短微加工电泳装置上高性能DNA测序的优化。

Optimization of high-performance DNA sequencing on short microfabricated electrophoretic devices.

作者信息

Salas-Solano O, Schmalzing D, Koutny L, Buonocore S, Adourian A, Matsudaira P, Ehrlich D

机构信息

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.

出版信息

Anal Chem. 2000 Jul 15;72(14):3129-37. doi: 10.1021/ac000055j.

DOI:10.1021/ac000055j

PMID:10939377

Abstract

We have examined the parametric performance of short microfabricated electrophoresis devices that operate with a replaceable linear poly(acrylamide) (LPA) solution for the application of DNA sequencing. A systematic study is presented of the dependence of selectivity, separation efficiency, and resolution of sequencing fragments on buffer composition, LPA concentration, LPA composition, microdevice temperature, electric field, and device length. A specific optimization is made for DNA sequencing on 11.5-cm devices. Using a separation matrix composed of 3.0% (w/w) 10 MDa plus 1.0% (w/w) 50 kDa LPA, elevated microdevice temperature (50 degrees C), and 200 V/cm, high-speed DNA sequencing of 580 bases on standard M13mp18 was obtained in only 18 min with a base-calling accuracy of 98.5%. Read lengths of 640 bases at 98.5% accuracy were achieved in approximately 30 min by reducing the electric field strength to 125 V/cm. We believe that this constitutes matrix-limited performance for microdevices of this length using LPA sieving matrix and this buffer chemistry. In addition, it was confirmed, that shorter devices are rather impractical for production sequencing applications when LPA is used as sieving matrix.

摘要

我们研究了用于DNA测序的、采用可替换线性聚丙烯酰胺（LPA）溶液运行的短微加工电泳装置的参数性能。本文对测序片段的选择性、分离效率和分辨率对缓冲液组成、LPA浓度、LPA组成、微装置温度、电场和装置长度的依赖性进行了系统研究。针对11.5厘米装置上的DNA测序进行了具体优化。使用由3.0%（w/w）10 MDa加1.0%（w/w）50 kDa LPA组成的分离基质、升高的微装置温度（50摄氏度）和200 V/cm，在标准M13mp18上仅用18分钟就实现了580个碱基的高速DNA测序，碱基识别准确率为98.5%。通过将电场强度降低到125 V/cm，在大约30分钟内实现了640个碱基、准确率为98.5%的读长。我们认为，这构成了使用LPA筛分基质和这种缓冲液化学的该长度微装置的基质限制性能。此外，还证实了，当使用LPA作为筛分基质时，较短的装置对于生产测序应用而言相当不实用。