Method for determining DNA sequences

• US 5,119,316 A
• Filed: 06/29/1990
• Issued: 06/02/1992
• Est. Priority Date: 06/29/1990
• Status: Expired due to Term

• Current Assignee: PerkinElmer Life Sciences, Inc.
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Dam, Rudy J., Hochberg, Alan M., Prober, James M., Davis, James E.
• Primary Examiner(s): Lall, Parshotam S.
• Assistant Examiner(s): ZANELLI, MICHAEL J
• Application Number:
  US07/545,746
• Time in Patent Office: 704 Days
• Field of Search: 364/498, 364/484, 364/485, 364/486, 364/487, 364/555, 364/554, 364/551.01, 364/550, 364/571.01, 364/571.02, 364/571.05, 364/497, 364/496, 364/413.1 , 364/413.07-413.09, 250/458.1, 324/77 R, 324/77 A, 324/77 B, 435/6, 935/77, 73/23.36, 73/61.1 C
• US Class Current: 702/20
• CPC Class Codes: G01N 27/44717 Arrangements for investigating the separated zones, e.g. localising zones

Abstract

The method of this invention obtains the underlying peak shapes of two signal spectrums whose ratios are representative of different species. The underlying peak shapes are fitted to the signal spectrums in a way that yield accurate ratios. The ratios are accurate even when the location of the peaks are not precisely known.

Claims

1. In a method for obtaining signals characteristic of different species following the species separation in time and/or space and determining the identity of such species, the method including generating a first signal that varies in amplitude in a first sense to form peaks as a function of the nature of the species, generating a second signal that varies in amplitude in a second sense different than the first sense to form peaks as a function of the nature of the species, any peak of the first and second signal being approximately representable by the sum of three or more peak templates, and obtaining a third signal corresponding to the ratio of the first and second signals, the amplitude of the third signal being indicative of the identity of each of the species, the improvement wherein the third signal is obtained by the steps:

finding a sequence of peaks XYX in each of the first and second signals where X represents one species and Y represents another species,

subtracting a fraction of the first signal from the second signal from approximately the peak center of the first X to approximately the peak center of the second X in each sequence of peaks to obtain differences such that the amplitude of the X peaks in each difference is substantially zero, thereby obtaining a prototypical template for a peak, and

fitting the respective prototypical template to the respective first and second signals to obtain the peak amplitude of the first and second signals for obtaining the third signal from the ratio of such peak amplitudes.

2. The method set forth in claim 1 further characterized by:

differentiating the prototypical templates of the first and second signals one or more times, and

fitting by least squares the sum of the prototypical templates for each signal and their corresponding derivatives to each first and second signals.

3. The method set forth in claim 1 further characterized by:

shifting the prototypical templates from each signal forward and backward with respect to the preceding and succeeding peak position in order to reduce the summed squared difference between each signal and a weighted sum of the templates.

4. The method set forth in claim 3 achieved by:

fitting each signal to a weighted sum of shifted prototypical templates and their derivatives, so that the derivatives terms minimize the summed squared differences between each signal and the corresponding summed templates by compensating for errors in the choices of initial prototypical template positions.

5. The method set forth in claim 4 further characterized by differentiating the prototypical and shifted templates of the first and second signal spectrums before the least squares fitting.

6. In a method for obtaining signals characteristic of different species following the species separation in time and/or space and determining the identity of such species, the method including generating a first signal that varies in amplitude in a first sense to form peaks as a function of the nature of the species, generating a second signal that varies in amplitude in a second sense different than the first sense to form peaks as a function of the nature of the species, any peak of the first and second signal being approximately representable by the sum of three successive peak templates, and obtaining a third signal corresponding to the ratio of the peaks in the first and second signal, the amplitude of the third signals being indicative of the identity of each of the species, the improvement wherein the third signal is obtained by the steps:

obtaining a template for fitting to each of the signals, and

fitting the templates to the respective first and second signals to obtain the peak amplitude of the first and second signals for obtaining the third signal from the ratio of such peak amplitudes.

7. The method set forth in claim 6 further characterized by:

differentiating the template for fitting the first and second signals, and

fitting by least squares the sum of the templates and their derivatives to each first and second signals.

8. The method of claim 7 further characterized by finding the combination of templates and their derivatives that fit each of the first and second signals, thereby to determine the first and second signal amplitudes for each peak.