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.

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Claims: What is claimed is:

A method for genotyping comprised of the steps:
1. obtaining nucleic acid material from a genome;
2. amplifying locations of the material to generate a reproducible pattern;
3. labeling the amplified material with labels;
4. converting the labels with a sensing device which produces a first electricalsignal;
5. operating on the first electrical signal to form a third electrical signal; and
6. producing from the third electrical signal a genotype of the material at thelocations.
A method as described in claim 1 wherein the reproducible pattern corresponds toa PCR stutter artifact of the location.
A method as described in claim 1 wherein the step of operating on the firstelectrical signal includes the step of a deconvolution procedure.
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.
A method as described in claim 1 wherein the step of operating on the firstelectrical signal includes the step of deconvolving with matrix processing of theelectrical signals.
A method as described in claim 4 wherein the second electrical signal is formed bythe steps:
1. obtaining nucleic acid material from a genome;
2. amplifying locations of the material to generate a reproducible pattern;
3. labeling the amplified material with labels;
4. converting the labels with a sensing device which produces a first electricalsignal; and
5. operating on the first electrical signal produced from the amplified material toproduce a second electrical signal corresponding to a reproducible pattern.
A method as described in claim 1 wherein the obtaining step pools nucleic acidmaterial from one or more individuals.
A method as described in claim 1 wherein the amplifying step includes more thanone polynucleotide repeat location.
A method as described in claim 1 wherein the amplifying step includes more thanone polynucleotide repeat location, and the size properties of these locations mayoverlap.
A method as described in claim 1 wherein the amplifying step uses more thanone location, the size properties of these locations are not necessarily disjoint, and thefirst set of electrical signals shows concentrations of the amplified material fromdifferent locations having the same size.
A method as described in claim 1 wherein the amplifying step uses more thanone location, the size properties of these locations are not necessarily disjoint, the firstset of electrical signals shows concentrations of the amplified material from differentlocations having the same size, and the PCR stutter patterns of the different locationsprovide the primary mechanism for genotyping the locations.
A method as described in claim 1 wherein the operating step makes use of asecond set of electrical signals corresponding to response patterns of the locations.
A system for genotyping comprising:
1. means for obtaining nucleic acid material from a genome;
2. means for amplifying locations of the material to generate a reproduciblepattern, a reaction vessel within said amplifying means in contact with the nucleicacid material;
3. means for assaying the amplified material, said assaying means in contactwith the reaction vessel;
4. means for converting the assayed amplified material into a first electricalsignal, said converting means in communication with the assaying means; and
5. means for operating on the first electrical signal with a second electricalsignal to form a third electrical signal, said operating means in communicationwith the electrical signals.
A system as described in claim 13 wherein:
1. the amplifying means includes polymerase chain reaction, or harvestingcloned cells;
2. the assaying means includes gel or ultrathin gel electrophoresis, or massspectroscopy, or denaturing gradient gel electrophoresis, or differentialhybridization, or sequencing by hybridization;
3. the converting means employs labeling with detection including radioactivity,or fluorescence, or phosphorescence, or chemiluminescence, or visible light, orions, or pH, or electricity, or resistivity, or biotinylation, or antibodies; andincludes the detecting means or mechanism which includes a photomultipliertube; a radioactivity counter, a resistivity sensor, a pH meter, or an opticaldetector; and
4. the operating means includes statistical moment determinations, or Fouriertransformation, or optimal filtering, or polynomial calculations, or matrixcomputations.
A method for analyzing genetic material of an organism comprising the steps of:
1. obtaining nucleic acid material from the organism;
2. amplifying locations of the material to generate a reproducible pattern;
3. labeling the amplified material with labelsl;
4. converting the labels with a sensing device which produces a first electricalsignal; and
5. producing the genotype of an amplified location of the genetic material in anelectronic acquisition system comprising a region having a radius of less thanfive feet at a rate exceeding 100 genotypes per hour.
A method as described in claim 1 wherein the step of operating on the firstelectrical signal includes the step of determining a numerical value corresponding to anerror.
A method as described in claim 1 wherein the step of producing the genotypeincludes the step of determining a numerical value corresponding to the genotypeaccuracy.
A method as described in claim 1 wherein the step of operating on the firstelectrical signal includes forming a first third electrical signal and a second thirdelectrical signal, and the step of producing a genotype includes a comparison of the firstand second third electrical signals.
A method of determining the risk of genetic disease comprising the steps of:
1. producing a genotype of related individuals according to the method of claim 1
2. determining the phenotypic risk of disease for an individual by using thegenotype as a linked genetic marker; and
3. presenting the results of the risk determination.
A method of constructing a genetic linkage map comprising the steps of:
1. determining a genotype according to the method of claim 1;
2. entering the genotype into a computer device with memory; and
3. applying a computer program to the entered genotype to build the geneticlinkage map.
A method of localizing a gene expressing a phenotype comprising the steps of:
1. determining a genotype according to the method of claim 1;
2. entering the genotype into a computer device with memory; and
3. applying a computer program to the entered genotype to localize the geneexpressing the phenotype.
A method of positionally cloning a gene comprising the steps of:
1. determining a genotype according to the method of claim 1;
2. entering the genotype and pedigree information into a computer device withmemory;
3. applying a computer program to the entered genotype and the pedigreeinformation to obtain localization information for the gene relative to a geneticlinkage map; and
4. positionally cloning the gene using the localization information.
A method as described in claim 22 where after the step of positionally cloning thegene there is the step of determining the sequence of the cloned gene.
A method as described in claim 23 where after the step of determining the genesequence there is the step of constructing a diagnostic test related to the sequence ofthe gene.
A method as described in claim 23 where after the step of determining the genesequence there is the step of developing a molecule that binds to a protein product ofthe gene sequence.
A method of observing a cancerous material comprising the steps of:
1. determining a genotype of the cancerous material according to the method ofclaim 1;
2. entering the genotype into a computer device with memory; and
3. comparing the entered genotype with a reference genotype to form anobservation.
A method of genetic fingerprinting comprising the steps of:
1. determining a genotype of an individual according to the method of claim 1;
2. entering the genotype into a computer device with memory; and
3. recording the genotype together with an identifier of the individual to form agenetic fingerprint.
A method as described in claim 27 where after the step of forming the geneticfingerprint there is the step of comparing the genetic fingerprint with a reference geneticfingerprint.
A method of identifying a genetic pattern of inheritance in a populationcomprising the steps of:
1. determining genotypes of a plurality of individuals according to the method ofclaim 1;
2. entering the genotypes into a computer device with memory; and
3. comparing the genotypes to identify a genetic pattern of inheritance in thepopulation.
A method of assessing genetic risk of disease in individuals comprising the stepsof:
1. determining a genotype of an individual according to the method of claim 1;
2. entering the genotype into a computer device with memory; and
3. comparing the genotype with reference information that relates genotype anddisease risk to assess risk of disease in the individual.