| ForewordHaptoglobin ( Hp ) is an α2 - sialoglycoprotein with hemoglobin ( Hb ) -binding capacity that is being in serum and other body fluid of human and all mammals. Heritable normal variations in human were first reported by Smithies and Walker. In 1955, by using starch gel electrophoresis, Smithies discovered the heredity polymorphism of Hp and divided into three major phenotype by such : Hp1 , Hp2 -1 and Hp2,and made it become a serum protein that there was e-lectrophoresis polymorphism. Hp coding gene consists of two alleles gene: Hp and Hp2 that being situated long arm (16q22.1 ) of human chromosome of 16th , and presents the general dominance heredity.A great lot of population datum show that Hp phenotype has the better frequency distribution in most populations. There is a high DP and EPP in Hp system, thereby it becomes an important genetic maker for personal identification and paternity testing in forensic medicine. Using application researching and practicing , people discover that Hp have so much heredity variation pheno-types. The phenotype of HpO is the most familiar phenotype and people attach importance to it. Hp0 include two status that is anhaptoglobinemia and hypohap-toglobinemia. In these people, their serum concentration of Hp is exceedingly low or being lacked so that we can not examine the being of Hp by the way of the general electrophoresis means , hence people called this two kinds of situation by a common name HpO. In the past few years , the reports that have something to do with HpO day by day grow in number , even to the extent that the frequency of HpO achieves some degree of genetic polymorphism level in some areas . But the genetic mechanism can not be explained by Meng Deer heredity law,people pay more attention to it.For the moment , there is no research report about the genetic mechanism of HpO and gene analysis, and there are more and more cases of HpO in our parentage testing. Being aimed at this problem , native is researched to select the samples of HpO and their stirps in the northern colony of Chinese Han, and examine the gene spot of Hp allele , and decide whether there are gene deletion,mutation or other abnormity in the Hp gene spot, and determine the genotype ofHpO by ways of molecular biology, and discriminates attempt to supply the genetics base for the personal identification and parentage testing in forensic medicine.Materails and MethodsNative is researched to use noncontinuous PAGE, and the electrophoresis u-nite the phenotype of Hp that the benzidine colour means is examined out the northern Han nationality individual of 210 instance Chinese , and select out HpO individual. In view of these individuals adopting PCR compositely to co - amplify Hpdel gene ,Hp2gene and HpMOn3 gene on Hp gene spot ,the electrophoresis with polypropylene acyl amine is used to separate the PCR products uniting silver dyed that the means carries through the interpretation of the result . Application of PCR amplify the HpdeI gene on Hp gene locus, and the electrophoresis with agarose uniting the EB dyed,we investigate the gene frequency of Hpdel in number of 210 northern Han nationality individual of China that there is no cognation relationship.ResultsBy using means of the general electrophoresis we examine the Hp phenotype of 210 serum specimens and obtain the result that there are 20 samples of phenotype 1,66 samples of phenotype 2 -1,120 samples of phenotype 2,4 samples of HpO. By examining genome DNA of 210 samples we find that there is Hpdel gene in 10 samples and in 4 samples of HpO ,2 samples of Hpl ,4 samples of Hp2. Weobtain the gene frequency of Hpdelthat is 0.0262 showing genetic polymorphism. By means of multi - amplifing the Hpdelgene ,Hp2gene and Hpexon3 gene on Hp gene spot we can exactly distinguish three genotype of HpO that are Hp 1/Hp del Hp2/Hpdel and Hpdel/HpdeI. When the annealing temperature is 66℃ ,we can obtain the best result of multi - PCR and the electrophoresis spectrum is clear and bright and attain optimum gene amplify effect. Using this method,we determine the genotype of 4 samples HpO that the genotype of 3 samples is Hp 2/Hp del and of 1 sample is Hpdel/Hpdel. The genetic mechanism of HpdeI gene accord with Meng Deer heredity law.DiscussionThis study is the first report that adjust northern Chinese Han nationality colony HpO individual through the gene level to analyse and clarify that the form mechanism of HpO is identical with that of Japanese population reported by Ko-da. DNA sequence analysis of Hp gene show that the deleted allele of the hapto-globin gene ( Hpdel) is the cause. The genotype of Hpdel/Hpdeldetermine the phe-notype of anhaptoglobinemia and Hp2/Hpdel determine the phenotype of hypohap-toglobinemia. The gene frequency of Hpdel is 0. 0262 in northern Chinese Han comparing with that of Japan and Korea. The result indicates that there is no difference in evidence and the distribution of Hpdel in the populations has homogeneity. It provide some degree basal data for the study of forensic medicine and human evolution genetics.For the sake of distinguishing the genotype of HpO exactly, this study establish a multi - PCR way that using three primer pairs to amplify specific gene of Hpdel and Hp2 and the mutual exon 3 of Hp1 and Hp2, then by examining the products to determine the genotype of HpO. The annealing temperature is 69 ℃, but amplify by self when attaining Hpdel gene , the annealing temperature is 60 °C , hence compositely amplify when gaining , and selecting an appropriate annealing temperature reaching to stretch the key that matter concentration gain successfully than becoming the best effect. The way can not distinguish the genotype of Hp 2/ Hp2 and Hp2/ Hp1, but can exactly distinguish the genotype of...
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