Apparatus and method for the generation, separation, detection, and recognition of biopolymer fragments

• US 6,218,121 B1
• Filed: 04/26/1999
• Issued: 04/17/2001
• Est. Priority Date: 05/09/1995
• Status: Expired due to Fees

• Current Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Original Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Inventors: Simpson, John W., Went, Gregory T., Rothberg, Jonathan Marc
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): TUNG, JOYCE
• Application Number:
  US09/300,163
• Time in Patent Office: 722 Days
• Field of Search: 435/6, 435/91.2, 435/89, 435/91.21, 935/77
• US Class Current: 435/6.13
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00783 Laminate assemblies, i.e. the reactor comprising a stack of plates
• CPC Class Codes: B01J 2219/00828 Silicon wafers or plates
• CPC Class Codes: B01J 2219/00831 Glass
• CPC Class Codes: B01J 2219/00833 Plastic
• CPC Class Codes: B01J 2219/00873 Heat exchange
• CPC Class Codes: B01J 2219/00995 Mathematical modeling
• CPC Class Codes: G01N 27/44708 Cooling
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44782 of a plurality of samples
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention is an integrated instrument for the high-capacity electrophoretic analysis of biopolymer samples. It comprises a specialized high-voltage, electrophoretic module in which the migration lanes are formed between a bottom plate and a plurality of etched grooves in a top plate, the module permitting concurrent separation of 80 or more separate samples. In thermal contact with the bottom plate is a thermal control module incorporating a plurality of Peltier heat transfer devices for the control of temperature and gradients in the electrophoretic medium. Fragments are detected by a transmission imaging spectrograph which simultaneously spatially focuses and spectrally resolves the detection region of all the migration lanes. The spectrograph comprises a transmission dispersion element and a CCD array to detect signals. Signal analysis comprises the steps of noise filtering, comparison in a configuration space with signal prototypes, and selection of the best prototype. Optionally post-processing is done by a Monte-Carlo simulated annealing algorithm to improve results. Optionally, an array of micro-reactors can be integrated into the instrument for the generation of sequencing reaction fragments directly from crude DNA samples.

Claims

1. A method for generating sequencing reaction fragments of a DNA in one reaction chamber, the method comprising the sequential steps of:

(a) performing a polymerase chain reaction amplification step on said DNA with dUTP rich PCR primers, to produce an amplified DNA fragment;

(b) fragmenting the dUTP rich PCR primers with Uracil DNA Glycosylase into primer fragments, said primer fragments being ineffective as DNA polymerase primers; and

(c) performing Sanger sequencing reactions to generate said DNA sequencing reaction fragments without performing a step to separate said primer fragments from said amplified DNA fragment.

2. The method of claim 1 wherein the dUTP rich PCR primers have dUTP residues spaced no more than approximately six bases apart.

3. The method of claim 1 performed in an array of micro-reactors for ejection onto a biopolymer separation apparatus.