Methods for detection of genetic disorders

• US 7,718,370 B2
• Filed: 12/28/2006
• Issued: 05/18/2010
• Est. Priority Date: 03/01/2002
• Status: Expired due to Fees

• Current Assignee: Ravgen, Inc.
• Original Assignee: Ravgen, Inc.
• Inventors: Dhallan, Ravinder S.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US11/648,778
• Publication Number:
  US 20070122835A1
• Time in Patent Office: 1,237 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• 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 2527/125 Specific component of sample, medium or buffer
• 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 the presence or absence of a fetal chromosomal abnormality, said method comprising:

(a) determining the sequence of alleles of a locus of interest from an RNA template in a sample obtained from a pregnant female comprising maternal and fetal nucleic acid, wherein said sample is selected from the group consisting of blood, serum and plasma and further wherein said sample is obtained from the pregnant female when the fetus is at a gestational age selected from the group consisting of;

5-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, 40-44, 44-48, and 48-52 weeks; and

(b) quantitating a ratio of the relative amount of alleles of said locus of interest from said RNA, wherein said ratio indicates the presence or absence of a fetal chromosomal abnormality.

2. The method of claim 1, wherein said fetal chromosomal abnormality is selected from the group consisting of trisomy 13, trisomy 18, and trisomy 21.

3. The method of claim 1, wherein alleles of multiple loci of interest are sequenced and their relative amounts quantitated and expressed as a ratio.

4. The method of claim 3, wherein said multiple loci of interest are on multiple chromosomes

5. The method of claim 4, wherein said ratio of alleles at a loci of interest on a chromosome are summed and compared to the ratio of alleles at loci of interest on a different chromosome, wherein a difference in ratios indicates the presence of a chromosomal abnormality.

6. The method of claim 5, wherein the chromosomes that are compared are human chromosomes selected from the group consisting of:

chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, and Y.

7. The method of claim 5, wherein the ratio of the alleles at loci of interest of chromosomes 13, 18, and 21 are compared.

8. The method of claim 1, wherein said locus of interest is a single nucleotide polymorphism.

9. The method of claim 1, wherein said locus of interest is a mutation.

10. The method of claim 1, wherein said fetal chromosomal abnormality is on an X chromosome or on a Y chromosome.

11. The method of claim 1, wherein said fetal chromosomal abnormality is trisomy 21.

12. The method of claim 1, wherein said fetal chromosomal abnormality is trisomy 13.

13. The method of claim 1, wherein said fetal chromosomal abnormality is trisomy 18.