36. DNA Sequencing Using Capillary Array Electrophoresis 

Indu Kheterpal¹, Gary T. Wedemeyer¹, Yuping Cai², Alexander N. Glazer², and Richard A. Mathies^1 
1Departments of Chemistry and ²Molecular and Cellular Biology, University of California, Berkeley, CA 94720 
indu@zinc.cchem.berkeley.edu 

Capillary array electrophoresis has emerged as a valuable tool for DNA analysis. We are developing methods for obtaining high quality sequencing separations using confocal fluorescence CAE instruments^1,2 and energy transfer (ET) primers³. In practice replaceable separation matrices and base calling programs have been evaluated and optimized for high throughput sequencing separations of genomic DNA fragments from the cyanobacterium Anabaena. 

We have evaluated the available replaceable gels using the same sequencing samples, temperature, detection system, injection and separation conditions. These gels can be pumped into the capillaries allowing the use of capillaries for potentially 100 runs. The three gels evaluated were linear polyacrylamide (LPA), hydroxyethylcellulose (HEC) and a mixture of HEC and polyethylene oxide (PEO). We have found LPA to provide the best sequencing separations with the longest read lengths of 1000 bases in the least amount of time. We have also evaluated several base calling packages for their ease-of-use, ability to batch process and base-calling performance. BaseFinder ⁴ has emerged as the leading program for our data and is being used for all of the sequencing data analysis. 

We are currently validating methods by incorporating them into our Anabaena sequencing project performed by undergraduates. Several 6-15 kbp libraries of Anabaena genome potentially involved in the biosynthesis and control of phycobiliproteins have been constructed. The templates for sequencing are prepared by partial digestion of the genomic DNA utilizing restriction enzymes cocktails. Fragments in ~0.5 kb range are cloned into pUC 19 for bidirectional sequencing. The sequencing samples are generated using the Sanger dideoxy method and cycle sequencing. The separations are performed using our planar CAE instruments² and replaceable gels. The data are analyzed using BaseFinder and assembled using Phred, Phrap and Consed. Two libraries p69 and p74 have now been completely assembled and sequencing on five other libraries is near completion. These libraries total over 70,000 bases and the fragments are being sequenced with ~5-fold redundancy to ensure complete and accurate assembly. 

¹ Huang, X. C.; Mathies, R. A. (1992) Nature (London), 359, 167-169. 

² Kheterpal, I.; Scherer, J. R.; Clark, S. M.; Radhakrishnan, A; Ju, J.; Ginther, C. L.; Sensabaugh, G. F. and Mathies, R. A. (1996) Electrophoresis, 17, 1852-1859. 

³ Ju, J.; Glazer, A. N.; Mathies, R. A. (1996) Nature Medicine, 2, 246-249. 

⁴ Giddings, M. C.; Severin, J; Westphall, M; Wu, J. Z; and Smith, L. M. (1998) Genome Research 8, 644-665.