The Detection Of RHDψ Pseudogene And RHD-CE-D Hybridization Gene And Medicine Application In Kunming RH-negative Ones

Objective:To have a study of D gene polymorphism of RHD-negative blood group of Kunming resident population with PCR technology for gene typing.To study the allele with its molecular basis and the population frequence among Chinese people.Meanwhile,the topic research is to survey the distribution state and find more D variants.In the end,PCR detecting method for RHD gene is to be quickly established and perfected,and provide information for antenatal diagnosis and clinic blood-transfusion.Method:To select healthy population of Kunming as research object.Firstly,screening 46 samples of RHD-negative with serum method.Secondly,detecting out Del phenotype with absorption and elution test.Then extracting genome DNA as template of PCR with classic method of phenol-chloroform-isoamyl alcohol and analyze the D gene polymorphism through PCR technology.That is,have an amplification of all the samples and their D exons formulations using the exon-sequence specificity primers.For the ones of D gene part existion,they are to be amplified with the intron-sequence specificity primers.Then PAGE electrophoresis and AgNO3 silver staining with banding.At last,RHD_ψand RHD-CE-D are to be examined out by DNA sequencing.Results:Among 46 RHD(-)samples,The number of D gene to be absent wholely is 29,that is true RHD(-);Del is 12,they have a complete D gene;Of the 5 ones for D gene part existing,there are 2 only existing D10 exon,1 is to be D7,D9,D10,both D4,D6 and only D6 are eachly one.With DNA sequencing results displaying,2 samples of only containing D10 are both RHD-CE(3-9)-D,the sample with only D4 and D6 is the newly discovered variant allele,that is RHD-CE(2-3,5,7-9)-D.RHD_ψis not found in my experiment.Conclusion:1.There is high frequency of Del and RHD-CE(3-9)-D in Kunming.2.A new variant allele of RHD-CE(2-3,5,7-9)-D is first found in Kunming area.