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首页> 外文期刊>Journal of Pathology: Journal of the Pathological Society of Great Britain and Ireland >Nested polymerase chain reaction-based HLA class II typing for the unique identification of formalin-fixed and paraffin-embedded tissue.
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Nested polymerase chain reaction-based HLA class II typing for the unique identification of formalin-fixed and paraffin-embedded tissue.

机译：基于巢式聚合酶链反应的HLA II类分型，用于福尔马林固定和石蜡包埋组织的独特鉴定。

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摘要

Human leukocyte antigen (HLA) genotyping is routinely performed prior to organ transplantation using peripheral blood leukocyte-derived DNA. In addition, polymerase chain reaction (PCR)-based methods have permitted HLA genotyping using DNA extracted from formalin-fixed and paraffin-embedded tissue, with proven applications in HLA-disease association studies and surgical biopsy identification. The utility of current techniques may be limited by the poor yield of intact DNA from such paraffin biopsies. This paper describes a new nested PCR-based HLA class II genotyping method which reliably detects HLA DRB alleles within DNA extracted from even extremely small paraffin biopsies. This method comprises initial PCR amplification of exon II sequences of the HLA DRB1, 3, 4, and 5 genes using generic PCR primers. Identification of the HLA DRB1 alleles and detection of the DRB3, 4, and 5 genes is then performed using a series of separate individual second-round PCR reactions, each of which contains PCR primer pairs detecting a single HLA DRB allele or group of alleles (PCR-SSP). The ability of this method to detect 19 individual HLA DRB1 alleles or groups of alleles, covering all common DRB1 specificities, was confirmed via concordant results when compared with 'direct' (single amplification step) PCR-SSP analysis of one cell line-derived and nine peripheral blood-derived DNA samples, and with five DNA samples extracted from paraffin biopsies. The technique was then successfully applied to 11 further paraffin biopsy-derived DNA samples, of which ten were untypable by 'direct' PCR-SSP analysis, from five cases in which doubt existed as to the individual origin of the tissues.
机译：人类白细胞抗原（HLA）基因分型通常在器官移植之前使用外周血白细胞衍生的DNA进行。此外，基于聚合酶链反应（PCR）的方法已允许使用从福尔马林固定和石蜡包埋的组织中提取的DNA进行HLA基因分型，已在HLA疾病关联研究和手术活检鉴定中得到了证实的应用。当前技术的实用性可能受到来自这种石蜡活组织检查的完整DNA收率不佳的限制。本文介绍了一种基于嵌套式PCR的新型HLA II类基因分型方法，该方法可可靠地检测从非常小的石蜡活检样品中提取的DNA中的HLA DRB等位基因。该方法包括使用通用PCR引物对HLA DRB1、3、4和5基因的外显子II序列进行初始PCR扩增。然后使用一系列单独的第二轮PCR反应进行HLA DRB1等位基因的鉴定和DRB3、4和5基因的检测，每个反应均包含检测单个HLA DRB等位基因或等位基因组的PCR引物对（ PCR-SSP）。当与“直接”（单扩增步骤）PCR-SSP分析一个细胞系和一个细胞系的结果进行比较时，通过一致的结果证实了该方法检测19个单独的HLA DRB1等位基因或等位基因组的能力，涵盖所有常见的DRB1特异性。 9个外周血DNA样本，以及5个从石蜡活组织检查中提取的DNA样本。然后，该技术成功应用于另外11个石蜡活检来源的DNA样品，其中有10个通过“直接” PCR-SSP分析无法分型，其中有5个对组织的个体来源存在疑问。

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