Polymerase chain reaction with sequence‐specific primers‐based genotyping of the human Dombrock blood group DO1 and DO2 alleles and the DO gene frequencies in Chinese blood donors

Abstract

The Dombrock blood group system (ISBT 014, DO) was discovered 36 years ago and has been associated with haemolytic transfusion reactions [1,2]. Two common antigens, DO1 (Do a ) and DO2 (Do b ), and other three high-incidence antigens -DO3 (Gy a ), DO4 (Hy) and DO5 (Jo a ) -were identified using serological methods [1,[3][4][5]. Usually it is difficult to obtain monospecific DO-typing reagents and there are only limited DO gene-frequency studies, especially in the Chinese population [2,6]. As serological DO typing has severe limitations, establishing a DNA-based DO genotyping technique appears to be essential. Recently in a linkage study the DO locus was assigned to chromosome 12p12.3-p13.2 (chromosome 12, short arm, region 1, band 2, sub-band 3, through band 3, sub-band 2) [7]. More recently, the DO gene has been successfully cloned, ending a long period of searching for the molecular basis of the DO1 / DO2 polymorphism [8]. Homology studies suggested that the DO molecule is a member of the adenosine 5 ′ -diphosphate (ADP)-ribosyltransferase ectoenzyme gene family [8]. DO1 and DO2 alleles are the result of a single nucleotide substitution causing an amino acid change within an encoded arginine-glycine-aspartic acid (RGD) motif of the molecule [8]. On the basis of these findings, we have developed, for the first time, a polymerase chain reaction with sequence-specific primers (PCR-SSP)-based DO1 and DO2 genotyping method using newly designed allele-specific primers.The DO gene comprises 3 exons spanning 13 743 base pairs (bp) and predicts a peptide of 314 amino acid residues. A single nucleotide change from A to G at nucleotide position 892 (numbering according to the DOK1 clone, GenBank acc. no.: AF29004) within exon 2 results in the substitution of asparagine (N) for aspartic acid (D) at position 265 of the protein sequence. Serological testing indicated that N265 and D265 corresponded to the phenotypes DO:1,-2 [DO(a+b-) ] and DO:-1,2 [DO(a-b+) ], respectively [8]. In order to detect A892G substitution, two allele-specific reverse primers and a single forward-consensus primer were designed according to the DOK1 clone sequence and the human

DNA‐based typing of Kell, Kidd, MNS, Dombrock, Colton, and Yt blood group systems in the French Basques

“…The following alleles were explored according to previously published genotyping procedures: KEL*1 specific thymidine and KEL*2 specific cytosine at nucleotide 698 for the KEL gene (Hessner et al, 1996); JK*1 specific guanidine and JK*2 specific adenine at nucleotide 838 for the SCLA14A1 gene (Irshaid et al, 1998); MNS*1 specific guanidine and thymidine and MNS*2 specific adenine and guanidine at positions 71 and 72, respectively, of the GYPA gene; MNS*3 specific thymidine and MNS*4 specific cytosine at position 143 of the GYPB gene for the MNS system (Eshleman et al, 1995;Huang et al, 1991); DO*1 specific adenine and DO*2 specific guanidine at nucleotide 793 for the ART4 gene (Wu et al, 2001); CO*1 specific cytosine and the CO*2 specific thymidine at nucleotide 134 for the AQP1 gene (Joshi et al, 2001); YT*A specific cytosine and YT*B specific adenine at nucleotide 1057 for the ACHE gene (Yan et al, 2005) (Table 1).…”

Membrane Blood Group Antigens and Antibodies

“…This is particularly useful when antibodies are not available or are weakly reactive. A good example is the Dombrock blood group polymorphism, where DNAbased assays [112][113][114] are used to type patients and donors for Do a and Do b to overcome the problem of not having reliable typing reagents. Furthermore, the newer DNA technologies have the potential for screening pools of DNA for rare blood types and thereby increasing the number of donors that can be tested.…”

Recurrent acute hemolytic transfusion reactions by antibodies against Doa antigens, not detected by cross‐matching

“…Polymerase chain reaction with sequence-specific priming (PCR-SSP) was adapted from the method of Wu and colleagues. 3 DOA/DOB PCR-SSP was performed in a thermal cycler (PE 9700, Perkin Elmer, Norwalk, CT) on 100 ng of gDNA in a total volume of 50 µL (range of input, 50-500 ng of DNA). Reaction mixtures contain 15 pmol of each primer (DOAR, DOBR, DOF, HGHF, and HGHR), 0.2 mmol per L each dNTP, 1.5 mmol per L MgCl 2 , 2.0 U of Taq DNA polymerase (Promega, Leiden, Netherlands) in the appropriate buffer.…”