Method for the simultaneous determination of blood group and platelet antigen genotypes

Abstract

RBC and platelet (Plt) alloimmunization requires antigen-matched blood to avoid adverse transfusion reactions. Some blood collection facilities use unregulated Abs to reduce the cost of mass screening, and later confirm the phenotype with government approved reagents. Alternatively, RBC and Plt antigens can be screened by virtue of their associated single nucleotide polymorphisms (SNPs). We developed a multiplex PCR-oligonucleotide extension assay using the GenomeLab SNPStream platform to genotype blood for a plurality of blood group antigen-associated SNPs, including but not limited to: RhD (2), RhC/c, RhE/e, S/s, K/k, Kp a/b , Fya/b, FY0, Jk a/b , Di a/b , and HPA-1a/b.

1 .- 19 . (canceled) 
     
     
         20 . A probe having (i) at least one of the nucleotide sequence of nucleotide positions 1 to 20 of any one of SEQ ID NO: 25 to 35 and nucleotide position 1 to 18 of SEQ ID NO: 6 and (ii) the nucleotide sequence of nucleotide positions 21 to 45 of as set forth in SEQ ID NO: 25. 
     
     
         21 . The probe of  claim 20  for use as an extension probe for the detection of a C/T SNP in an exon 4 of a RHD gene. 
     
     
         22 . A blood group/platelet antigen typing kit comprising a first oligonucleotide having a nucleotide sequence as set forth in SEQ ID NO: 1, a second oligonucleotide having a nucleotide sequence as set forth in SEQ ID NO: 2 and a probe having the nucleotide sequence as defined in  claim 20 . 
     
     
         23 . A method of detecting a RhD/RhCE blood group antigen in a sample, said method comprising:
 (a) providing genomic DNA from said sample;   (b) submitting the genomic DNA of step (a) to a PCR amplification with at the first oligonucleotide as defined in claim  3  and the second oligonucleotide as defined in claim  3  to obtain at least one amplification product; and   (c) analyzing the at least one amplification product of step (b) to detect the blood group antigen in the sample.   
     
     
         24 . The method of  claim 23 , wherein the at least one amplification product is digested with a restriction enzyme. 
     
     
         25 . The method of  claim 24 , wherein said restriction enzyme is Exonuclease I or shrimp alkaline phosphatase. 
     
     
         26 . The method of  claim 23 , further comprising, in step (c), generating at least one extension product from the at least one amplification product. 
     
     
         27 . The method of  claim 26 , further comprising hybridizing the at least one extension production with a probe having the sequence of nucleotide positions 21 to 45 of as set forth in SEQ ID NO: 25. 
     
     
         28 . The method of  claim 27 , wherein the probe has the sequence as defined in  claim 20 . 
     
     
         29 . The method of  claim 27 , wherein said extension product is hybridized to a tag-arrayed microplate.