Methods for detection of genetic disorders

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.

1 . A method for determining a sequence of a locus of interest comprising: (a) amplifying a locus of interest on template DNA, wherein the template DNA comprises a mixture of fetal DNA and maternal DNA and is obtained from a sample from a pregnant female, using a first and second primers, wherein the second primer contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5′ overhang containing the locus of interest; (b) digesting the amplified DNA with the restriction enzyme that recognizes the recognition site on the second primer; (c) incorporating a nucleotide into the digested DNA of (b) by using the 5′ overhang containing the locus of interest as a template; and (d) determining the sequence of the locus of interest by determining the sequence of the DNA of (c). 
     
     
         2 . The method of  claim 1 , wherein said sample is selected from the group consisting of blood, serum, plasma, saliva, urine, tears, vaginal secretion, sweat, lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascitic fluid, fecal matter, and body exudates. 
     
     
         3 . The method of  claim 1 , wherein said sample is blood. 
     
     
         4 . The method of  claim 3 , 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. 
     
     
         5 . The method of  claim 3 , wherein said template DNA is obtained from plasma from said blood. 
     
     
         6 . The method of  claim 3 , wherein said template DNA is obtained from serum from said blood. 
     
     
         7 . The method of  claim 1 , wherein the restriction enzyme cuts DNA at a distance from the recognition site. 
     
     
         8 . The method of  claim 1 , wherein the recognition site is for a Type IIS restriction enzyme. 
     
     
         9 . The method of  claim 8 , wherein the Type IIS restriction enzyme is selected from the group consisting of: Alw I, Alw26 I, Bbs I, Bbv I, BceA I, Bmr I, Bsa I, Bst71 I, BsmA I, BsmB I, BsmF I, BspM I, Ear I, Fau I, Fok I, Hga I, Pie I, Sap I, SSfaN I, and Sthi32 I. 
     
     
         10 . The method of  claim 1 , wherein said method of amplification is PCR. 
     
     
         11 . The method of  claim 10 , wherein an annealing temperature for cycle 1 of PCR is about the melting temperature of the portion of the 3′ region of the second primer that anneals to the template DNA. 
     
     
         12 . The method of  claim 11 , wherein an annealing temperature for cycle 2 of PCR is about the melting temperature of the portion of the 3′ region of the first primer that anneals to the template DNA. 
     
     
         13 . The method of  claim 12 , wherein an annealing temperature for the remaining cycles of PCR is at about the melting temperature of the entire second primer. 
     
     
         14 . The method of  claim 1 , wherein determining the sequence comprises a method selected from the group consisting of allele specific PCR, mass spectrometry, hybridization, primer extension, fluorescence resonance energy transfer (FRET), sequencing, Sanger dideoxy sequencing, DNA microarray, southern blot, slot blot, dot blot, and MALDI-TOF mass spectrometry. 
     
     
         15 . A method for determining a sequence of alleles of a locus of interest comprising: (a) amplifying alleles of a locus of interest on a template DNA, wherein the template DNA comprises a mixture of fetal DNA and maternal DNA and is obtained from a sample from a pregnant female: using a first and second primers, wherein the second primer contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5′overhang containing the locus of interest; (b) digesting the amplified DNA with the restriction enzyme that recognizes the recognition site on the second primer; (c) incorporating nucleotides into the digested DNA of (b), wherein; (i) a nucleotide that terminates elongation, and is complementary to the locus of interest of an allele, is incorporated into the 5′ overhang of said allele, and (ii) a nucleotide complementary to the locus of interest of a different allele is incorporated into the 5′ overhang of said different allele, and said terminating nucleotide, which is complementary to a nucleotide in the 5′ overhang of said different allele, is incorporated into the 5′ overhang of said different allele; (d) determining the sequence of the alleles of a locus of interest by determining the sequence of the DNA of (c). 
     
     
         16 . The method of  claim 15 , wherein said sample is selected from the group consisting of blood, serum, plasma, saliva, urine, tears, vaginal secretion, sweat, lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascitic fluid, fecal matter, and body exudates. 
     
     
         17 . The method of  claim 15 , wherein said sample is blood. 
     
     
         18 . The method of  claim 17 , wherein said template DNA is obtained from plasma from said blood. 
     
     
         19 . The method of  claim 17 , wherein said template DNA is obtained from serum from said blood. 
     
     
         20 . The method of  claim 15 , wherein the recognition site is for a Type IIS restriction enzyme. 
     
     
         21 . The method of  claim 20 , wherein the Type IIS restriction enzyme is selected from the group consisting of: Alw I, Alw26 I, Bbs I, Bbv I, BceA I, Bmr I, Bsa I, Bst71 I, BsmA I, BsmB I, BsmF I, BspM I, Ear I, Fau I, Fok I, Hga I, Pie I, Sap I, SSfaN I, and Sthi32 I.