DNA Sequencing apparatus

• US 5,122,345 A
• Filed: 07/03/1990
• Issued: 06/16/1992
• Est. Priority Date: 07/12/1988
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Tabor, Stanley, Richardson, Charles C.
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Trembley, Theresa A.
• Application Number:
  US07/547,870
• Time in Patent Office: 714 Days
• Field of Search: 422/116, 422/103, 422/68.1, 422/69, 422/82.05, 436/89, 935/88, 204/182.8, 204/299 R
• US Class Current: 422/116
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/125 Methyl transferase, i.e. methylase
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• 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 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an

This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

Claims

1. An automated DNA sequencing apparatus comprising:

a reactor comprising reagents which provide at least two series of DNA products formed from a single primer and a DNA strand, each said DNA product of a said series differing in molecular weight and having a chain terminating agent at one end,

separating means for separating said DNA products along one axis of the separator to form a series of bands, the intensity of substantially all nearby bands in a series being approximately the same, and the intensity of substantially all nearby bands in any one series being different from those of other series,

band reading means for determining the position and intensity of each said band after said separating along said axis, and

computing means that determines the DNA sequence of said DNA strand solely from said position and intensity of said bands along said axis and not from the wavelength of emission of light from any label that may be present in the separating means.

2. The apparatus of claim 1 wherein said reagent comprises a manganese or iron ion.

3. The apparatus of claim 1 wherein said reagent comprises T7-type polymerase and a manganese or iron ion.

4. The apparatus of claim 1, 2 or 3, said reagent further comprising a chelator.

5. The apparatus of claim 4, said chelator being citrate or isocitrate.

6. An automated DNA sequencing apparatus comprising:

a reactor comprising reagents which provide a series of DNA products formed from a primer and a DNA strand, wherein said reactor comprises a manganese ion,

a separator for separating said DNA products along one axis of the separator to form a series of bands,

a band reader for determining the position and intensity of each said band along said axis after said separating, and

a computer that determines the DNA sequence of said DNA strand solely from said position and said intensity of said bands along said axis and not from the wavelength of emission of light from any label that may be present in the separating means.

7. The apparatus of claim 6 wherein said reagent comprises a T7-type DNA polymerase.

8. The apparatus of claim 6 or 7 wherein said reagent further comprises a chelator.

9. The apparatus of claim 8 wherein said chelator is citrate or isocitrate.

10. An automated DNA sequencing apparatus, comprising:

a separator for separating DNA products along one axis of the separator, said products being formed from a primer and a DNA strand in the presence of a manganese ion, to form a series of bands,

a band reader for determining the position and intensity of each said band along said axis after said separating; and

computer that determines the DNA sequence of said DNA strand solely from both said position and intensity of said bands along said axis and not from the wavelength of emission of light from any label that may be present in the separating means.

11. The apparatus of claim 10 wherein said separator consists essentially of a single lane containing four separate series of DNA products with each said DNA product being labelled with the same label.

12. An automated DNA sequencing apparatus comprising a computer that determines the DNA sequence of a DNA strand solely from the position and intensity of bands formed from DNA products separated along one axis of a separator and not from the wavelength of emission of light from any label that may be present in the separator, said DNA products being produced by a DNA sequencing method from said DNA strand.

13. An automated DNA sequencing apparatus comprising a computer means that compute a DNA sequence solely from intensity and position data obtained from at least two series of DNA products labeled with an identical label and produced by a sequencing reaction performed in the presence of manganese ions after separating said DNA products in the same lane of a separator to form a series of bands and not from the wavelength of emission of light from the label that is present in the separator.