HLA基因序列电泳多态性分析及其在骨髓移植中的应用

利用聚合酶链反应和聚丙烯酰胺凝胶电泳技术建立了一套人白细胞抗原配型技术，包括ＨＬＡ－ＤＲ单链构象多态性，异二聚体分析和ＨＬＡ－ＤＲ，ＤＰ－ＤＮＡ链构象多态性分析技术。对６３例骨髓移植供，受体进行了分析，其中经血清学确定为ＨＬＡ相合的１５对ＰＣＲ－ＳＣＰ分析完全相合；５对因病人血细胞数目和功能异常而供，受体血清不方法无法分型，ＰＣＲ－ＳＣＰ分析确定３对相合，２对不相合，病人临床缓解后重复血清学ＨＬＡ     

用PCR－F、PCR－SSCP对骨髓移植植入状态的观察

应用ＨＬＡ－ＤＱＡ、ＤＲＢ、ＤＰＢ、ＤＱＢ位点聚合酶链反应一指纹图（ＰＣＲ－Ｆ）和聚合酶链反应一单链构象多态性（ＰＣＲ－ＳＳＣＰ）方法，对５例ＨＬＡ血清学配型一致的异基因骨髓移植病人，分别于移植后３５ｄ左右进行了植入状态的观察。此方法不受供、受体性别和血型的影响，具有灵敏、准确、快速、简便等优点，其中４例以特殊带型为异基因骨髓移植提供了直接的植入证据．     

创面异体移植培养表皮细胞后存活的判定

应用４种方法判定烧伤创面异体移植的培养表皮细胞存活情况：１．聚合酶链反应（ＰＣＲ）查Ｙ染色体特异性ＤＮＡ片段；２．ＰＣＲ扩增ＰＭＣＴ１１８长度多态性基因位点；３．ＰＣＲ／ＳＳＯＨＬＡ－ＤＱａ分型；４．ＤＮＡ指纹。共检测１６例病人的２７个活检标本，均证实有供者的ＤＮＡ，最长时间为移植后９２天。初步实验结果表明：培养表皮异体移植后可存活较长时间。     

在骨髓移植中人类白细胞抗原三维结构的差异引发移植物抗宿 …

为进一步探索移植物抗宿主病（ＧＶＨＤ）的发病机制，提高临床移植疗效，采用人类白细胞抗原（ＨＬＡ）分型方法，包括血清－细胞学，基因和基因亚型分型，血清－细胞学分型采用国际通用的微量淋巴细胞毒试验和混合淋巴细胞培养；基因分型采用ＰＣＲ－ＳＳＰ方法，对基因亚型进行ＤＮＡ测序，再进行ＨＬＡ三维模型模拟。     

创伤后多器官功能不定综合征与HLA—DRB1基因的相关性研究

目的 研究ＨＬＡ－ＤＲＢ１基因与创伤后多器官功能不全综合征（ＭＯＤＳ）的相关性。方法 采用多聚酶链反应－序列特异性引物分析法（ＰＣＲ－ＳＳＰ）,对３７例创伤后ＭＯＤＳ患者ＨＬＡ－ＤＲＢ１基因进行分型,用酶联免疫吸附试验（ＥＬＩＳＡ）进行血浆白细胞介素－１０（ＩＬ－１０）测定,并与相应健康人５２例结果比较。结果 创伤后ＭＯＤＳ患者ＨＬＡ－ＤＲＢ１＊１５０１￣ＤＲＢ１＊１５０２（编码ＨＬＡ－ＤＲ２抗原     

PCR直接扩增与套式扩增用于HLA—DR基因分型的研究

人类ＨＬＡ抗原分型在人类学、法医学、疾病易感性及器官组织移植等研究中有重要的意义。在骨髓移植中，进行供者、受者ＨＬＡ配型，选择相合的供体，减少ＧＶＨＤ发生，是移植成功的关键。     

HLA—DR基因的克隆及其在小鼠黑色素瘤细胞中的表达

获得表达ＨＬＡ－ＤＲ基因的小鼠墨色素瘤细胞，方法应用ＲＴ－ＰＣＲ技术从人Ｂ淋巴瘤细胞Ｒａｊｉ中克隆了ＨＬＡ－ＤＲ３α和β链ｃＤＮＡ，并经测序证实。将其分别插入至真核表达载体ｐＬＸＳＮ和ｐＣＥＰ４中，用脂质体转上鼠黑色素瘤细胞Ｂ１６，然后用Ｇ４１８和Ｈｙｇ双重筛选。     

聚合酶链反应快速诊断白色念珠菌菌血症

目的：应用聚合酶链反应（ＰＣＲ）技术诊断白色念珠菌菌血症。方法：设计的引物特异性扩增编码白色念珠菌细胞色素Ｐ４５０Ｌ１Ａ１的基因，其长度为２４３ｂｐ。ＥＤＴＡ抗凝血用去污剂溶解，用ＤＮａｓｅＩ去除人体白细胞和污染的细菌ＤＮＡ；念珠菌的细胞壁用溶胞酶消化并用ＳＤＳ和蛋白酶Ｋ裂解。用酚／氯仿／异戊醇提取模板ＤＮＡ。用ＰＣＲ技术扩增。结果：与细菌、病毒及人体细胞ＤＮＡ无非特异扩增。检出血中白色葡萄的阈值     

应用聚合酶链反应技术检测类风湿关节炎患者HLA－DR1和－DR4基因

应用聚合酶链反应技术检测类风湿关节炎患者ＨＬＡ－ＤＲ１和－ＤＲ４基因１００８６３北京解放军总医院袁国华，施桂英，栗占国，丁玉珍中国图书资料分类号Ｒ５９３．２２许多国家的调查发现，ＨＬＡ－ＤＲ抗原与类风湿关节炎ＲＡ相关。传统的ＨＬＡ－ＤＲ分型方法多采用...     

FAS抗原胞外区片段GST融合蛋白 产生

获得得重组ＦＡＳ抗原，用于抗体制备及进一步的功能分析，方法用ＰＣＲ技术从完整的ＦＡＳｃＤＮＡ克隆上扩增其编码胞外区的部分，将ＰＣＲ产物直接克隆到ｐＧＥＭ－Ｔ载体系统，ＤＮＡ序列分析证实序列完全正确；用ＥｃｏＲⅠ和ＳａｌⅠ将ＦＡＳ胞外区片段切出，     

A simple ABO genotyping by PCR using sequence-specific primers with mismatched nucleotides

In forensics, the specific ABO blood group is often determined by analyzing the ABO gene. Among various methods used, PCR employing sequence-specific primers (PCR-SSP) is simpler than other methods for ABO typing. When performing the PCR-SSP, the pseudo-positive signals often lead to errors in ABO typing. We introduced mismatched nucleotides at the second and the third positions from the 3′-end of the primers for the PCR-SSP method and examined whether reliable typing could be achieved by suppressing pseudo-positive signals. Genomic DNA was extracted from nail clippings of 27 volunteers, and the ABO gene was examined with PCR-SSP employing primers with and without mismatched nucleotides. The ABO blood group of the nail clippings was also analyzed serologically, and these results were compared with those obtained using PCR-SSP. When mismatched primers were employed for amplification, the results of the ABO typing matched with those obtained by the serological method. When primers without mismatched nucleotides were used for PCR-SSP, pseudo-positive signals were observed. Thus our method may be used for achieving more reliable ABO typing.     

Rapid PCR of STR markers: Applications to human identification

Multiplex PCR with fluorescently labeled primers has been an essential method for the amplification of short tandem repeats used in human identify testing. Within the STR workflow of extraction, quantitation, amplification, separation, and detection, multiplex PCR is commonly identified as the bottleneck in the process. The time requirement of up to three hours to complete 28–30 cycles of multiplex PCR for STR genotyping is the greatest amount of time required for a single step within the process. The historical use of commercially available thermal cyclers and heat stable polymerases may have given the impression that large multiplex will always require long PCR cycling times to ensure that all of the varying sized targets (typically 100–400 bp) can be amplified in a balanced manner throughout the multiplex. However, with the advent of improved polymerases and faster thermal cyclers the time required for the amplification of large STR multiplexes is no longer on the order of three hours, but as little as 14 min. Faster amplification times can be performed while retaining the balance and integrity of large multiplex PCRs by implementation of alternate polymerases and thermal cyclers. With the reduction in PCR cycling times there has also been an impact on the development of integrated and microfluidics devices which employ the use of reduced or rapid thermal cycling protocols as part of their integration. Similarly, PCR inhibitor resistant polymerases can also reduce overall STR processing times for reference samples by eliminating the need for DNA extraction and purification that is additionally implemented within the development of integrated DNA typing devices.     

DNA identification of formalin-fixed organs is affected by fixation time and type of fixatives: using the AmpF l STR(R) Identifiler(R) PCR Amplification Kit

Personal identification using DNA typing of formalin-fixed tissue is very important in the forensic sciences. However, few studies have been conducted to determine the detection limit of DNA typing of formalin fixation time in samples using the AmpF?STR(?) Identifiler(?) PCR Amplification Kit (Identifiler Kit). We collected samples from five cadavers submitted for forensic autopsies, and fixed them either in a 10% formalin solution, or in a 10% neutral-buffered formalin solution. The amount of template DNA for polymerase chain reaction (PCR) amplification and the detection limit of DNA typing for the Identifiler Kit were determined. When tissues were fixed in 10% formalin, 10 ng of DNA template was required for successful genotyping even after three-hour fixation and 100 ng was required after one-week fixation for PCR amplification. However, when tissues were fixed in 10% neutral-buffered formalin, the required amount of DNA template was 1 ng for a fixation time of three hours to three days and 125 ng for three months. Fixation time in neutral-buffered formalin was longer for successful PCR than that in formalin solution. Dropout was more common with increasing formalin fixation time. These results suggest that neutral-buffered formalin is preferred to formalin for fixation of tissues if they are to be subjected to DNA typing and that tissues fixed with neutral-buffered formalin can be used for DNA typing using the Identifiler Kit unless the fixation time exceeds one month.     

布鲁菌基因分型研究进展

布鲁菌是引起人畜共患传染性布鲁菌病的病原体。基因分型作为布鲁菌病溯源的主要方法,目前以多位点VNTR分析技术备受关注,其他分型技术如随机扩增多态性和扩增片段长度多态性、多位点序列分型等也在布鲁菌基因分型中得到应用,本文综述了近年来布鲁菌基因分型的研究进展。     

A one step multiplex PCR assay for rapid screening of East Asian mtDNA haplogroups on forensic samples

The mitochondrial DNA (mtDNA) haplogroup typing has become an essential tool to study human evolutionary history and to infer the matrilineal bio-geographic ancestry. In forensic field, the screening of mtDNA haplogroups by genotyping of mtDNA single nucleotide polymorphisms (SNPs) can help guarantee the quality of mtDNA sequence data as well as can reduce the need to sequence samples that do not match. Here, a multiplex mutagenically separated (MS) polymerase chain reaction (PCR) system was developed for simultaneous rapid detection of 14 coding region SNPs and one deletion motif representing common mtDNA haplogroups of East Asia. The multiplex MS PCR system we developed has the advantage of being a one step procedure that requires only a single PCR amplification with allele-specific primers and allowing straightforward designation of haplogroups along the branches of the phylogenetic tree. Therefore, it would be a simple, rapid, and reliable detection method useful for large-scale screening of mtDNA variations to determine East Asian mtDNA haplogroups.     

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpF?STR(?) Identifiler(?) amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpF?STR(?) Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency.     

不完全相合造血干细胞移植中GVHD的预测研究:模拟HLA抗原结构的比较与应用

采用通用的微量淋巴细胞毒实验和序列测定方法对拟进行造血干细胞移植的患者及供体进行HLA分型；用计算机分子模拟手段，从HLA分子三维结构方面探讨预测移植物抗宿主病（GVHD）发生程度，以预测移植后GVHD的发生情况。结果发现，8例供受体中，3例为半匹配移植，其中2例发生了Ⅳ度GVHD，1例发生了Ⅱ度GVHD；3例为两个抗原不相合移植，其中1例发生了Ⅱ度GVHD，2例发生了Ⅱ度GVHD；2例口才为一个抗原不相合移植，分别发生了Ⅰ度、Ⅱ度GVHD；GVHD的发生程度与HLA不相合抗原RMSD差异的大小呈正相关。提示异基因造血干细胞移植时，GVHD发生程度与HLA分子三维结构的差异有密切关系，三维结构分子模拟进行GVHD预测可以为临床医生提供较好的预后指标。     

Differences of PCR efficiency between two-step PCR and standard three-step PCR protocols in short tandem repeat amplification

The effects on STR typing results, using a two-step PCR protocol that combines the annealing and extension steps, were determined using samples at various DNA concentrations. DNA samples ranging from 62.5 to 500 pg were amplified using the reagents contained within the AmpFlSTR Identifiler PCR Amplification Kit. At 250–500 pg of DNA template, the rates of detecting alleles were close to those found when using three-step PCR (the standard Identifiler protocol) and two-step PCR protocols. The two-step PCR increased by 8.1% and 64.2% the average number of amplified alleles compared with the three-step PCR at 125 pg and 62.5 pg of template DNA, respectively. At 62.5–500 pg of DNA, the average peak heights were 23.3–65.0% higher than heights using the three-step PCR protocol. When two different PCR protocols were applied to old bones, the average number of amplified loci was similar. These results suggested that the two-step PCR protocol can generate more STR alleles in low template DNA samples; however, the method may be limited with samples of very low-quality, i.e., degraded DNA, compared with the three-step protocol. A better understanding of the effects of the two-step PCR protocol on amplification conditions might help researchers improve STR typing.     

Mitochondrial DNA heteroplasmy or artefacts--a matter of the amplification strategy?

We compared two different PCR strategies for the amplification of mtDNA hypervariable region 1 (HV1) with regard to the detection and interpretation of point mutation heteroplasmy in human hair roots. We monitored the level of detected heteroplasmy using direct sequence analysis. PCR amplifications were performed in duplicate on each hair root, using 62 cycles of nested PCR versus 35 cycles of direct PCR. As a previous publication reported different sensitivities of heteroplasmy detection based on the number of PCR cycles used, we were interested in whether and how different PCR amplification strategies would impact sequence quality and the detection of point heteroplasmy. We identified 12 out of 93 hair roots as heteroplasmic (7 out of 31 persons) with direct PCR, whereas 2 of these heteroplasmic events could not be identified with the nested PCR approach. Generally, the quality of the sequence electropherograms in terms of background noise was significantly lower for the nested PCR amplification strategy, leading to ambiguous results in some of the nucleotide positions. Thus, the ability to clearly distinguish a genuine mixture of two nucleotides from background noise at a heteroplasmic position was substantially greater with direct PCR amplification, which generally resulted in higher quality sequence electopherograms.