ABSTRACT

DNA sequencing is a necessary technique for almost all molecular biology studies including DNA cloning, characterization, mutagenesis, DNA recombination, regulation of gene expression,1-3 and protein expression. There are several well-established methods for nucleic acid sequencing, and in recent years, advances in automated sequencing have made DNA sequence analysis an easy and routine process. The present chapter describes in detail the preparation of DNA for sequencing.4-6 DNA sequencing by dideoxynucleotides chain termination2,3 and cycle sequencing.1,4-6 These techniques are the foundation of most sequencing applications; however, with the advent of automated

7.1 Preparation of DNA for Sequencing ..................................................................................... 142 7.1.1 Preparation of Double-Stranded Plasmid DNA........................................................ 142 7.1.2 Preparation of Single-Stranded Template DNA ....................................................... 142

7.1.2.1 Preparation of Single-Stranded M13 DNA ................................................ 142 7.1.2.2 Preparation of Single-Stranded DNA Using Helper Phage R408 .............. 143

7.1.3 Puri cation of Double-Stranded Lambda DNA ....................................................... 143 7.1.4 Preparation of Double-Stranded DNA Fragments ................................................... 144

7.2 DNA Sequencing by Dideoxynucleotide Chain Termination............................................... 144 7.3 Dye Cycle Sequencing and Automated Sequencing ............................................................. 145

7.3.1 Selection of Oligonucleotides ................................................................................... 145 7.3.2 Dye Cycle Sequencing and Automated Sequencing ................................................. 146 7.3.3 DNA Sequencing Using an Automated Sequencer .................................................. 148

7.3.3.1 Sequencing Reactions ................................................................................ 148 7.3.3.2 Sample Preparation for Injection ............................................................... 150 7.3.3.3 Analysis of Sequence Chromatograms ...................................................... 151

7.4 Troubleshooting Guide ......................................................................................................... 151 7.4.1 Weak Signals ............................................................................................................ 151 7.4.2 Extensions Appear Short (Read Length Limited to Less than 350 Bases) .............. 153 7.4.3 Chromatogram Anomalies ....................................................................................... 153

7.5 Importance of Genome Sequencing ..................................................................................... 153 7.5.1 Shotgun Sequencing ................................................................................................. 155 7.5.2 Next Generation Sequencing Platforms .................................................................... 156

7.5.2.1 Solexa Sequencing ..................................................................................... 158 7.5.2.2 Other Sequencing Technologies ................................................................ 158

7.6 Sequence Assembly and Annotation .................................................................................... 159 7.6.1 Assembly Algorithms ............................................................................................... 159 7.6.2 Scaffolding ................................................................................................................ 160 7.6.3 Missing Segments and Repeats ................................................................................ 160 7.6.4 Finishing ................................................................................................................... 160 7.6.5 Gene Identi cation and Annotation .......................................................................... 161

References ...................................................................................................................................... 162

sequencing and the availability of automated liquid handlers, we also brie§y describe how the newer machines and sources have greatly increased the speed of DNA sequence analysis. The following techniques are routinely used in our respective labs.