Automated DNA sequencing technique

• US 5,171,534 A
• Filed: 10/15/1990
• Issued: 12/15/1992
• Est. Priority Date: 01/16/1984
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Hood, Leroy E., Smith, Lloyd M., Hunkapiller, Michael W., Hunkapiller, Tim J.
• Primary Examiner(s): Johnston, Jill A.
• Application Number:
  US07/558,312
• Time in Patent Office: 792 Days
• Field of Search: 422/116, 422/82.05, 435/6, 436/94, 935/77-78, 935/85-87, 204/299 R, 204/182.8
• US Class Current: 204/612
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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 2563/107 fluorescence
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A system for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations comprising: a source of chromophore or fluorescent tagged DNA fragments; a zone for contacting an electrophoresis gel; means for introducing said tagged DNA fragments to said zone; and photometric means for monitoring said tagged DNA fragments as they move through said gel.

Claims

1. A novel system for the electrophoretic analysis of oligonucleoide fragments produced in sequencing operations comprising:

a source of chromophore of fluorescent tagged oligonucleotide fragments, said chromophore or fluorophore being distinguishable by its spectral characteristics,

a zone for containing an electrophoresis medium,

means for introducing said tagged oligonucleotide fragments to said zone; and

photometric means for monitoring and distinguishing said tagged DNA fragments upon separation in said medium.

2. The novel system of claim 1 wherein the photometric means is an absorption photometer.

3. The novel system of claim 1 wherein the photometric means is a fluorescent photometer.

4. The novel system of claim 1 wherein the oligonucleotide fragments are labeled with an amino group which is coupled to a dye molecule.

5. The novel system of claim 1 wherein a set of four chromophores or fluorophores are present to tag said oligonucleotide.

6. The novel system of claim 1 wherein said source of tagged oligonucleotide fragments is positioned at one end of said zone, and said detector is positioned in proximity to the opposite end of said zone.

7. A novel system for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations by the chain termination method, comprising:

a source of chromophore or fluorescent tagged DNA fragments from sequencing operations wherein the primer oligonucleotide from each of the four sequencing reactions A, C, G, and T has a different chromophore or fluorescent tag attached to it, each tag being distinguishable from the others by its spectral characteristics.

a zone containing an electrophoresis medium;

means for introducing said tagged DNA fragments to said zone; and

photometric means for monitoring and distinguishing said tagged DNA fragments upon separation in said medium.