Methods and compositions for flow cytometric determination of DNA sequences

• US 6,057,107 A
• Filed: 04/06/1998
• Issued: 05/02/2000
• Est. Priority Date: 10/11/1995
• Status: Expired due to Term

• Current Assignee: Luminex Corporation (DiaSorin S.p.A.)
• Original Assignee: Luminex Corporation (DiaSorin S.p.A.)
• Inventors: Fulton, R. Jerrold
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/055,329
• Time in Patent Office: 757 Days
• Field of Search: 422/68.1, 435/6, 435/91.1, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: G01N 15/1012 Calibrating particle analysers; References therefor
• CPC Class Codes: G01N 15/1456 without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1014 Constitution of reference particles
• CPC Class Codes: G01N 2015/1477 Multiparameters
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2015/1488 Methods for deciding
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form

Abstract

A method for the analysis of DNA sequences and PCR products comprises the steps of constructing an oligonucleotide-labeled beadset, and labeled complementary probe, and exposing the beadset and probe to a DNA fragment or PCR product under hybridizing conditions and analyzing the combined sample/beadset by flow cytometry. Flow cytometric measurements are used to classify beads within an exposed beadset to determine the presence of identical or nonidentical sequences within the test sample. The inventive technology enables the rapid analysis of DNA sequences and detection of point mutations, deletions and/or inversions while also reducing the cost and time for performing genetic assays.

Claims

1. A method of detecting a non-mutated genetic sequence in a nucleic acid sample comprising the steps of:

(a) selecting a detector probe suitable for detecting the non-mutated genetic sequence;

(b) preparing a fluorescent nucleic acid probe complementary to the detector probe;

(c) coupling the selected probe to each bead of a plurality of beads to form a bead aliquot;

(d) amplifying, by PCR, the non-mutated genetic sequence of the nucleic acid sample;

(e) mixing the bead aliquot, the nucleic acid sample, and the fluorescent probe to form a mixture;

(f) incubating the mixture under a competitive hybridization condition;

(g) measuring a fluorescence of the each bead; and

(h) detecting the non-mutated genetic sequence, or absence thereof, as a function of the measured fluorescence.

4. The method according to claim 1, wherein the nucleic acid sample includes a RNA.

5. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid sample includes an exon sequence.

6. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid sample includes an intron sequence.

7. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid includes a part of an exon sequence and a part of an intron sequence.

8. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid includes a genomic sequence.

9. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid belongs to a gene of an infectious agent selected from the group consisting of bacteria, viruses, rickettsia, fungi, mycoplasma, chlamydia, and protozoa.

10. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid belongs to a polymorphic gene.

11. The method according to claim 10, wherein the polymorphic gene comprises MHC genes.

12. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid belongs to a gene selected from the group consisting of cystic fibrosis gene, multiple endocrine neoplasia type 2a (MEN2a), multiple endocrine neoplasia type 2b (MEN2b), multiple endocrine neoplasia type 1 (MEN 1), ret proto-oncogene, low density lipoprotein (LDL) receptor, neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2), breast and ovarian cancer susceptibility type 1 (BRCA1), breast and ovarian cancer susceptibility type 2 (BRCA2), breast and ovarian cancer susceptibility type 3 (BRCA3), adenomatous polyposis coli (APC), adenosine deaminase, xeroderma pigmentosum group A correcting gene (XPAC), excision repair cross complementing rodent repair deficiency complementation group 6 (ERCC6), fragile X mental retardation protein 1 (fmr1), Duchenne muscular dystrophy gene, myotonic dystrophy protein kinase, androgen receptor, Huntington'"'"'s disease associated gene, hypoxanthine-guanine phosphoribotransferase (HPRT), apolipoprotein E, beta-hexosaminidase alpha chain (HEXA), steroid 21-hydroxylase, angiotensin, human nodular mixed lymphocytic and histiocytic cell mismatch repair (hNMLH1 and 2), retinoblastoma susceptibility (Rb), transformation-associated protein 53 (p53), breakpoint cluster region/tyrosine-protein kinase (bcr/abl), B-cell leukemia/lymphoma 2 (bcl-2), genes encoding ion transporters, and combinations thereof.

13. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid belongs to a gene comprising a ras gene.

14. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid belongs to a gene associated with diseases and clinical disorders selected from the group consisting of human myotonia, paramyotonia congenita, hyperkalemic periodic paralysis, hypertrophic cardiomyopathy, hereditary ovalocytic red blood cells, hereditary spherocytosis, glucose/galactose malabsorption, familial hypercholesterolemia, tuberous sclerosis, severe combined immunodeficiency, autoimmune disease, insulin-dependent diabetes mellitus, Cockayne'"'"'s syndrome, spinal and bulbar muscular atrophy, Peutz-Jegher'"'"'s syndrome, Lesh-Nyhan syndrome, Tay-Sachs disease, Alzheimer'"'"'s disease, congenital adrenal hyperplasia and hypertension, essential hypertension, hereditary non-polyposis colon cancer, hereditary colon cancer, colon cancer, familial retinoblastoma, Li-Fraumeni syndrome, chronic myelogenous leukemia, follicular and diffuse lymphoma, malignant lymphoma, skin cancer, lung cancer, pancreatic cancer, and combination thereof.

15. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid belongs to a gene selected from the group consisting of gene sequences provided in GENBANK.

16. The method according to claim 1, wherein said fluorescence measuring step (f) at least in part is performed by a flow cytometry.

17. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid includes a partially non-mutated sequence.

18. The method according to claim 1, wherein the nucleic acid sample includes a mutated sequence.

19. The method according to claim 18, wherein the mutated sequence results from a genetic mutation including at least one of a point mutation, a substitution, an insertion, a deletion, and an inversion.

20. The method according to claim 18, wherein the mutated sequence results from at least one of a translocation and a transposition.

21. The method according to claim 18, wherein the mutated sequence results from a somatic mutation.

22. The method according to claim 18, wherein the mutated sequence results from a germinal mutation.

23. The method according to claim 18, wherein the mutated sequence results from an induced mutation.

24. The method according to claim 18, wherein the mutated sequence results from a spontaneous mutation.

25. The method according to claim 18, wherein the mutated sequence results from a conservative mutation.

26. The method according to claim 18, wherein the mutated sequence results from a degenerate mutation.

27. The method according to claim 1, wherein the non-mutated genetic sequence of the nucleic acid belongs to an allele.

2. A method of detecting a non-mutated genetic sequence in a non-mutated genetic sequence comprising the steps of:

(a) selecting a detector probe suitable for detecting the non-mutated genetic sequence;

(b) preparing a fluorescent nucleic acid probe complementary to the detector probe;

(c) coupling the selected probe to each bead of a plurality of beads to form a bead aliquot;

(d) amplifying, by PCR, the non-mutated genetic sequence of the nucleic acid sample;

(e) mixing the bead aliquot, the nucleic acid sample, and the fluorescent probe to form a mixture;

(f) incubating the mixture under a competitive hybridization condition;

(g) measuring a fluorescence of the each bead; and

(h) detecting the non-mutated genetic sequence, or absence thereof, as a function of the measured fluorescence,

wherein the nucleic acid sample includes a DNA.

3. The method according to claim 2, wherein the DNA includes a satellite DNA.