Methods for detection of genetic disorders

• US 7,727,720 B2
• Filed: 08/26/2005
• Issued: 06/01/2010
• Est. Priority Date: 05/08/2002
• Status: Active Grant

• Current Assignee: Ravgen, Inc.
• Original Assignee: Ravgen, Inc.
• Inventors: Dhallan, Ravinder S.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US11/212,812
• Publication Number:
  US 20060121452A1
• Time in Patent Office: 1,740 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention provides a method useful for detection of genetic disorders. The method comprises determining the sequence of alleles of a locus of interest, and quantitating a ratio for the alleles at the locus of interest, wherein the ratio indicates the presence or absence of a chromosomal abnormality. The present invention also provides a non-invasive method for the detection of chromosomal abnormalities in a fetus. The invention is especially useful as a non-invasive method for determining the sequence of fetal DNA. The invention further provides methods of isolation of free DNA from a sample.

Claims

1. A method for detecting a free nucleic acid, wherein said method comprises:

(a) isolating free nucleic acid from a non-cellular fraction of a sample, wherein said sample comprises an agent that impedes cell lysis, if cells are present, and wherein said agent is selected from the group consisting of membrane stabilizer, cross-linker, and cell lysis inhibitor; and

(b) detecting the presence or absence of the free nucleic acid.

2. The method of claim 1, wherein said sample is obtained from a source selected from the group consisting of bacteria, viruses, fungi, mycobacteria, protozoa, molds, yeasts, plants, humans, non-humans, multi-cellular parasite, animals, and archeabacteria.

3. The method of claim 2, wherein the sample is obtained from a human source.

4. The method of claim 1, wherein the sample is obtained from a source selected from the group consisting of:

tissue, blood, serum, plasma, saliva, urine, tear, vaginal secretion, umbilical cord blood, chorionic villi, amniotic fluid, embryonic tissue, lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascitic fluid, fecal matter, and body exudates.

5. The method of claim 4, wherein said sample is blood.

6. The method of claim 5, wherein said sample is obtained from plasma from said blood.

7. The method of claim 1, wherein said nucleic acid contains at least one mutation.

8. The method of claim 7, wherein said mutation is selected from the group consisting of:

single point mutation, multiple point mutations, an insertion, a frameshift, a truncation, a deletion, a duplication, and a transversion.

9. The method of claim 7, wherein said mutation is in a gene selected from the group consisting of:

BRCA1, BRCA2, MSH6, MSH2, MLH1, RET, PTEN, ATM, H-RAS, p53, ELAC2, CDH1, APC, AR, PMS2, MLH3, CYP1A1, GSTP1, GSTM1, AXIN2, CYP19, MET, NAT1, CDKN2A, NQ01, trc8, RAD51, PMS1, TGFBR2, VHL, MC4R, POMC, NROB2, UCP2, PCSK1, PPARG, ADRB2, UCP3, glur1, cart, SORBS1, LEP, LEPR, SIM1, TNF, IL-6, IL-1, IL-2, IL-3, IL1A, TAP2, THPO, THRB, NBS1, RBM15, LIE, MPL, RUNX1, Her-2, glucocorticoid receptor, estrogen receptor, thyroid receptor, p21, p27, K-RAS, N-RAS, retinoblastoma protein, Wiskott-Aldrich (WAS) gene, Factor V Leiden, Factor II (prothrombin), methylene tetrahydrofolate reductase, cystic fibrosis, LDL receptor, HDL receptor, superoxide dismutase gene, SHOX gene, genes involved in nitric oxide regulation, genes involved in cell cycle regulation, tumor suppressor genes, oncogenes, genes associated with neurodegeneration, and genes associated with obesity.

10. The method of claim 1, wherein said free nucleic acid is from a species different from the species from which the sample was taken.

11. The method of claim 10, wherein said different species is from a group consisting of bacteria, viruses, fungi, mycobacteria, protozoa, molds, yeasts, plants, humans, non-humans, multi-cellular parasite, animals, and archeabacteria.

12. The method of claim 11, wherein said different species is a bacteria.

13. The method of claim 12, wherein said bacterial species is a gram-positive or gram-negative bacteria.

14. The method of claim 13, wherein said bacteria is selected from the group consisting of:

Acidaminococcus, Acinetobacter, Acinetobacter Iwoffi, Aeromonas, Alcaligenes, Bacteroides, Bordetella, Branhamella, Brucella, Calyrnmatobacterium, Campylobacter, Cardiobacterium, Chromobacterium, Citrobacter, Citrobacter freundii, Cotiform group, Edwardsiella, Enterobacter, Enterobacter sakazaki, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter agglomerans, Enterococcus, Enterococcus faecalis, Enterococcus faecium, Escherichia, Escherichia coli, Escherichia coli-O157, Flavobacterium, Francisella, Fusobacterium, Haemophilus, Hafnia alvei, Kiebsiella, Kiebsiella oxytoca, Kiebsiella pneumoniae, Legionella, Moraxella, Morganella, Morganella morganii, Neisseria, Pasturella, Plesiomonas, Proteus, Providencia, Proteus mirabilis, Pseudomonas, Pseudomonas aeruginosa, Salmonella, Salmonella typhimurium, Serratia, Serratia marcescens, Shigella, Shigella flexneri, Streptobacillus, Veillonella, Vibrio, Vibrio cholera, Yersinia, Yersinia entercolitica, Xanthomanas maltophiia, Staphylococcus, Staphylococcus albus, Staphylococcus aureus, Streptococcus, Streptococcus dysgalacticae, Micrococcus, Peptococcus, Peptostreptococcus, Bacillus, Bacillus cereus, Clostridium, Lactobacillus, Listeria, Listeria monocytogenes, Erysipelothrix, Propionibacterium, Eubacterium, and Corynebacterium.

15. The method of claim 11, wherein said different species is a virus.

16. The method of claim 15, wherein said virus is a DNA virus or an RNA virus.

17. The method of claim 15, wherein said virus is selected from the group consisting of:

retrovirus, pathogenic virus, non-pathogenic virus, drug-resistant virus, drug-sensitive virus, adeno-associated virus, bird flu virus, cauliflower mosaic virus, cytomegalovirus (CMV), dengue virus, Epstein-Barr virus, feline leukemia virus, flavivirus, haemophilus influenza, hemorrhagic fever viruses, hepatitis virus including hepatitis A, B, C, and B, viruses, herpes simplex virus, human herpesvirus type A and B, human immunodeficiency virus (HIV), human papilloma virus, human T-cell lymphotrophic virus, HTLV Type I, HTLV Type II, influenza virus, Japanese encephalitis virus, moraxella catarrhalis, non-typeable haemophilus, reovirus, parainfluenza, parvovirus, papova virus, Respiratory syncytial virus, Rubella virus, rotavirus, SARS, tomato bushy stunt virus, varicella-zoster virus, and vaccinia virus.

18. The method of claim 11, wherein said different species is a fungus.

19. The method of claim 18, wherein said fungus is a drug-sensitive fungus or a drug-resistant fungus.

20. The method of claim 18, wherein said fungus is selected from the group consisting of:

Candida, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida stellatoidea, Candida krusei, Candida parakrusei, Candida lusitanae, Candida pseudotropicalis, Candida guilliennondi, Candida glabrata, Aspergillus, Aspergillus fumigatis, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Cryptococcus, Histoplasma, Coccidioides, Paracoccidioides, Blastomyces, Basidiobolus, Conidiobolus, Rhizopus, Rhizomucor, Mucor, Absidia, Mortierella, Cunninghamella, Saksenaea, Pseudallescheria, Sporotrichosis, Fusarium, Trichophyton, Trichosporon, Microsporum, Epidennophyton, Scytalidium, Malassezia, Actinomycetes, Sporothrix, Penicillium, Saccharomyces and Pneumocystis.

21. The method of claim 1, wherein isolation of free nucleic acid comprises a centrifugation step.

22. The method of claim 21, wherein the centrifugation step is performed with the centrifuge braking power set to zero.

23. The method of claim 21, wherein the centrifugation step is performed at a speed selected from the group consisting of 0-50 rpm, 50-100 rpm, 100-200 rpm, 200-300 rpm, 300-400 rpm, 400-500 rpm, 500-600 rpm, 600-700 rpm, 700-800 rpm, 800-900 rpm, 900-1000 rpm, 1000-2000 rpm, 2000-3000 rpm, 3000-4000 rpm, 4000-5000 rpm, 5000-6000 rpm, 6000-7000 rpm, 7000-8000 rpm, and greater than 8000 rpm.

24. The method of claim 1, wherein said method is used to detect, diagnose, or monitor a disease.

25. The method of claim 24, wherein said disease is cancer.

26. The method of claim 25, wherein said cancer is selected from the group consisting of:

carcinoma of the bladder, breast, bronchial, colon, kidney, liver, lung, esophagus, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin;

small cell lung cancer, squamous cell carcinoma, hematopoietic tumors of lymphoid lineage, leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin'"'"'s lymphoma, non-Hodgkin'"'"'s lymphoma, hairy cell lymphoma, Burkett'"'"'s lymphoma, hematopoietic tumors of myeloid lineage, acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia, tumors of mesenchymal origin, fibrosarcoma and rhabdomyosarcoma, tumors of the central and peripheral nervous system, astrocytoma, neuroblastoma, glioma and schwannomas, melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi'"'"'s sarcoma.

27. The method of claim 1, wherein said method is used to monitor response to treatment.

28. The method of claim 27, wherein the treatment is selected from the group consisting of surgery, radiation, lifestyle change, dietary protocol, supplementation and administration of a drug.

29. The method of claim 28, wherein the treatment is administration of a drug.

30. The method of claim 29, wherein said drug is selected from the group consisting of:

chemotherapeutic agents, anti-bacterial agents, anti-viral agents, anti-fungal agents, targeted-cancer drugs, cytoxoic agents, cytostatic agents, anti-proliferative agents, Avastin, altretamine, busulfan, chlorambucil, cyclophosphamide, Erbitux, Rituxan, ifosfamide, mechlorethamine, melphalan, thiotepa, cladribine, fluorouracil, floxuridine, gemcitabine, thioguanine, pentostatin, methotrexate, 6-mercaptopurine, cytarabine, carmustine, lomustine, streptozotocin, carboplatin, cisplatin, oxaliplatin, iproplatin, tetraplatin, lobaplatin, JM216, JM335, fludarabine, aminoglutethimide, flutamide, goserelin, leuprolide, megestrol acetate, cyproterone acetate, tamoxifen, anastrozole, bicalutamide, dexamethasone, diethylstilbestrol, prednisone, bleomycin, dactinomycin, daunorubicin, doxirubicin, idarubicin, mitoxantrone, losoxantrone, mitomycin-c, plicamycin, paclitaxel, docetaxel, CPT-11, epothilones, topotecan, irinotecan, 9-amino camptothecan, 9-nitro camptothecan, GS-211, etoposide, teniposide, vinblastine, vincristine, vinorelbine, procarbazine, asparaginase, pegaspargase, methotrexate, octreotide, estramustine, and hydroxyurea.