Methods for detection of genetic disorders

• US 20060160105A1
• Filed: 08/26/2005
• Published: 07/20/2006
• Est. Priority Date: 05/08/2002
• Status: Active Grant

• Current Assignee: Ravgen, Inc.
• Original Assignee: Ravgen, Inc.
• Inventors: Dhallan, Ravinder S.
• Granted Patent: US 7,442,506 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2533/101 Primer extension

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 preparing a sample for analysis, comprising isolating free nucleic acid from a sample that contains nuleic acid, wherein formalin has been added to said sample to a concentration selected from the group consisting of:

0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, and 2-3%.

2. The method of claim 1, wherein said sample is obtained from a source selected from the group consisting of human, non-human, mammal, reptile, cattle, cat, dog, goat, swine, pig, monkey, ape, gorilla, bull, cow, bear, horse, sheep, poultry, mouse, rat, fish, dolphin, whale, and shark.

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:

a cell, fetal cell, 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, or body exudates.

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

6. The method of claim 5, wherein said blood is from a pregnant female.

7. The method of claim 6, wherein said blood is obtained from a human pregnant female when the fetus is at a gestational age selected from the group consisting of:

0-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, 40-44, 44-48, 48-52, and more than 52 weeks.

8. The method of claim 7, wherein said sample is obtained from plasma from said blood.

9. The method of claim 1, wherein the concentration of formalin in the sample is 0.1%.

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

11. The method of claim 10, wherein the centrifugation step is performed with the centrifuge braking power set to zero.

12. The method of claim 11, 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.

52. The method of claim 51, wherein the cell lysis inhibitor is formalin at a percentage selected from the group consisting of:

0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, and 2-3%.

53. The method of claim 52, wherein the concentration of formalin in the sample is 0.1%.

13. A method for detecting a chromosomal abnormality in a sample, said method comprising:

(a) determining the sequence of alleles of a locus of interest in a sample from template DNA, wherein determining the sequence of the alleles comprises;

(1) amplification of the locus of interest;

(2) hybridization of amplified loci to GeneCHIP array

(3) washing GeneCHIP array;

(4) staining the GeneCHIP array with detectable reagents; and

(5) scanning GeneCHIP array.

14. The method of claim 13, wherein the amplification method in (a)(1) is selected from the group consisting of:

polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, and splice overlap extension polymerase chain reaction.

15. The method of claim 14, wherein said method of amplification is by PCR.

16. The method of claim 13, wherein said staining method comprises streptavidin phycoerhthrin and biotinylated anti-streptavidin.

51. The method of claims 13, 17, 24, 25, 29, 37, and 47, wherein a cell lysis inhibitor has been added to the sample.

17. A method for detecting a chromosomal abnormality in a sample, said method comprising:

(a) determining the sequence of alleles of a locus of interest from template DNA,

wherein determining the sequence of the alleles comprises;

(1) amplification of the locus of interest;

(2) amplicon fragmentation;

(3) hybridization of fragmented amplicons to CodeLink Arrays;

(4) extension reaction to incorporate a nucleotide; and

(5) detection of incorporated nucleotides.

18. The method of claim 17, wherein the amplification method is selected from the group consisting of:

polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, and splice overlap extension polymerase chain reaction.

19. The method of claim 18 wherein said method of amplification is by PCR.

20. The method of claim 17, wherein said amplicon fragmentation is by exonuclease digestion.

21. The method of claim 17, wherein said incorporated nucleotide is a dideoxynucleotide or deoxynucleotide.

22. The method of claim 21, wherein said incorporated nucleotide is labeled with a molecule selected from the group consisting of radioactive molecule, fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate, biotin, derivative of biotin, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, and moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity.

23. The method of claim 22, wherein the labeled nucleotide is labeled with a fluorescent molecule.

24. A method for detecting a chromosomal abnormality in a sample, said method comprising:

(a) determining the sequence of alleles of a locus of interest from template DNA, wherein determining the sequence of the alleles comprises using BeadArray Technology.

25. A method for detecting a chromosomal abnormality in a sample, said method comprising:

(a) determining the sequence of alleles of a locus of interest from template DNA, wherein determining the sequence of the alleles comprises;

(1) amplification of the locus of interest;

(2) dephosphorylation of the unused reagents in (a);

(3) in vitro transcription reaction of the products of (b);

(4) RNase A cleavage of the products of (c);

(5) mixing the products of (d) with CleanResin;

(6) transfer products of (e) to SpectroCHIP; and

(7) analysis of the SpectroCHIP.

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

polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, and splice overlap extension polymerase chain reaction.

27. The method of claim 26, wherein said method of amplification is by PCR.

28. The method of claim 25, wherein said dephosphorylation reaction is with shrimp alkaline phosphatase.

29. A method for detecting a chromosomal abnormality in a sample, said method comprising:

(a) determining the sequence of alleles of a locus of interest from template DNA, wherein determining the sequence of the alleles comprises;

(1) amplification of a locus of interest;

(2) dephosphorylation of the unused reagents in (a);

(3) hybridization of a primer to the locus of interest;

(4) incorporation of a nucleotide;

(5) mixing the products of (d) with CleanResin;

(6) transfer products of (e) to SpectroCHIP; and

(7) analysis of the SpectroCHIP.

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

polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, and splice overlap extension polymerase chain reaction.

31. The method of claim 30, wherein said method of amplification is by PCR.

32. The method of claim 29, wherein said dephosphorylation reaction is with shrimp alkaline phosphatase.

33. The method of claim 29, wherein said hybridization of primer is adjacent to the locus of interest.

34. The method of claim 29, wherein said incorporated nucleotide is a dideoxynucleotide or deoxynucleotide.

35. The method of claim 34, wherein said incorporated nucleotide is labeled with a molecule selected from the group consisting of radioactive molecule, fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate, biotin, derivative of biotin, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, and moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity.

36. The method of claim 35, wherein the labeled nucleotide is labeled with a fluorescent molecule.

37. A method for detecting a chromosomal abnormality in a sample, said method comprising:

(a) determining the sequence of alleles of a locus of interest from template DNA, wherein determining the sequence of the alleles comprises;

(1) amplification of the locus of interest;

(2) exonuclease treatment of the products of (1);

(3) single stranded DNA of (2) is annealed to an oligonucleotide;

(4) incorporation of a nucleotide using the annealed template and primer of (3);

(5) detection of the incorporated nucleotide.

38. The method of claim 37, wherein the amplification method is selected from the group consisting of:

polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, and splice overlap extension polymerase chain reaction.

39. The method of claim 38, wherein said method of amplification is by PCR.

40. The method of claim 37, wherein said primer hybridizes adjacent to the locus of interest.

41. The method of claim 37, wherein said incorporated nucleotide is a dideoxynucleotide or deoxynucleotide.

42. The method of claim 37, wherein said incorporation reaction comprises two terminating nucleotides and two non-terminating nucleotides.

43. The method of claim 41, wherein said incorporated nucleotide is labeled with a molecule selected from the group consisting of radioactive molecule, fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate, biotin, derivative of biotin, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, and moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity.

44. The method of claim 42, wherein said terminating nucleotides are labeled with a molecule selected from the group consisting of radioactive molecule, fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate, biotin, derivative of biotin, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, and moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity.

45. The method of claim 43, wherein the labeled nucleotide is labeled with a fluorescent molecule.

46. The method of claim 44, wherein the terminating nucleotides are labeled with a fluorescent molecule.

47. A method for detecting a chromosomal abnormality in a sample, said method comprising:

(a) determining the sequence of alleles of a locus of interest from template DNA, wherein determining the sequence of the alleles comprises;

(1) amplification of the locus of interest, wherein the amplification reaction comprises a forward primer, a reverse primer, and a probe that anneals to the locus of interest, which is within the region of the amplicon, and further, wherein the probe contains a reporter dye at one end of the probe, and a quenching dye at the other end of the probe; and

(2) detection of the PCR products, wherein the amount of PCR product is used to determine the presence or absence of a specific genetic sequence.

48. The method of claim 47, wherein the amplification is by PCR.

49. The method of claim 47, wherein the probe contains a reporter dye at the 5′

end and the 3′

end contains a quenching dye.

50. The method of claim 47, wherein the PCR products are detected using the ABI 7700 Sequence Detection System.

54. A composition comprising fetal DNA and matemal DNA, wherein the percentage of free fetal DNA in the total free DNA of the composition is selected from the group consisting of:

about 15-16% fetal DNA, about 16-17% fetal DNA, about 17-18% fetal DNA, about 18-19% fetal DNA, about 19-20% fetal DNA, about 20-21% fetal DNA, about 21-22% fetal DNA, about 22-23% fetal DNA, about 23-24% fetal DNA, about 24-25% fetal DNA, about 25-35% fetal DNA, about 35-45% fetal DNA, about 45-55% fetal DNA, about 55-65% fetal DNA, about 65-75% fetal DNA, about 75-85% fetal DNA, about 85-90% fetal DNA, about 90-91% fetal DNA, about 91-92% fetal DNA, about 92-93% fetal DNA, about 93-94% fetal DNA, about 94-95% fetal DNA, about 95-96% fetal DNA, about 96-97% fetal DNA, about 97-98% fetal DNA, about 98-99% fetal DNA, and about 99-99.7% fetal DNA.

55. A composition comprising fetal DNA and maternal DNA, wherein the percentage of free fetal DNA in the total free DNA of the composition is selected from the group consisting of:

about 15-16% fetal DNA, about 16-17% fetal DNA, about 17-18% fetal DNA, about 18-19% fetal DNA, about 19-20% fetal DNA, about 20-21% fetal DNA, about 21-22% fetal DNA, about 22-23% fetal DNA, about 23-24% fetal DNA, about 24-25% fetal DNA, about 25-35% fetal DNA, about 35-45% fetal DNA, about 45-55% fetal DNA, about 55-65% fetal DNA, about 65-75% fetal DNA, about 75-85% fetal DNA, about 85-90% fetal DNA, about 90-91% fetal DNA, about 91-92% fetal DNA, about 92-93% fetal DNA, about 93-94% fetal DNA, and about 94-95% fetal DNA.

56. A prenatal diagnostic method comprising analyzing a composition comprising fetal DNA and maternal DNA, wherein the percentage of free fetal DNA in the total free DNA of the composition is selected from the group consisting of:

about 15-16% fetal DNA, about 16-17% fetal DNA, about 17-18% fetal DNA, about 18-19% fetal DNA, about 19-20% fetal DNA, about 20-21% fetal DNA, about 21-22% fetal DNA, about 22-23% fetal DNA, about 23-24% fetal DNA, about 24-25% fetal DNA, about 25-35% fetal DNA, about 35-45% fetal DNA, about 45-55% fetal DNA, about 55-65% fetal DNA, about 65-75% fetal DNA, about 75-85% fetal DNA, about 85-90% fetal DNA, about 90-91% fetal DNA, about 91-92% fetal DNA, about 92-93% fetal DNA, about 93-94% fetal DNA, and about 94-95% fetal DNA.