Automated DNA profiling by fluorescent labeling of PCR products.
Automated DNA profiling by fluorescent labeling of PCR products.
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通过 PCR 产物荧光标记进行自动化 DNA 分析。
DOI：
10.1101/gr.2.1.34
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Julius S. Youngner
中科院分区：
文献类型：
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作者：
Hiromichi Yazyu;Yosuke Kishimoto;Takao Sekiya;Helmut Zarbl;P. Keohavong;Gerald F. Joyce;Kevin M. Sullivan;James M. Robertson;Julius S. Youngner
DNA 分析已通过扩增可变数目串联重复 (VNTR) 位点的荧光标记实现自动化。这是通过使用荧光标记引物扩增 10 ng 基因组 DNA，并在电泳过程中对产物进行激光检测来实现的。 PCR 产物通过与每个样品混合的标准尺寸梯子共电泳来确定尺寸，从而消除了泳道间迁移率差异导致的尺寸估计误差。这提高了 VNTR 表征的精度，并能够解析因单个 15 bp 重复而不同的等位基因。该系统具有高通量能力：在 6 小时内对 24 个样品进行电泳和分析。此外，由于有四种不同的染料可用，因此可以使用用作内标的第四种染料同时表征三个不同的位点。对大约 100 名不相关的英国白种人的 D1S80、D17S5 和 ApoB 位点进行了分析。两个无关个体在这三个位点偶然匹配的概率（pM）被确定为分别为0.065、0.040和0.069，总pM为1.8 x 10(-4)。
DNA profiling has been automated by the fluorescent tagging of amplified variable number tandem repeat (VNTR) loci. This was achieved by the use of fluorescently labeled primers in the amplification of 10 ng of genomic DNA, coupled with laser detection of the products during electrophoresis. The PCR products are sized by co-electrophoresing a standard size ladder mixed with every sample, thereby eliminating errors in size estimation caused by lane-to-lane differences in migration rate. This increases the precision of VNTR characterization and enables alleles that differ by a single 15-bp repeat to be resolved. The system is capable of high throughput: Twenty-four samples are electrophoresed and analyzed within 6 hr. Also, because four different dyes are available, three different loci can be simultaneously characterized with the fourth dye used for the internal standard. Approximately 100 unrelated British caucasians were analyzed at the loci D1S80, D17S5, and ApoB. The probabilities of two unrelated individuals matching by chance (pM) at these three loci were determined to be 0.065, 0.040, and 0.069, respectively, with a combined pM of 1.8 x 10(-4).