Partial genotyping by differential hybridization

Abstract

In a method of detecting the number of repeat units in a selected short tandem repeat (STR) in a genomic sample, the genomic sample is amplified by PCR and a single stranded target DNA is selected and separated for use in differential hybridization experiments. Subsequent partial genotyping comprises the steps of admixing to the target DNA at least one fluorescent labeled STR probe oligonucleotide and one different fluorescent labeled reference probe oligonucleotide allowing hybridization to the single stranded target DNA in a hybridization experiment. Measurement of the fluorescence intensities of the probes that are bound to the repeat units of the selected STR and normalizing the fluorescence intensity of the STR probes on the base of the reference probe intensity reveals a relative fluorescence signal representing the result of the differential hybridization experiment. Also disclosed are kits for carrying out partial genotyping by differential hybridization.

What is claimed is: 
     
         1 . A method of detecting the number of repeat units in a selected short tandem repeat (STR) in a genomic sample, the method comprising the steps of:
 a) providing at least one:
 a1) genomic sample containing the selected STR; 
 a2) set of polymerase chain reaction—(PCR) oligonucleotides for carrying out PCR amplification of the selected genomic sample with the short STR sequence; 
 a3) STR probe oligonucleotide (P1,P1′) with individual fluorescent label; 
 a4) reference probe oligonucleotide (P2) with a different fluorescent label; 
   b) amplifying the genomic sample with the selected STR sequence provided in step a1) by PCR using the set of PCR oligonucleotides provided in step a2), denaturing the amplified double stranded sample DNA for generating single stranded DNA, selecting and separating a single stranded target DNA for use in hybridization experiments;   c) carrying out partial genotyping by differential hybridization that comprises the steps:
 c1) mixing an amount of the single stranded target DNA selected and separated in step b) with the STR probe oligonucleotides provided in step a3) and the reference probe oligonucleotide (P2), and allowing hybridization to the single stranded target DNA in a hybridization experiment; 
 c2) measuring the intensity of the fluorescence provided by the labeled STR probe oligonucleotides that are bound to the repeat units of the selected STR; 
 c3) measuring the intensity of the different fluorescence provided by the labeled reference probe oligonucleotide (P2) that is bound to a flanking sequence of the target DNA; and 
 c4) normalizing the intensity measured of the STR probe fluorescence in step c2) based on the intensity measured of the different fluorescence in step c3) and revealing a relative fluorescence signal representing the result of the differential hybridization experiment. 
   
     
     
         2 . The method of  claim 1 ,
 wherein a first STR probe oligonucleotide (P1) is a 16-mer probe to a 5′-ATCT-3′ repeat.   
     
     
         3 . The method of  claim 1 ,
 wherein in step a3) a second STR probe oligonucleotide (P1′) is provided in addition.   
     
     
         4 . The method of  claim 3 ,
 wherein the second STR probe oligonucleotide (P1′) is a 20-mer probe to a 5′-ATCT-3′ repeat.   
     
     
         5 . The method of  claim 1 , further comprising the step:
 a5) providing a set of flanking oligonucleotides (F1,F2);   wherein in step c1), the set of flanking oligonucleotides (F1,F2) provided in step a5) is admixed to a first amount of the single stranded target DNA, to the STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a first differential hybridization experiment (FF).   
     
     
         6 . The method of  claim 1 , further comprising the step:
 a6) providing a blocking oligonucleotide (B2) and a flanking oligonucleotide (F1);   wherein in step c1), the blocking oligonucleotide (B2) and the flanking oligonucleotide (F1) provided in step a5) are admixed to a second amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a second differential hybridization experiment (FB).   
     
     
         7 . The method of  claim 1 , further comprising the step:
 a7) providing a blocking oligonucleotide (B1) and a flanking oligonucleotide (F2);   wherein in step c1), the blocking oligonucleotide (B1) and the flanking oligonucleotide (F2) provided in step a7) are admixed to a second amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in an alternative second differential hybridization experiment (BF).   
     
     
         8 . The method of  claim 1 , further comprising the step:
 a8) providing a set of blocking oligonucleotides (B1,B2);   wherein in step c1), the set of blocking oligonucleotides (B1,B2) provided in step a8) is admixed to a first amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a third differential hybridization experiment (BB).   
     
     
         9 . The method of  claim 1 , further comprising the steps of:
 a5) providing a set of flanking oligonucleotides (F1,F2);   wherein in step c1), the set of flanking oligonucleotides (F1,F2) provided in step a5) is admixed to a first amount of the single stranded target DNA, to the STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a first differential hybridization experiment (FF);   a6) providing a blocking oligonucleotide (B2) and a flanking oligonucleotide (F1);   wherein in step c1), the blocking oligonucleotide (B2) and the flanking oligonucleotide (F1) provided in step a5) are admixed to a second amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a second differential hybridization experiment (FB);   a8) providing a set of blocking oligonucleotides (B1,B2);   wherein in step c1), the set of blocking oligonucleotides (B1,B2) provided in step a8) is admixed to a first amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a third differential hybridization experiment (BB);   wherein the steps c2), c3), and c4) are carried out for the three differential hybridization experiments; and   wherein three relative fluorescence signals representing the individual results of the three differential hybridization experiments (FF,FB,BB) are achieved.   
     
     
         10 . The method of  claim 1 , further comprising the steps of:
 a5) providing a set of flanking oligonucleotides (F1,F2);   wherein in step c1), the set of flanking oligonucleotides (F1,F2) provided in step a5) is admixed to a first amount of the single stranded target DNA, to the STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a first differential hybridization experiment (FF);   a7) providing a blocking oligonucleotide (B1) and a flanking oligonucleotide (F2);   wherein in step c1), the blocking oligonucleotide (B1) and the flanking oligonucleotide (F2) provided in step a7) are admixed to a second amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in an alternative second differential hybridization experiment (BF);   a8) providing a set of blocking oligonucleotides (B1,B2);   wherein in step c1), the set of blocking oligonucleotides (B1,B2) provided in step a8) is admixed to a first amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a third differential hybridization experiment (BB);   wherein the steps c2), c3), and c4) are carried out for the three differential hybridization experiments; and   wherein three relative fluorescence signals representing the individual results of the three differential hybridization experiments (FF,BF,BB) are achieved.   
     
     
         11 . The method of  claim 1 , further comprising the steps of:
 a5) providing a set of flanking oligonucleotides (F1,F2);   wherein in step c1), the set of flanking oligonucleotides (F1,F2) provided in step a5) is admixed to a first amount of the single stranded target DNA, to the a STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a first differential hybridization experiment (FF);   a6) providing a blocking oligonucleotide (B2) and a flanking oligonucleotide (F1);   wherein in step c1), the blocking oligonucleotide (B2) and the flanking oligonucleotide (F1) provided in step a5) are admixed to a second amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a second differential hybridization experiment (FB);   a7) providing a blocking oligonucleotide (B1) and a flanking oligonucleotide (F2);   wherein in step c1), the blocking oligonucleotide (B1) and the flanking oligonucleotide (F2) provided in step a7) are admixed to a second amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in an alternative second differential hybridization experiment (BF);   a8) providing a set of blocking oligonucleotides (B1,B2);   wherein in step c1), the set of blocking oligonucleotides (B1,B2) provided in step a8) is admixed to a first amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a third differential hybridization experiment (BB);   wherein the steps c2), c3), and c4) are carried out for the four differential hybridization experiments; and   wherein four relative fluorescence signals representing the individual results of the three differential hybridization experiments (FF,FB,BF,BB) are achieved.   
     
     
         12 . The method of  claim 1 ,
 wherein the reference probe (P2) is labeled with Cy5 at the 3′-end of the oligonucleotide.   
     
     
         13 . The method of  claim 2 ,
 wherein the first STR probe (P1) is labeled with FAM at the 3′-end of the oligonucleotide.   
     
     
         14 . The method of  claim 3 ,
 wherein the second STR probe (P1′) is labeled with Cy3 at the 3′-end of the oligonucleotide.   
     
     
         15 . A kit (XX) for carrying out the method of partial genotyping by differential hybridization of  claim 1 , the kit (XX) comprising:
 one STR probe oligonucleotide (P1); and   one reference probe oligonucleotide (P2).   
     
     
         16 . A kit for carrying out the method of partial genotyping by differential hybridization of  claim 3 , the kit comprising:
 one first STR probe oligonucleotide (P1);   one second STR probe oligonucleotide (P1′);   one reference probe oligonucleotide (P2); and one of:
 two flanking oligonucleotides (F1,F2); or 
 one blocking oligo (B1) and one flanking oligo (F2); or 
 one flanking oligo (F1) and one blocking oligo (B2); or 
 two blocking oligonucleotides (B1,B2). 
   
     
     
         17 . The kit of  claim 16 , further comprising:
 one insert probe oligonucleotide (P3).   
     
     
         18 . A kit (FF) for carrying out the method of partial genotyping by differential hybridization of  claim 5 , the kit (FF) comprising:
 one STR probe oligonucleotide (P1);   one reference probe oligonucleotide (P2); and   two flanking oligonucleotides (F1,F2).   
     
     
         19 . A kit (FB) for carrying out the method of partial genotyping by differential hybridization of  claim 6 , the kit (FB) comprising:
 one STR probe oligonucleotide (P1);   one reference probe oligonucleotide (P2);   one flanking oligonucleotide (F1); and   one blocking oligonucleotide (B2).   
     
     
         20 . A kit (BF) for carrying out the method of partial genotyping by differential hybridization of  claim 7 , the kit (BF) comprising:
 one STR probe oligonucleotide (P1);   one reference probe oligonucleotide (P2);   one blocking oligonucleotide (B1); and   one flanking oligonucleotide (F2).   
     
     
         21 . A kit (BB) for carrying out the method of partial genotyping by differential hybridization of  claim 8 , the kit (BB) comprising:
 one STR probe oligonucleotide (P1);   one reference probe oligonucleotide (P2); and   two blocking oligonucleotides (B1,B2).