Chan K C, Muschik G M, Issaq H J, Garvey K J, Generlette P L
Chemical Synthesis and Analysis Laboratory, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702, USA,
Anal Biochem. 1996 Dec 1;243(1):133-9. doi: 10.1006/abio.1996.0491.
In an effort to develop capillary electrophoresis (CE) for high-throughput polymerase chain reaction (PCR) molecular diagnostics, a method was developed to rapidly screen small PCR products of similar molecular weights. The assay of interest required the separation of two PCR products (375 and 400 bp) in an assay of TGF-beta 1 knockout mice to determine the genotype of neonates. Using a commercially available CE instrument, the two PCR products were separated in 12 min with a replaceable gel buffer, a 20-cm effective length DB-17 capillary, and 185 V/cm field strength. With the coinjection of a 20-bp ladder, the sizes of the PCR products were determined from the electropherogram without using a calibration plot and curve-fitting program. Faster separation was obtained using the combination of a short effective length capillary and high field strength. The two PCR products were separated in 82 s with a 7-cm effective length capillary and 556 V/cm. A 60% buffer further reduced the separation time in about a minute. This high-speed separation, with minimum postrun data processing, is highly desirable for the high-throughput screening of PCR products using a single-capillary CE system.
为了开发用于高通量聚合酶链反应（PCR）分子诊断的毛细管电泳（CE）技术，人们研发了一种方法来快速筛选分子量相近的小PCR产物。在一项检测转化生长因子β1基因敲除小鼠的实验中，需要分离两种PCR产物（375和400碱基对）以确定新生小鼠的基因型，这正是我们感兴趣的检测项目。使用市售的CE仪器，通过可更换的凝胶缓冲液、一根有效长度为20厘米的DB - 17毛细管以及185伏/厘米的场强，在12分钟内分离出了这两种PCR产物。通过同时注入一条20碱基对的梯状条带，无需使用校准图和曲线拟合程序，就能从电泳图中确定PCR产物的大小。采用短有效长度毛细管和高场强相结合的方式可实现更快的分离。使用一根有效长度为7厘米的毛细管和556伏/厘米的场强，在82秒内就分离出了这两种PCR产物。60%的缓冲液能在大约一分钟内进一步缩短分离时间。这种高速分离且运行后数据处理最少的方式，对于使用单毛细管CE系统进行PCR产物的高通量筛选非常理想。
