Signal processing method for determining base sequence of nucleic acid

• US 4,885,696 A
• Filed: 03/25/1987
• Issued: 12/05/1989
• Est. Priority Date: 03/26/1986
• Status: Expired due to Term

• Current Assignee: Fuji Photo Film Co Limited (Fujifilm Holdings Corporation)
• Original Assignee: Fuji Photo Film Co Limited (Fujifilm Holdings Corporation)
• Inventors: Hara, Makoto
• Primary Examiner(s): Lall, Parshotam S.
• Assistant Examiner(s): Teska, Kevin J.
• Application Number:
  US07/030,062
• Time in Patent Office: 986 Days
• Field of Search: 364/413.01, 364/496, 364/497, 435/6, 250/303, 250/484.1 B, 250/327.2 A, 250/327.2 C, 250/327.2 D, 935/77, 436/94
• US Class Current: 702/20
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: G01N 27/44717 Arrangements for investigating the separated zones, e.g. localising zones
• CPC Class Codes: G01T 1/2942 using autoradiographic methods
• CPC Class Codes: Y10S 250/909 Methods and apparatus ancillary to stimulable phosphor systems
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A signal processing method for determining the base sequence of nucleic acids from data on an autoradiograph is provided. An autoradiograph including a plurality of resolved rows is formed by resolving a mixture of base-specific DNA fragments or base-specific RNA fragments that have been labeled with a radioactive element in a one-dimensional direction on a support medium. The method detects bands along each resolved row corresponding to the nucleic acid bases from digital signal data representing position and signal level for each row. The digital signal data corresponding to the autoradiograph may be photoelectrically detected as stimulated emission from the recording of the autoradiograph of the resolved rows on a phosphor sheet or as visible images from the recording by the autoradiograph of the resolved rows on a radiosensitive material.

Claims

1. A method for determining the base sequence of a nucleic acid by obtaining digital signals corresponding to an autoradiograph of a resolved pattern including plural resolved rows consisting of four rows formed by resolving each of four groups of a mixture of base-specific nucleic acid fragments on a support medium labeled with a radioactive element that have migrated in a one-dimensional migration direction on the support medium and processing the digital signals thus obtained to determine the nucleic acid base sequence, wherein the digital signal processing method includes the steps of:

(a) generating from said digital signals a waveform including peaks representative of base position in the resolving direction and the signal level for each resolved row;

(b) selecting a search-fiducial point, an interval adjacent to said search-fiducial point, and a signal level on the waveform;

(c) defining a plurality of domains within said interval;

(d) first determining a mean signal level value of the waveform signal levels in said domains and then selecting a threshold signal value based on the mean signal signal level value;

(e) searching the interval to locate a peak having a signal level greater than the threshold signal value and, if a peak is located, identifying a band corresponding to a nucleic acid base at the position of the peak, wherein the located peak is then selected to be the next search-fiducial point and steps (b)-(e) are repeated to identify all of said bands on the resolved rows;

or, if no peak is located, selecting a new search-fiducial point a predetermined distance from said original search-fiducial point; and

(f) determining the nucleic acid base sequence from the sequence of the bands identified in step (e).

2. The method as claimed in claim 1, wherein said given interval and said given level in the step (b), said threshold value in the step (c) and said given distance in the step (e) are set depending on the position on the one-dimensional waveform, respectively.

3. The method as claimed in claim 1, wherein said digital signals corresponding to the autoradiograph are obtained by placing the support medium and a stimulable phosphor sheet comprising a stimulable phosphor together in layers to record the autoradiograph of the resolved rows on the phosphor sheet as an energy-stored image, irradiating said phosphor sheet with stimulating rays and photoelectrically detecting the autoradiograph as stimulated emission.

4. The method as claimed in claim 1, wherein said digital signals corresponding to the autoradiograph are obtained by placing the support medium and a radiosensitive material together in layers to record the autoradiograph of the resolved rows on the radiosensitive material as a visible image and photoelectrically reading out the autoradiograph visualized on said radiosensitive material.

5. The method as claimed in claim 1, wherein the nucleic acid is DNA and the four groups of base-specific fragments consist of:

(1) guanine-specific DNA fragments;

(2) adenine-specific DNA fragments;

(3) thymine-specific DNA fragments; and

(4) cytosine-specific DNA fragments.

6. The method as claimed in claim 1, wherein the search-fiducial point selected in step (b) is located at the greatest distance along the one-dimensional migration direction and step (e) is conducted in the direction opposite to the resolution direction.

7. The method as claimed in claim 1, wherein the peak nearest to the selected search-fiducial point becomes the next search-fiducial point.

8. A signal processing method for determining the base sequence of a nucleic acid by obtaining digital signals corresponding to an autoradiograph of a resolved pattern including plural resolved rows consisting of four rows formed by resolving each of four groups of a mixture of base-specific nucleic acid fragments on a support medium labeled with a radioactive element in a one-dimensional direction on the support medium and processing the digital signals thus obtained to determine the nucleic acid base sequence, wherein said digital signal processing method includes the steps of:

(a) generating from said digital signals at least two adjacent waveforms representative of base position in the resolving direction and the signal level for each resolved row;

(b) identifying the peaks on each of the waveforms;

(c) selecting one peak on one of the waveforms;

(d) searching a given domain on either side of the selected peak to determine the existence of a peak on an adjacent waveform having a center corresponding to the center of the selected peak;

(e) repeating step (d) to search each adjacent waveform generated in step (a);

(f) determining the existence or nonexistence of a band corresponding to a nucleic acid base based on the presence or absence, respectively, of a series of corresponding peaks located on adjacent waveforms in steps (d) and (e); and

(g) repeating sequentially steps (c), (d), (e) and (f) until all of the bands on a resolved row have been detected.

9. The signal processing method as claimed in claim 8, wherein said digital signals corresponding to the autoradiograph are obtained by placing the support medium and a stimulable phosphor sheet comprising a stimulable phosphor together in layers to record the autoradiograph of the resolved rows on the phosphor sheet as an energy-stored image, irradiating said phosphor sheet with stimulating rays and photoelectrically detecting the autoradiograph as stimulated emission.

10. The signal processing method as claimed in claim 8, wherein said digital signals corresponding to the autoradiograph are obtained by placing the support medium and a radiosensitive material together in layers to record the autoradiograph of the resolved rows on the radiosensitive material as a visible image and photoelectrically reading out the autoradiograph visualized on said radiosensitive material.

11. The signal processing method as claimed in claim 8, wherein the nucleic acid is DNA and the four groups of base-specific fragments consist of:

(1) guanine-specific DNA fragments;

(2) adenine-specific DNA fragments;

(3) thymine-specific DNA fragments; and

(4) cytosine-specific DNA fragments.