van Schie R C, Wilson M E
Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo 14214-3092, USA.
J Immunol Methods. 1997 Oct 13;208(1):91-101. doi: 10.1016/s0022-1759(97)00132-4.
Genetic polymorphisms of low-affinity IgG Fc receptors (Fc gamma R) have been found to influence binding of human IgG subclass antibodies, and may influence susceptibility to certain types of infectious and autoimmune diseases. Phenotypic and/or genotypic analyses of Fc gamma R polymorphisms have traditionally employed peripheral venous blood as a source of leukocytes or genomic DNA, respectively. The present study was undertaken to determine whether human salivary DNA is a suitable alternative to DNA extracted from blood for genetic analysis of FC gamma R allelic polymorphisms. Genomic DNA was extracted from whole saliva of 69 healthy adult volunteers using a commercial DNA purification kit. The average quantity of genomic DNA isolated per ml of saliva was 19.2 +/- 14.1 micrograms. To assess intrasubject variation in yield of salivary DNA, ten saliva samples were collected from a single donor over a 3-month period. The average yield of DNA recovered from these samples was 25.2 +/- 13.7 micrograms. Volumes of saliva as small as 100 microliters, as well as saliva samples stored at -70 degrees C for prolonged periods (up to 6 years), provided DNA in amounts sufficient for PCR-based genetic analysis. Two comparative PCR assays were performed using DNA extracted from both peripheral blood and saliva from a number of individuals. The assays were able to detect a single nucleotide substitution (G-->A) in the Fc gamma RIIA gene, as well as two codominant alleles encoding the NA polymorphism in Fc gamma RIIIB, respectively. Furthermore, Fc gamma RIIA and Fc gamma RIIIB genotype results were confirmed by quantitative flow cytometry using specific monoclonal antibodies. Complete concordance was achieved between the typing results of our salivary DNA, and blood DNA-based assays. Therefore, saliva appears to be an excellent source of DNA for studies of Fc gamma RIIA and Fc gamma RIIIB polymorphisms.
已发现低亲和力IgG Fc受体（FcγR）的基因多态性会影响人IgG亚类抗体的结合，并且可能影响对某些类型的感染性疾病和自身免疫性疾病的易感性。传统上，FcγR多态性的表型和/或基因型分析分别采用外周静脉血作为白细胞或基因组DNA的来源。本研究旨在确定人唾液DNA是否是用于FcγR等位基因多态性基因分析的、从血液中提取的DNA的合适替代物。使用商用DNA纯化试剂盒从69名健康成年志愿者的全唾液中提取基因组DNA。每毫升唾液分离出的基因组DNA的平均量为19.2±14.1微克。为了评估唾液DNA产量的个体内变异，在3个月的时间里从一名供体收集了10份唾液样本。从这些样本中回收的DNA的平均产量为25.2±13.7微克。低至100微升的唾液体积，以及在-70℃下长期保存（长达6年）的唾液样本，都能提供足以用于基于PCR的基因分析的DNA量。使用从多个个体的外周血和唾液中提取的DNA进行了两项比较PCR分析。这些分析能够分别检测FcγRIIA基因中的单核苷酸取代（G→A），以及编码FcγRIIIB中NA多态性的两个共显性等位基因。此外，使用特异性单克隆抗体通过定量流式细胞术证实了FcγRIIA和FcγRIIIB基因型结果。我们的唾液DNA分型结果与基于血液DNA的分析结果完全一致。因此，唾液似乎是用于研究FcγRIIA和FcγRIIIB多态性的极佳DNA来源。
