Richards B, Skoletsky J, Shuber A P, Balfour R, Stern R C, Dorkin H L, Parad R B, Witt D, Klinger K W
Department of Genetic Disease Research, Integrated Genetics, Framingham, MA 01701.
Hum Mol Genet. 1993 Feb;2(2):159-63. doi: 10.1093/hmg/2.2.159.
Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.
传统上，用于基于聚合酶链反应（PCR）的诊断分析的DNA来源于从全血中分离出的白细胞。尽管这种方法能产生大量的DNA，但该过程存在一些缺点，包括采血不便、接触血源性病原体的风险、液体样本处理以及提取过程有些繁琐。另外，用于基因诊断的DNA也来源于手指采血样本、发根、颊部刮片和尿液样本。口腔盐水冲洗液也被广泛用作收集颊上皮细胞作为DNA来源的一种方式。然而，这种方法仍然需要处理液体样本。在此，我们展示了我们的研究结果，即从细胞学刷和拭子上收集的颊细胞中快速提取DNA用于PCR反应，特别是对囊性纤维化跨膜传导调节因子（CFTR）基因内的5个外显子进行多重扩增。以这种方式收集的颊细胞中分离出的DNA质量足以可重复地支持多重扩增。颊细胞样本以及按此处所述从中制备的DNA非常稳定。从颊细胞制备的DNA上进行PCR扩增的成功率为99%。在一项盲法研究中，对464名个体的血液和颊细胞样本中的DNA扩增的多重产物中导致囊性纤维化的12种突变进行分析比较，血液和颊细胞DNA的结果相关性为100%，验证了从细胞学刷上收集的颊细胞中提取的DNA用于基因检测的可行性。
