HLA-DQalpha Typing by in situ PCR

原位聚合酶链式反应需要对样品进行前处理，以使聚合酶链式反应成分渗透到细胞内，并允许目标序列被获取以进行扩增。必须仔细控制通透性，以保持组织形态，并且较大的扩增物不会扩散出去。最常用的蛋白水解酶K前处理取决于标本类型和固定方法。以颊癌细胞为模型，对Y染色体上的DYS389II基因座进行了扩增。口腔拭子用蒸馏水洗涤后，在水浴中煮沸5min后，在微管中用生物素标记的引物进行扩增。用碱性磷酸酶标记的链霉亲和素对扩增产物进行检测。或者，我们使用福尔马林固定的口腔细胞。在男性样本中，我们能够检测到典型的阳性细胞，其细胞核被强烈染色。但部分细胞的胞浆也被染色。另一方面，女性样本没有显示出积极的信号。用福尔马林固定的细胞也得到了类似的结果。这些发现表明，煮沸固定/破碎作为原位PCR的前处理是有效的。我们花了大部分时间进行初步实验。虽然我们未能达到最初的目的，即用原位聚合酶链式反应进行人类白细胞抗原-DQα基因分型，但我们成功地简化了样品的前处理。项目期满后，我们将致力于建立原位聚合酶链式反应进行人类白细胞抗原-DQα基因分型。

In situ PCR requires pre-treatment of samples in order to get penetration of the PCR components into the cells and allow the target sequences to be accessed for amplification. The permeabilization must be carefully controlled so that tissue morphology is maintained and the larger amplicons do not diffuse out. The most popular pre-treatment with proteinase K depends on the type of specimen and the fixation method. We explored for simple and reproducible pre-treatment without enzyme digestion.We used buccal cells as a model and amplified the DYS389II locus on the Y chromosome. After washing buccal swabs with distilled water and boiling them on a waterbath for 5 min., we amplified the locus with biotin-labeled primers in a microtube. We detected PCR products with alkaline phosphatase-conjugated streptavidin. Alternatively, we used formalin-fixed buccal cells.In male samples, we were able to detect typical positive cells whose nuclei were strongly stained. However, the cytoplasm of some cells was also stained. On the other hand, female samples showed no positive signal. Cells fixed with formalin gave similar results. These findings indicate that fixation/disruption by boiling is effective as pre-treatment for in situ PCR.We have spent most of the time for preliminary experiments. Although we were not able to achieve the original purpose, HLA-DQ alpha typing by an situ PCR, we successfully simplified pre-treatment of samples in a decisive manner. After the term of the project, we will make, an effort to develop HLA-DQ alpha typing by in situ PCR.