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

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 [1,3–5]. 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 chromosome 12 working draft sequence (BAC clone, GenBank acc. no.: AC007655). The primer sequences, primer mixes and corresponding PCR products are shown in Table 1. Primer pairs DOF/DO1R and DOF/DO2R were used to identify DO1 and DO2 alleles, respectively. Primers HGHF and HGHR were included in all PCR reactions to amplify the internal positivecontrol PCR product, a 427-bp fragment from the human growth hormone (HGH) gene.

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 chromosome 12 working draft sequence (BAC clone, GenBank acc. no.: AC007655). The primer sequences, primer mixes and corresponding PCR products are shown in Table 1. Primer pairs DOF/DO1R and DOF/DO2R were used to identify DO1 and DO2 alleles, respectively. Primers HGHF and HGHR were included in all PCR reactions to amplify the internal positivecontrol PCR product, a 427-bp fragment from the human growth hormone (HGH) gene. The initial PCR was carried out with 1 µ l of DNA sample (0·15 -0·5 µ g), 1 µ l of diluted Taq polymerase (0·25 -0·33 U) and 8 µ l of DO typing PCR mix in a final 10-µ l reaction volume. After denaturation for 5 min at 95 ° C, samples were subjected to 30 cycles of PCR in a DNA thermal cycler. Each cycle comprised 95 ° C for 30 s, 60 ° C for 30 s and 72 ° C for 1·5 min, and was followed by a final extension at 72 ° C for 5 min. PCR products were analysed by electrophoresis on a 2% agarose gel containing 0·5 µ g/ml ethidium bromide and then visualized using UV transillumination. The DO typing PCR mix contained 10 m M Tris-HCl (pH 8·3), 50 m M KCl, 1·5 m M MgCl 2 , 0·001% (wt/vol) gelatin, 0·2 m M dNTPs, 0·5 µ M of each DO forward and reverse primer, and 0·2 µ M of each HGHF and HGHR primer. All PCR mixes were prepared in advance as a 'ready-to-use' kit (G & T Biotech, Rockville, MD). Eight microlitres of the mix was stored at -20 ° C under 8 µ l of mineral oil either in 0·2-ml PCR tubes or in 96-well PCR plates.
A total of 292 DNA samples from unrelated healthy Chinese blood donors were typed for DO1 and DO2 using the assay described above. The genomic DNA was isolated from 0·3 ml of EDTA or ACD anticoagulated blood using a DNA purification kit (G & T Biotech). The gene frequencies were 0·1027 for DO1 and 0·8973 for DO2 , showing a good fit to the Hardy-Weinberg equilibrium ( Table 2).
Validation criteria are essential to check the reliability and specificity of the typing method. Thirty DNA samples randomly selected from 292 donors were repeatedly tested and a concordance rate of 100% was observed. In addition, the validity of this method was verified by sequencing analysis. The complete DO exon 2 regions of two DO homozygous samples, DO1/1 and DO2/2 (Fig. 1a, donor 1 and donor 2), was first amplified by PCR using flanking primers 51979F and 51122R (Table 1) and then sequenced. A total of 782 bp was sequenced around exon 2 of the DO gene (GenBank acc. nos: AF340233 for DO1/1 and AF340234 for DO2/2 ). Three single-nucleotide differences between DO1/1 and DO2/2 individuals were found. Two changes, TAC to TAT encoding a Tyr at codon 126 and CTT to CTC encoding a Leu at codon 208, were silent substitutions. The third substitution, A to G at nucleotide position 892 (Fig. 1b), corresponded to DO1 and DO2 alleles, as reported previously [8]. Sequence alignment analysis indicated that the DNA sequences of these two Chinese samples are identical to the DOK1 and BAC clone sequences (data not shown), further supporting the validity of this typing method.
In conclusion, here we described a simple, accurate and inexpensive method of DO genotyping, which does not require the additional steps of probe hybridization or restriction enzyme digestion. The typing results can be visualized on a single photograph within 3 h, making this reliable method suitable for large-scale typing of potential blood donors without serological backup.