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Cell-free fetal DNA in maternal plasma of pregnant woman, originated from fetal and / or placental cells undergoing apoptosis, is mainly the short-sized DNA fragments of less than 313 base pairs in length for the sake of nuclear endonuclease selectively cleaving fetal DNA during the apoptosis process. The mean cell-free circulating fetal DNA in maternal plasma accounted for 3.4% and 6.2% of plasma total DNA during the early and the late gestation, respectively. Owing to its relative abundance, circulating fetal DNA in maternal plasma has now become the important DNA source for non-invasive prenatal molecular genetic diagnosis and it is widely used in fetal sex-determination, detection of fetal Rh (D) sequence in the plasma of the rhesus-negative woman, fetal aneuploidy detection, fetal STR genotyping and other clinical applications. Cell-free fetal DNA source, concentration, purity, size, distributions and postnatal clearance of fetal DNA in maternal plasma as well as the reported clinical applications are summarized and discussed in this paper. Based on the molecular characteristics of cell-free fetal DNA and the target gene, the using of appropriate molecular diagnosis strategy and experimental design as well as reducing the fragment size of PCR product and adjusting the PCR conditions to the optimum enable the improvement of non-invasive prenatal diagnosis accuracy.

In order to study the genetic status of a rare chimeric family, some samples of A(3)B(3) family were identified by sequencing of ABO gene; flow-rSSO and PCR-SSP were used to detect loci of HLA-A, B, DRB1 genes, and multiplex amplifying with fluorescence-dye were performed for 16 short tandem repeat (STR) loci. The results indicated that two individuals from A(3)B(3) family contained more than two alleles at ABO gene, HLA-B, DRB1 and some STR loci. In conclusion, analysis of chimeric blood group by using genotyping techniques clearly demonstrating genetic status of this rare chimeric blood group promotes further elucidation of the existing state of specific genetic status.

This study was aimed to investigate the polymorphism of D2S1338 and D19S433 loci in Southern Chinese Han nationality population, and to evaluate its forensic application. The DNA was extracted by chromatography on che-lex-100. Fifteen STR loci (D2S1338, D19S433 etc.) were amplified by Identifiler kit and analyzed by 3100 genetic analyzer. The softwares of analysis were GeneScan 3.0 and Genetype 3.7. The results indicated that the allele frequencies of H, DP, PIC, PE of D2S1338 and D19S433 loci were obtained. The allele frequency of samples were in Hardy-Weinberg equilibrium by chi2 test. No mutation was found on D2S1338 and D19S433 loci in 370 cases of meiosis. It is concluded that the D2S1338 and D19S433 loci are high polymorphic and their mutation rates in Southern Chinese Han nationality population are low. Their good distribution is suitable for forensic individual identification and paternity testing.

Objective: To study the genetic polymorphisms of 9 Y-chromosome specific STR loci that the allele size is less than 180 bp in length in the southern Chinese Han population, and to utilize the studied result to forensic science., Methods: Nine Y-STR loci were amplified by single multiplex PCR, and the PCR products were sequenced by using ABI Prism 3100 DNA Sequencer. The allele and haplotype frequencies at 9 Y-STR loci were determined in a total of 213 unrelated males from southern Chinese Han population. Eighty-four father/son pairs with demonstrated paternity and thirty-six non-paternity father/son pairs were detected by using our Y-STR multiplex system., Results: Three Y-STR alleles for DYS426, five alleles for DYS393, DYS460, DYS391 and DYS389 I respectively, six alleles for DYS456, seven alleles for both H4 and DYS388, and eight alleles for DYS458 loci were detected in 213 unrelated male individuals. Except for the DYS426 locus with a low GD value of 0.1489, the GD values for other 8 Y-STR loci ranged from 0.5064 to 0.9133. A total of 178 haplotypes were found at 9 Y-STR loci, of which 154 haplotypes were observed only once, and the haplotype diversity was 0.9983. None of Y-STR allele mutation was observed in the 84 father/son pairs with demonstrated paternity. Among the 36 non-paternity father/son pairs, two cases could get the paternity exclusions at 2 Y-STR loci; and the paternity of 33 cases could be ruled out by 3 or more Y-STR loci; only one case was found no exclusion of paternity regardless of detecting 9 Y-STR loci., Conclusion: This result indicates that the 9 Y-STR loci with short fragment size alleles are highly polymorphic. The fluorescent multiplex amplification system that we developed is suitable for personal identification and paternity testing.

Objective: To study the ABO allele molecular characteristics of Ael blood subgroup., Methods: Five individuals of diagnosed as Ael blood subgroup were subjected to PCR amplify ABO alleles using four pairs of sequence-specific primers. Exon 6 and exon 7 at ABO locus of all samples were sequenced. An individual with AelB phenotype was chosen for further analysis of transcript structure of ABO gene., Results: Sequence analysis indicated one Ael phenotype sample with reported Ael01 allele, one Ael phenotype sample with an Ael05 allele, and two AelB and one Ael individuals did not contain referred A allele, but contain O01 or O02 allele with 261G deletion., Conclusion: Molecular bases for the Ael have highly polymorphism. The mechanism responsible for the express weak A antigen of O allele with 261G deletion awaits to be elucidated.

To study the correlation between acute lymphoblastic leukemia (ALL) and HLA-A, B and DRB1 gene in southern Chinese Han population and to investigate the susceptible HLA gene to ALL, a total of 4707 healthy volunteer bone marrow donors from southern Chinese Han population were used as a control group, 201 patients diagnosed as patient group from southern Han individuals were genotyped at HLA-A, B and DRB1 loci by PCR-SSP, PCR-SSOP and SBT. HLA allele frequency and its distribution of ALL patient group were compared with the control group by using chi(2) test, and calculated the statistic value of relative risk (RR), pathogenicity score (EF) and preventive score (PF). The results showed that in comparison with the control group, the gene frequence of HLA-A26, B56 and DR9 increased significantly, but the gene frequence of HLA-A30, A33 and B58 allele frequency decreased significantly for patients with ALL. It is concluded that HLA-A26, B56 and DR9 gene have a high correlation with ALL and seem to contribute the genetic susceptibility to ALL in southern Chinese Han populations. However, HLA-A30, A33 and B58 gene seem to have protective role for southern Han individuals suffered from ALL.

To study four A(3) subgroup samples identified by serologic tests, among which two belong to a family, three were A(3) subgroup, one was A(3)B subgroup. All four samples were genotyped by PCR-SSP method, and the nucleotide sequences of Exon 6, Exon 7 and part introns at the ABO locus for these samples were detected by ABI Prism 3100 DNA sequencer. Comparison with the consensus of A101 was performed. The results showed that haplotypes of two A(3) subgroups were common A102 allele and O1-2 allele, and haplotypes of one A(3) subgroup were common A102 allele and rare O(1v)-4 allele. Unexpectedly, a synonymous substitution 838C-->T had been found in A allele of the A(3)B subgroup sample, which predict a Leu280Phe alteration. The results suggested that molecular genetic background of the A(3) phenotypes is polymorphic. Possibly, the missense mutation 838C-->T is the molecular genetic basis of A(3)B subgroup that lead to low activity of the glycosyltransferases.

To set up the simultaneous genotyping of human platelet antigens of 1,2,3,4,5,6 system by PCR-SSP assay and use the genotyping method for the study of platelet antigens. In this study, 18 sequence-specific primers were designed and synthesized. The annealing temperature for all sequence-specific primer pair, the concentration of each primer pair and the concentration of Mg2+ were adjusted to the optimum so that HPA-1 to 6 systems could be amplified simultaneously under the same PCR cycling parameters. The electrophoresis of PCR products was conducted simultaneously on the same agarose gel. Control DNA samples that genotypes known were used to confirm the sensitivity and specificity of each sequence-specific primer. 15 coded samples (including 2 blood samples and 13 DNA samples) distributed by 10TH Platelet Genotyping and Serology Workshop of the International Society of Blood Transfusion (ISBT) were typed for HPA-1 to 6 systems by this method. A concordance rate of 100 percent was observed between the results of control DNA samples typed by our PCR-SSP assay and the data of known specificity of control DNA. The results of 15 coded samples tested by our method agreed well with the results provided by ISBT report.

Objective: To analyze human leukocyte antigen (HLA) polymorphism and search for new alleles in Chinese Han population bone marrow registry donors., Methods: DNA-based HLA genotyping methods were used including PCR-SSP, BST and molecular cloning., Results: A total of 6965 unrelated donors, 4707 from South China origin and 2258 from north, were typed for HLA-A, B, and DRB1 loci. Seventy-two specificities of HLA alleles were identified. The HLA-A25, A34, A74, B41, B42, B53, B73 and B81 that were rarely reported in previously Chinese population studies were identified in this study. Estimation of gene frequency indicated that the blank gene frequency was less than 0.2% for HLA-A, 0.25% for HLA-B and 0.70% for HLA-DRB1 loci. Three novel alleles were identified and officially assigned by the World Health Organization (WHO) Nomenclature Committee as A*0253N, A*1114 and B*5610., Conclusion: Large-scale DNA-based HLA genotyping used in bone marrow registry donors is highly accurate and reliable for estimating gene frequency and searching for new alleles. The discrepancy of HLA gene distribution between South and North China Han population showed the necessity of setting the more regions in South and North China to screen the bone marrow registry donors for bone marrow transplant.

To study the genetic polymorphisms of six Y-chromosome specific STR loci in the southern Chinese Han population and apply it in forensic science, six Y-STR loci were amplified by multiple PCR and the PCR products were detected by using ABI Prism 377 Sequencer. The haplotype frequencies at 6 Y-STR loci were determined in a total of 204 unrelated males from southern Han population of China. Ninety-three father/son pairs with demonstrated paternity and thirty-eight non-paternity father/son pairs were detected by using our Y-STR system. As a result, the number of alleles for DYS393, DYS19, DYS389 II, DYS390, DYS391 and DYS385 were 5, 6, 8, 6, 4 and 44, respectively. A total of 176 haplotypes at 6 Y-STR loci were found. Two father/son pairs with single Y-STR mutation were observed in the 93 father/son pairs with demonstrated paternity. Among the 38 non-paternity father/son pairs, one case with one Y-STR exclusion of paternity, one case with two Y-STR exclusions and 35 cases with 3 or more Y-STR exclusions were observed. Non-exclusion of paternity at 6 Y-STR loci was found only in one case. This result indicated that the six Y-STR loci were highly polymorphic and are suitable for personal identification and paternity testing.

The purpose of this research was to monitor quantitatively and study the dynamic changes and development rules of engraftment, chimera types, as well as relative amount of donor cells after allogeneic transplantation of mixed umbilical-cord blood from two units. An adult patient with acute myeloid leukemia received two units HLA one locus mismatched unrelated umbilical cord blood transplantation (2.5 x 10(7)/kg karyocytes in umbilical cord blood unit 1, and 1.53 x 10(7)/kg karyocytes in umbilical cord blood unit 2). Nine STR loci of the blood sample before and after transplantation were determined by quantitative detecting technique with fluorescence labeling polymerase chain reaction, while the engraftment and chimera types were qualitatively evaluated by comparing differential loci between the recipient and the donors. Then the relative proportion of chimera from two units of umbilical-cord blood in the patient after transplantation was calculated according to the differential gene peak areas of two donors on 377XL DNA sequencer after fluorescence scanning, and the engraftment level and the development rules of donor cells were analyzed. In addition, the results were also compared with that of HLA loci distinct analysis for engraftment. The results showed that two umbilical cord blood units at 15 days after transplantation were engrafted simultaneously and revealed a complete chimerism of the two. The relative amounts of chimera from unit 1 vs that of unit 2 were 51.3% vs 48.7%; subsequently relative amounts of chimera from unit 1 went up to 70.0% at 30 days, and that from unit 2 declined to 30.0%. However, at 52 days, only the genotype of umbilical cord blood unit 1 was detected, so that the engraftment turned to a complete chimerism of a single donor type. The one with fewer karyocytes was rejected and the one with more karyocytes finally engrafted in long-term. It is concluded that quantitatively detecting STR chimera with fluorescence labeling polymerase chain reaction can depict precisely the engraftment level and the change course of two umbilical cord blood units. It provides an accurate and reliable experimental basis for clinical umbilical cord blood application and donor selection, and is proved to be feasible for adult transplantation by using dual unit of umbilical-cord blood with HLA one locus mismatched at the same time.