• Description and claims (html)
• Figures:
  • cover page
  • Fig 1
  • Fig 2 - 3
• The PATENT COOPERATION TREATY (PCT) has released the international patent application but they repeatedly have broken my links. Until they stabilize, you can use their prototype search engine ("simple search" page!) for:
  • Schneider Rubens (all words)
    
  • PCT/US00/23736 (This exact phrase) (This still gives two relevant links.)
  • Use simple search for 0116375 (ie WO0116375)
• The PCT Database has released the the international patent application (use Username: guest and Password: guest to log in, click on "view images" and then use the Next button). Patent number: WO0116375
• Because links to the PCT repeatedly fail I have put the PCT application above.

• Federal Register Links to the Federal Register fail. Search for page number and year: '"page 44804" in 2003'.
• Federal Register announcement Volume 65, Number 74, page 20472, Monday April 17, 2000, in pdf format
• Notice of Prospective Grant of Exclusive License to LI-COR Inc. was posted on the Federal Registry.
  Federal Register announcement (pdf) Federal Register, Vol. 68, No. 146, pages 44804, Wednesday, July 30, 2003
• Notice of Prospective Grant of Exclusive License to Helicos BioSciences Corporation was posted on the Federal Registry.
  Federal Register announcement (html) Volume 69, Number 149, page 47160-47161, August 4, 2004.
• Notice of Prospective Grant of Exclusive License to VisiGen Biotechnologies was posted on the Federal Registry.
  Federal Register announcement (html) Federal Register, Vol. 70, No. 19, page 4880, Monday, January 31, 2005.

Schneider Lab
origin: 2000 April 26
updated: 2013 Sep 11