US5876933

Method and system for genotyping

The present invention pertains to a process which can be fully automated for accurately determining the alleles of genetic markers. More specifically, the present invention is related to performing PCR amplification on locations of DNA to generate a reproducible pattern, labeling the PCR products, converting the labels into a signal, operating on the signal, and then determining the genotype of the location of the DNA. An amplification can include multiple locations from the DNA of one or more individuals. The invention also pertains to genetics applications and systems which can effectively use this genotyping information.

What is claimed is:

1. A method for genotyping comprised of the steps:

(a) obtaining nucleic acid material from a genome;
(b) amplifying locations of the material to generate a reproducible pattern;
(c) labeling the amplified material with labels;
(d) converting the labels with a sensing device which produces a first electrical signal;
(e) operating on the first electrical signal to form a third electrical signal; and
(f) producing from the third electrical signal a genotype of the material at the locations.

2. A method as described in claim 1 wherein the reproducible pattern corresponds to a PCR stutter artifact of the location.

3. A method as described in claim 1 wherein the step of operating on the first electrical signal includes the step of a deconvolution procedure.

4. A method as described in claim 1 wherein the step of operating on the first electrical signal includes the step of deconvolving with a second electrical signal related to the reproducible pattern of the amplification.

5. A method as described in claim 1 wherein the step of operating on the first electrical signal includes the step of deconvolving with matrix processing of the electrical signals.

6. A method as described in claim 4 wherein the second electrical signal is formed by the steps:

(a) obtaining nucleic acid material from a genome;
(b) amplifying locations of the material to generate a reproducible pattern;
(c) labeling the amplified material with labels;
(d) converting the labels with a sensing device which produces a first electrical signal; and
(e) operating on the first electrical signal produced from the amplified material to produce a second electrical signal corresponding to a reproducible pattern.

7. A method as described in claim 1 wherein the obtaining step pools nucleic acid material from one or more individuals.

8. A method as described in claim 1 wherein the amplifying step includes more than one polynucleotide repeat location.

9. A method as described in claim 1 wherein the amplifying step includes more than one polynucleotide repeat location, and the size properties of these locations may overlap.

10. A method as described in claim 1 wherein the amplifying step uses more than one location, the size properties of these locations are not necessarily disjoint, and the first set of electrical signals shows concentrations of the amplified material from different locations having the same size.

11. A method as described in claim 1 wherein the amplifying step uses more than one location, the size properties of these locations are not necessarily disjoint, the first set of electrical signals shows concentrations of the amplified material from different locations having the same size, and the PCR stutter patterns of the different locations provide the primary mechanism for genotyping the locations.

12. A method as described in claim 1 wherein the operating step makes use of a second set of electrical signals corresponding to response patterns of the locations.

13. A system for genotyping comprising:

(a) means for obtaining nucleic acid material from a genome;
(b) means for amplifying locations of the material to generate a reproducible pattern, a reaction vessel within said amplifying means in contact with the nucleic acid material;
(c) means for assaying the amplified material, said assaying means in contact with the reaction vessel;
(d) means for converting the assayed amplified material into a first electrical signal, said converting means in communication with the assaying means; and
(e) means for operating on the first electrical signal with a second electrical signal to form a third electrical signal, said operating means in communication with the electrical signals.

14. A system as described in claim 13 wherein:

(a) the amplifying means includes polymerase chain reaction, or harvesting cloned cells;
(b) the assaying means includes gel or ultrathin gel electrophoresis, or mass spectroscopy, or denaturing gradient gel electrophoresis, or differential hybridization, or sequencing by hybridization;
(c) the converting means employs labeling with detection including radioactivity, or fluorescence, or phosphorescence, or chemiluminescence, or visible light, or ions, or pH, or electricity, or resistivity, or biotinylation, or antibodies; and includes the detecting means or mechanism which includes a photomultiplier tube; a radioactivity counter, a resistivity sensor, a pH meter, or an optical detector; and
(d) the operating means includes statistical moment determinations, or Fourier transformation, or optimal filtering, or polynomial calculations, or matrix computations.

15. A method for analyzing genetic material of an organism comprising the steps of:

(a) obtaining nucleic acid material from the organism;
(b) amplifying locations of the material to generate a reproducible pattern;
(c) labeling the amplified material with labelsl;
(d) converting the labels with a sensing device which produces a first electrical signal; and
(e) producing the genotype of an amplified location of the genetic material in an electronic acquisition system comprising a region having a radius of less than five feet at a rate exceeding 100 genotypes per hour.

16. A method as described in claim 1 wherein the step of operating on the first electrical signal includes the step of determining a numerical value corresponding to an error.

17. A method as described in claim 1 wherein the step of producing the genotype includes the step of determining a numerical value corresponding to the genotype accuracy.

18. A method as described in claim 1 wherein the step of operating on the first electrical signal includes forming a first third electrical signal and a second third electrical signal, and the step of producing a genotype includes a comparison of the first and second third electrical signals.

19. A method of determining the risk of genetic disease comprising the steps of:

(a) producing a genotype of related individuals according to the method of claim 1
(b) determining the phenotypic risk of disease for an individual by using the genotype as a linked genetic marker; and
(c) presenting the results of the risk determination.

20. A method of constructing a genetic linkage map comprising the steps of:

(a) determining a genotype according to the method of claim 1;
(b) entering the genotype into a computer device with memory; and
(c) applying a computer program to the entered genotype to build the genetic linkage map.

21. A method of localizing a gene expressing a phenotype comprising the steps of:

(a) determining a genotype according to the method of claim 1;
(b) entering the genotype into a computer device with memory; and
(c) applying a computer program to the entered genotype to localize the gene expressing the phenotype.

22. A method of positionally cloning a gene comprising the steps of:

(a) determining a genotype according to the method of claim 1;
(b) entering the genotype and pedigree information into a computer device with memory;
(c) applying a computer program to the entered genotype and the pedigree information to obtain localization information for the gene relative to a genetic linkage map; and
(d) positionally cloning the gene using the localization information.

23. A method as described in claim 22 where after the step of positionally cloning the gene there is the step of determining the sequence of the cloned gene.

24. A method as described in claim 23 where after the step of determining the gene sequence there is the step of constructing a diagnostic test related to the sequence of the gene.

25. A method as described in claim 23 where after the step of determining the gene sequence there is the step of developing a molecule that binds to a protein product of the gene sequence.

26. A method of observing a cancerous material comprising the steps of:

(a) determining a genotype of the cancerous material according to the method of claim 1;
(b) entering the genotype into a computer device with memory; and
(c) comparing the entered genotype with a reference genotype to form an observation.

27. A method of genetic fingerprinting comprising the steps of:

(a) determining a genotype of an individual according to the method of claim 1;
(b) entering the genotype into a computer device with memory; and
(c) recording the genotype together with an identifier of the individual to form a genetic fingerprint.

28. A method as described in claim 27 where after the step of forming the genetic fingerprint there is the step of comparing the genetic fingerprint with a reference genetic fingerprint.

29. A method of identifying a genetic pattern of inheritance in a population comprising the steps of:

(a) determining genotypes of a plurality of individuals according to the method of claim 1;
(b) entering the genotypes into a computer device with memory; and
(c) comparing the genotypes to identify a genetic pattern of inheritance in the population.

30. A method of assessing genetic risk of disease in individuals comprising the steps of:

(a) determining a genotype of an individual according to the method of claim 1;
(b) entering the genotype into a computer device with memory; and
(c) comparing the genotype with reference information that relates genotype and disease risk to assess risk of disease in the individual.