US5580728

Method and system for genotyping

The present invention pertains to a process which can be fully automated for accurately determining the alleles of STR genetic markers. More specifically, the present invention is related to performing PCR amplification on a location of DNA, labelling the PCR products, converting the labels into a signal, removing a reproducible PCR stutter pattern from the signal by means of a computational device, and then determining the genotype of the location of the DNA. 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 DNA or RNA material from a genome;
(b) amplifying a location of the material, with the length of the location not exceeding fifty kilobases and the location containing a multinucleotide repeat region;
(c) labelling the amplified material with labels;
(d) converting the labels with a sensing device which produces a first electrical signal
(e) removing a reproducible pattern of the amplification from the first electrical signal using a program residing in the memory of a computer to form a third electrical signal:and
(f) producing from the third electrical signal a genotype of the material at the location.

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

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

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

5. A method as described in claim 1 wherein the deconvolution procedure includes the step of deconvolving using computed moment properties of the electrical signals, including moments of at least the second order.

6. A method as described in claim 1 wherein the removing step on the first electrical signal includes the step of deconvolving using Fourier techniques.

7. A method as described in claim 1 wherein the removing step on the first electrical signal includes the step of deconvolving using a noise tolerant procedure.

8. A method as described in claim 1 wherein the removing step on the first electrical signal includes the step of optimal (Wiener) filtering.

9. A method as described in claim 1 wherein the genotype corresponds to no more than two allele sizes.

10. A method as described in claim 1 wherein the amplification step is polymerase chain reaction using two oligonucleotide primers sufficiently complementary to non-repeated sequences that flank the multinucleotide repeat location.

11. A method as described in claim 1 wherein the amplified material is labeled.

12. A method as described in claim 1 wherein the oligonucleotide primers are obtained by direct synthesis.

13. A method as described in claim 1 wherein the oligonucleotide primers are end-labeled.

14. A method as described in claim 1 wherein more than one multinucleotide repeat location on a genome is characterized simultaneously.

15. A method as described in claim 1 used to establish the identity, pedigree, or biological relationship of an individual comprising comparison of the individual's genotype with the genotype of one or more distinct individuals.

16. A method as described in claim 1 wherein the sensing device includes the step of separating DNA molecules of distinct sizes, and determining a DNA concentration related to a size.

17. A method as described in claim 1 wherein the DNA or RNA material is genomic DNA or cloned DNA.

18. A system for genotyping comprising

(a) means for obtaining DNA or RNA material from a genome;
(b) means for amplifying a location of the material, with the length of the location not exceeding fifty kilobases and the location containing a multinucleotide repeat region, said amplifying means in communication with the DNA or RNA material;
(c) means for labelling the amplified material with labels, said labelling means in communication with amplified material;
(d) means for converting the labels with a sensing device which produces a first electrical signal, said converting means in communication with the labelling means;
(e) means for removing a reproducible pattern of the amplification from the first electrical signal using a program residing in the memory of a computer to form a third electrical signal, said removing means in communication with the first electrical signal;and
(f) means for producing from the third electrical signal a genotype of the material at the location, said producing means in communication with the removing means.

19. A system as described in claim 18 wherein:

(a) the amplifying means includes polymerase chain reaction;
(b) the converting means includes gel electrophoresis, or mass spectroscopy, or denaturing gradient gel electrophoresis, or differential hybridization, or sequencing by hybridization;
(c) the labelling means employs labels and related sensors that include 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 which includes a photomultiplier tube, a radioactivity counter, a resistivity sensor, a pH meter, or an optical detector; and
(d) the removing means includes statistical moment determinations, or Fourier transformation, or optimal filtering, or polynomial calculations, or matrix computations.

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

(a) obtaining DNA or RNA material from the organism;
(b) amplifying a location of the material, with the length of the location not exceeding fifty kilobases and the location containing a multinucleotide repeat region;
(c) labelling the amplified material with labels;
(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 per organism.

21. A method for identifying and isolating a gene comprising the steps of:

(a) obtaining DNA or RNA materials from genomes of a first set of individuals expressing a trait, and possibly of a second set of individuals whose members may be biologically related to a member of the first set of individuals;
(b) amplifying a location of the materials, with the length of the location not exceeding fifty kilobases and the location containing a multinucleotide repeat region;
(c) labelling the amplified material with labels;
(d) converting the labels with a sensing device which produces a first electrical signal;
(e) removing reproducible patterns of the amplifications from the first electrical signal using a program residing in the memory of a computer to form a third electrical signal;
(f) producing from the third electrical signal a genotype of the material at the location;
(g) identifying and isolating a gene linked to said trait by positional cloning;
(h) determining the DNA sequence of the gene;
(i) analyzing mutations in the genome of the first or second sets of individuals;
(j) verifying that the gene is causative or tightly linked to the trait by said analysis of mutations; and
(k) purifying derivatives of the gene that share at least 85% homology with the gene sequence.

22. A system for genotyping comprising:

(a) mechanism for obtaining DNA or RNA material from a genome;
(b) mechanism for amplifying a location of the material, with the length of the location not exceeding fifty kilobases and the location containing a multinucleotide repeat region, said amplifying mechanism in communication with the DNA or RNA material;
(c) mechanism for labelling the amplified material with labels, said labelling mechanism in communication with amplified material;
(d) mechanism for converting the labels with a sensing device which produces a first electrical signal, said converting mechanism in communication with the labelling mechanism;
(e) mechanism for removing a reproducible pattern of the amplification from the first electrical signal using a program residing in the memory of a computer to form a third electrical signal, said removing mechanism in communication with the first electrical signal; and
(f) mechanism for producing from the third electrical signal a genotype of the material at the location, said producing mechanism in communication with the removing mechanism.