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Research in Quantitative Analysis of Chimerism Post ABO Incompatible Hemopoietic Stem Cell Transplantation with ABO Alleles Associated Nucleotide Polymorphisms Sites

Author: LiuFeng
Tutor: HuLiHua
School: Huazhong University of Science and Technology
Course: Clinical Laboratory Science
Keywords: ABO alleles nucleotide polymorphisms sites (NPs) sequence specific primers (SSP) PCR albumin gene artificial mismatch primers screen real time quantitative PCR artificial chimerism 10-fold dilutions quantitatively detection linear ranges
CLC: R450
Type: PhD thesis
Year: 2011
Downloads: 16
Quote: 0
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Abstract


Part I Screen DNA templates of ABO common alleles associated nucleotide polymorphisms sites (NPs)Objective To screen DNA templates with ABO common alleles associated NPs containing of 261,467,802,803 and 1061, then use them to develop and assessment a SYBR green I-based real time quantitative PCR method for detecting chimerism post ABO incompatible HSCT.Methods Whole blood samples were collected from 76 cases of healthy examined people. ABO phenotyping by anti-A, anti-B, anti-A, B anti-H, ant-Al, and ABO reverse typing RBC reagents, including criteria were ABO blood grouping discrepancies and none Bombay type. The distribution of ABO blood group in 76 cases were 18 cases of A,27 cases of B,18 cases of 0,13 cases of AB. Extraction DNA templates from 76 cases whole blood samples then design sequence specific primers and develop PCR-SSP to screen heterozygote or homozygote of these NPs.Results The distribution of heterozygote or homozygote of these five NPs according to ABO phenotype were:7 cases of A (261 heterozygote、467normal homozygote),8 cases of A (261 heterozygote、467 heterozygote、1061 normal homozygote),3 cases of A (261 normal homozygote、467 heterozygote、1061 normal homozygote); 23 cases of B (261 heterozygote、803 heterozygote),4 cases of B (803 mutational homozygote),18 cases of 0 (all of 261 mutational homozygote), 10 cases of AB (467 heterozygote、803 heterozygote、1061 normal homozygote), 3 cases of AB (467 normal homozygote、803 heterozygote).467 mutational homozygote,802 heterozygote or mutational homozygote,1061 heterozygote or mutational homozygote could not be found.Conclusion Direct screen the 5 NPs from ABO common phenotype rather than first determination ABO genotype was feasible. We could not got 467 site mutational homozygote,802 site heterozygote or mutational homozygote,1061site heterozygote or mutational homozygote in view of the limited number of screen population and ethnic distribution of ABO alleles frequencies in Chinese, so these NPs could not be applied for development and assessment a SYBR greenⅠ-based real time quantitative PCR method for detecting chimerism post ABO incompatible HSCT.PartⅡDevelopment of a SYBR greenⅠ-based real time quantitative PCR method for detection common ABO alleles associated nucleotide polymorphisms sites (NPs)Objective To develop a SYBR greenⅠ-based real time quantitative PCR systems for detecting 261mutational site、261normal site、467 mutational site,803 mutational site and 803 normal site.Methods Use 261 site mutational or normal homozygote and heterozygote,467 site normal homozygote and heterozygote,803 site mutational or normal homozygote and heterozygote from sectionⅠof our study, develop a SYBR greenⅠ-based real time quantitative PCR methods for detecting these sites, including development of human albumin reference gene to calibrate input DNA amounts, setting up reaction parameters and contents of PCR systems, designing new primers with another artificial mismatched base adjoined to NPs. Determined the specificity and efficiency of the optimal primers by melting curve analysis and electrophoresis analysis of products andΔCt derived from Ct differentials of positive and negative DNA templates, and sequence analysis of products.Results Deduced by standard regression curve of human albumin reference gene, the formula of the linear relationship was y=-3.04x+33.95, R2 was 1.00. Besides, Ct value of the standard sample achieved from18.26 of 500ng to 23.66 of 7.81ng; the optimal primer of 261 mutational site was:261mut-260mismatch(T→C),ΔCt was 14.38. The optimal primer of 261 normal site was 261nor-260mismatch (T→C),ΔCt was 11.22. The optimal primer of 467 mutational site was 467mut-466mismatch(C→T),ΔCt was 10.2. The optimal primer of 803 mutational site was 803mut-804mismatch(G→A),ΔCt was 11.5. The optimal primer of 803 normal site was 803nor-804mismatch(G→A),ΔCt was 10.26. All the optimal primers were identical with expected results proved by sequence analysis.Conclusion The system of human albumin reference gene to calibrate input DNA amounts indicated the high amplification efficiency and the good linear relationship. All samples with unknown concentration from 7.81ng to 500ng can be accurately defined. A SYBR green I-based real time quantitative PCR methods for detecting 261mutational site、261normal site、467 mutational site,803 mutational site and 803 normal site were successfully developed with high specificity and efficiency.Part III quantitative assessment of the SYBR greenⅠ-based real time quantitative PCR method used for detection chimerism post ABO incompatible hemopoietic stem cell transplantationObjective To assess the quantitative range of the SYBR green I-based real time quantitative PCR methods for detecting chimerism status by 261mutational site、261normal site、467 mutational site,803 mutational site and 803 normal site.Methods DNA samples were extracted from 261sites、803 sites(mutational or normal homozygote and heterozygote)and 467 sites (heterozygote and normal homozygote), then their concentration were calibrated to 20ng/μ1(467sites to 40ng/μ1) by albumin reference gene. Then artificial mixed 261,467,803 normal or mutational positive with corresponding negative sites chimeric DNA templates ranged from 10-1、10-2、10-3、10-4、10-5 by 10-fold dilutions, as well as 100% positive and corresponding negative DNA templates were simultaneously detected by SYBR green I-based real time quantitative PCR. After amplification, melting curve was used to assure specificity of products. The values of Ct and actually copies of all 261,467,803 normal or mutational positive DNA templates were used to linear regression analysis by turns of point to point from low to high dilutions, and determined the linear quantitatively detecting range of all sites.Results All positive DNA templates were amplificated to specific products by analysis of melting curve. (1)The formula of the linear relationship in 10-fold dilution of 261 mutational homozygote with 261 normal homozygote DNA templates was y=-3.444x+38.83, R2 was1.00.The formula of the linear relationship in 10-fold dilution of 261 mutational heterozygote with 261 normal homozygote DNA templates was y=-3.386x+38.31, R2 was 1.00. The formula of the linear relationship in 10-fold dilution of 261 normal homozygote with 261 mutational homozygote DNA templates was y=-3.129x+37.51, R2 was 1.00. T he formula of the linear relationship in 10-fold dilution of 261 normal heterozygote with 261 mutational homozygote DNA templates was:y=-3.152x+37.66, R2 was1.00. In all of 261 sites, the linear quantitatively detecting ranges were no less than 10-3. (2) the formula of the linear relationship in 10-fold dilution of 467 mutational heterozygote with 467 normal homozygote DNA templates was y=-3.373x+41.02, R2 was 1.00. In 467 sites, the linear quantitatively detecting ranges were no less than 10-2.(3)the formula of the linear relationship in 10-fold dilution of 803 mutational homozygote with 803 normal homozygote DNA templates was y=-3.183x+40.26, R2 was0.99.The formula of the linear relationship in 10-fold dilution of 803 mutational heterozygote with 803 normal homozygote DNA templates was y=-3.244x+39.02, R2 was0.99.The formula of the linear relationship in 10-fold dilution of 803 normal homozygote with 803 mutational homozygote DNA templates was y=-3.162x+39.89, R2 was 0.99.The formula of the linear relationship in 10-fold dilution of 803 normal heterozygote with 803 mutational homozygote DNA templates was y=-3.136x+38.47, R2was 0.99. In all of 803 sites, the linear quantitatively detecting ranges were no less than 10"2.Conclusion The developed SYBR green I-based real time quantitative PCR methods for detecting 261mutational site、261normal site、467 mutational site,803 mutational site and 803 normal site have high sensitivity and linear quantitatively detecting ranges. At the same time, it is no different in the linear quantitatively detecting ranges from positive homozygote and positive heterozygote of 261 and 803 sites.

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