Griffin H G, Griffin A M
AFRC Institute of Food Research, Norwich Laboratory, Colney, England.
Appl Biochem Biotechnol. 1993 Jan-Feb;38(1-2):147-59. doi: 10.1007/BF02916418.
Determination of the sequence of DNA is one of the most important aspects of modern molecular biology. New sequencing methods currently being developed enable DNA sequence to be determined increasingly faster and more efficiently. One of the major advances in sequencing technology is the development of automated DNA sequencers. These utilize fluorescent rather than radioactive labels. A laser beam excites the fluorescent dyes, the emitted fluorescence is collected by detectors, and the information analyzed by computer. Robotic work stations are being developed to perform template preparation and purification, and the sequencing reactions themselves. Research is currently in progress to develop the technology of mass spectrometry for DNA sequencing. Success in this endeavor would mean that the gel electrophoresis step in DNA sequencing could be eliminated. A major innovation has been the application of polymerase chain reaction (PCR) technology to DNA sequence determination, which has led to the development of linear amplification sequencing (cycle sequencing). This very powerful yet technically simple method of sequencing has many advantages over conventional techniques, and may be used in manual or automated methods. Other recent innovations proposed recently to increase speed and efficiency include multiplex sequencing. This consists of pooling a number of samples and processing them as pools. After electrophoresis, the DNA is transferred to a membrane, and sequence images of the individual samples are obtained by sequential hybridizations with specific labeled oligonucleotides. Multiplex DNA sequencing has been used in conjunction with direct blotting electrophoresis to facilitate transfer of the DNA to a membrane. Chemiluminescent detection can also be used in conjunction with multiplex DNA sequencing to visualize the image on the membrane.
DNA序列的测定是现代分子生物学最重要的方面之一。目前正在开发的新测序方法能够越来越快速、高效地测定DNA序列。测序技术的一项重大进展是自动化DNA测序仪的开发。这些仪器使用荧光标记而非放射性标记。激光束激发荧光染料，探测器收集发射出的荧光，并由计算机分析信息。正在开发机器人工作站来进行模板制备、纯化以及测序反应本身。目前正在进行研究以开发用于DNA测序的质谱技术。这项工作的成功将意味着可以省去DNA测序中的凝胶电泳步骤。一项重大创新是将聚合酶链反应（PCR）技术应用于DNA序列测定，这导致了线性扩增测序（循环测序）的发展。这种非常强大但技术上简单的测序方法相对于传统技术有许多优点，可用于手动或自动化方法。最近提出的其他提高速度和效率的创新方法包括多重测序。这包括将多个样品混合并作为混合样本进行处理。电泳后，将DNA转移到膜上，并通过与特定标记的寡核苷酸进行顺序杂交获得各个样品的序列图像。多重DNA测序已与直接印迹电泳结合使用，以促进DNA转移到膜上。化学发光检测也可与多重DNA测序结合使用，以可视化膜上的图像。
