Method of irradiating biological specimens

• US 5,288,647 A
• Filed: 04/17/1991
• Issued: 02/22/1994
• Est. Priority Date: 05/02/1988
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Stratagene California
• Inventors: Sorge, Joseph A., Zimlich, William C. Jr.
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): SANTIAGO, AMALIA L
• Application Number:
  US07/686,491
• Time in Patent Office: 1,042 Days
• Field of Search: 422/186.3, 422/186.07, 422/186, 422/24, 436/174, 204/193, 250/372, 250/455.1, 250/492.1, 250/504 R, 435/6, 435/291, 536/27, 427/8, 427/10, 364/413.26, 364/188, 364/189, 356/51, 356/72
• US Class Current: 436/174
• CPC Class Codes: C07K 17/00 Carrier-bound or immobilised peptides carrier-bound or immobilised enzymes C12N11/00; Preparation thereof
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

A method of irradiating a biological specimen with ultraviolet, in particular a polynucleotide specimen selected from DNA or RNA, or optionally a protein. In the case where the specimen is DNA or RNA, or potentially proteins, the specimen is irradiated to cross-link the specimen to a substrate. In the case where the specimen is DNA, the specimen can also be irradiated to form thymine dimers. The method uses an apparatus which permits relatively precise control of the total ultraviolet dose received by the specimen, despite any changes of ultraviolet flux from the lamps which may occur from during any one experiment, or between a number of experiments. Thus, the method allows relatively highly reproducible results to be obtained.

Claims

1. A method of irradiating a polynucleotide specimen on a substrate, the method comprising:

providing an apparatus having;

(a) a chamber having a repository for the substrate;

(b) an ultraviolet lamp fixture disposed within the chamber, which fixture carries an ultraviolet lamp to illuminate the repository;

(c) an energy detector which can provide an indication of the total ultraviolet dose received on the repository, the energy detector having;

(i) a sensor disposed in a fixed location in the chamber, which provides a signal corresponding to the ultraviolet flux on the repository;

(ii) a detector circuit connected to the sensor, so as to provide an indication of the total ultraviolet dose received on the repository based on the signal received from the sensor over time;

(d) a control circuit connected to the detector circuit and the lamp fixture, which control circuit can de-energize the lamp fixture upon detection of a selected or predetermined total ultraviolet dose by the energy detector;

the method further comprising placing the substrate carrying the polynucleotide into the chamber, then energizing the lamp fixture so that the polynucleotide specimen will be irradiated with ultraviolet radiation from the lamp until the control circuit de-energizes the lamp upon detection of the selected or predetermined total ultraviolet dose.

2. A method of irradiating a polynucleotide specimen on a substrate, the method comprising:

providing an apparatus having;

(a) a chamber which is substantially opaque to ultraviolet light and having;

(i) a door allowing access to an interior of the chamber;

(ii) a repository for the substrate;

(b) an ultraviolet lamp fixture disposed within the chamber, which fixture carries an ultraviolet lamp to illuminate the repository;

(c) an energy detector which can provide an indication of the total ultraviolet dose received on the repository, the energy detector having;

(i) a sensor disposed in a fixed location in the chamber, which provides a signal corresponding to the ultraviolet flux on the repository;

(ii) a detector circuit connected to the sensor, so as to provide an indication of the total ultraviolet dose received on the repository based on the signal received from the sensor over time;

(d) a control circuit connected to the detector circuit and the lamp fixture, which control circuit can de-energize the lamp fixture upon detection of a selected or predetermined total ultraviolet dose by the energy detector, the control circuit comprising;

(i) a fixed memory which retains a fixed value corresponding to a fixed, predetermined total ultraviolet dose;

(ii) a variable memory which allows for the selection of a variable value corresponding to a variable, selected total ultraviolet dose;

(iii) a memory switch which engages either of the fixed or variable memories, such that the control circuit will de-energize the lamp fixture upon detection of the predetermined or selected total ultraviolet dose, respectively;

(e) a keypad on the outside of the chamber and connected to the control circuit and timer, the keypad carrying the memory switch and keys by which the variable value of the variable memory and timer setting can be selected;

the method further comprising placing the substrate carrying the polynucleotide in the chamber, entering on the keypad the setting of the memory switch, and further entering the setting of the variable memory if appropriate, then energizing the lamp fixture so that the polynucleotide specimen will be irradiated with ultraviolet radiation from the lamp until the control circuit de-energizes the lamp upon detection of the selected or predetermined total ultraviolet dose.

3. A method of irradiating a polynucleotide or protein specimen on a substrate, the method comprising:

providing an apparatus having;

(a) a chamber which is substantially opaque to ultraviolet light, and having;

(i) a door allowing access to an interior of the chamber;

(ii) a repository for the substrate;

(b) an ultraviolet lamp fixture disposed within the chamber, which fixture carries an ultraviolet lamp to illuminate the repository;

(c) an energy detector which can provide an indication of the total ultraviolet dose received on the repository, the energy detector having;

(i) a sensor disposed in a fixed location in the chamber, which provides a signal corresponding to the ultraviolet flux on the repository;

(ii) a detector circuit connected to the sensor, so as to provide an indication of the total ultraviolet dose received on the repository based on the signal received from the sensor over time;

(d) a control circuit connected to the detector circuit and the lamp fixture, which control circuit can de-energize the lamp fixture upon detection of a selected or predetermined total ultraviolet dose by the energy detector;

the method further comprising placing the substrate carrying the polynucleotide or protein into the chamber, then energizing the lamp fixture so that the polynucleotide specimen will be irradiated with ultraviolet light from the lamp until the control circuit de-energizes the lamp upon detection of the selected or predetermined total ultraviolet dose.

4. A method of irradiating a polynucleotide specimen on a substrate, the method comprising:

providing an apparatus having;

(a) a chamber which is substantially opaque to ultraviolet light, and having;

(i) a door allowing access to an interior of the chamber;

(ii) a repository for the substrate;

(b) an ultraviolet lamp fixture disposed within the chamber, which fixture carries an ultraviolet lamp to illuminate the repository;

(c) an energy detector which can provide an indication of the total ultraviolet dose received on the repository, the energy detector having;

(i) a sensor disposed in a fixed location in the chamber, which provides a signal corresponding to the ultraviolet flux on the repository;

(ii) a detector circuit connected to the sensor, so as to provide an indication of the total ultraviolet dose received on the repository based on the signal received from the sensor over time;

(d) a control circuit connected to the detector circuit and the lamp fixture, which control circuit can de-energize the lamp fixture upon detection of a selected or predetermined total ultraviolet dose by the energy detector, the control circuit comprising;

(i) a fixed memory which retains a fixed value corresponding to a fixed, predetermined total ultraviolet dose;

(ii) a variable memory which allows for the selection of a variable value corresponding to a variable, selected total ultraviolet dose;

(iii) a memory switch which engages either of the fixed or variable memories, such that the control circuit will de-energize the lamp fixture upon detection of the predetermined or selected total ultraviolet dose, respectively;

(e) a timer connected to the lamp fixture, which timer can de-energize the lamp fixture upon elapse of a variable, selected period during which the lamp fixture is energized

(f) a keypad on the outside of the chamber and connected to the control circuit and timer, the keypad carrying the memory switch and keys by

which the variable value of the variable memory

and timer setting can be selected;

the method further comprising placing the substrate carrying the polynucleotide into the chamber, entering on the keypad the setting of either one of the memory switch and the timer, and further entering the setting of the variable memory or timer if appropriate, then energizing the lamp fixture so that the polynucleotide specimen will be irradiated with ultraviolet light from the lamp until the control circuit de-energizes the lamp upon detection of the selected or predetermined total ultraviolet dose.

5. A method of exposing a polynucleotide specimen to UV radiation, comprising

providing apparatus having (a) a UV-opaque chamber, said chamber having a first region for disposing a specimen on a substrate, and having a second region for disposing a source of UV radiation, (b) a memory, and (c) a circuit for limiting an administered total UV dose to a desired UV dose, said circuit comprising (1) a UV sensor disposed at a fixed position in said chamber, (2) a first circuit coupled to said sensor capable of indicating a total UV dose received at said first region, and (3) a second circuit coupled to said first circuit and to said source of UV radiation, capable of de-energizing said source of UV radiation;

placing the specimen on a substrate in said first region;

retrieving from said memory either a first value corresponding to a predetermined desired total UV dose for said specimen or second value corresponding to a desired total UV dose which may be selected by an operator; and

irradiating said first region with UV radiation under control of said circuit.

6. A method as in claim 5, wherein said UV radiation wavelength is between about 200 nanometers and about 290 nanometers.