Development of an automated machine for HLA DNA typing

我们已经建立了一种灵敏、可靠的用于人类白细胞抗原II类基因分型的PCR-RFLP方法。在这项研究中，为了开发一种自动分型机器，我们分析了引物的荧光标记，并优化了使用自动DNA测序仪扫描聚合酶链式反应产物凝胶图谱的条件。对于DQA1和DQB1的5‘端标记，染料-酰胺比氨基键-2更可靠和稳定。用染料-酰胺荧光标记的DQA1引物和信息性限制性内切酶进行了PCR-RFLP分析。使用Genescan^&lt；TM&gt；672和Genotyper^&lt；TM&gt；软件试剂盒，根据带型和自动分配片段大小，可以很容易地确定4个人类白细胞抗原纯合子分型细胞(HTC)和14个异源健康个体的所有DQA1基因型。这些分型结果与常规的聚合酶链式反应-限制性片段长度多态性方法完全一致。即使PCR产物的数量只有1 ng，也可以准确地确定每个片段的大小。因此，这种新的聚合酶链式反应-限制性片段长度多态性方法在人类白细胞抗原II类基因分型中具有高度的敏感性和可靠性。在大小和定量分配的精确度上，区分扩增产物中的特异性片段和非特异性片段相对容易。还可以使用几种染料标记将电泳数据显示为多色数字化凝胶图案。因此，该系统对大量样本的应用具有很高的效率。

We have previously developed the PCR-RFLP method which is sensitive and reliable in HLA class II genotyping. In this study, to develop an automated typing machine, we analyzed for fluorescent labeling of primers and optimized the condition for scanning of gel patterns of PCR products using an automated DNA sequencer. Dye-amidide was more reliable and stable fluorescent than aminolink-2 for labeling of the 5'-end of DQA1 and DQB1 primers. The PCR-RFLP method was performed using DQA1 primers that was fluorescently labeled with dye-amidide, and informative restriction enzymes. Using Genescan^<TM>672 and Genotyper^<TM>software kits, all DQA1 genotypes of four HLA homozygous typing cells (HTC) and fourteen heterologous healthy individuals were easily defined from the band patterns and automatical fragment size assignment. These typing results were completely identical to those by the conventional PCR-RFLP method. Each fragment size can be determined precisely even if the quantity of PCR products is only 1 ng. Therefore, this new PCR-RFLP method is highly sensitive and reliable in HLA class II genotyping. Discrimination between specific and non-specific fragments in the PCR products was relatively easy for the precision of size and quantitative amount assignment. Electrophoretic data can be also displayd as a multi color digitized gel pattern using several dyes labeling. Therefore, this system is highly efficient for application to a lot of samples.