7] Random cloning and sequencing by the M13/dideoxynucleotide chain termination method

Abstract

This chapter discusses random cloning and sequencing by the M13/dideoxynucleotide chain termination method. The dideoxy chain terminator/Ml3 vector method of deoxyribonucleic acid (DNA) sequencing is considered to be the fastest method to determine the sequence of large fragments of DNA. Lengths of DNA are cloned into the bacteriophage M13 that provides a source of large quantities of single-stranded DNA. This single-stranded DNA can then be used as a template in a primer extension dideoxynucleotide sequence reaction. The chapter discusses the problems related to the simplest method of breaking a DNA molecule into subfragments, which uses restriction endonucleases. The easiest method of purifying the fragment to be sequenced from the cloning vector is cleavage with suitable restriction endonuclease(s) followed by fractionation of the restricted DNA and isolation from a low gelling-temperature agarose minigel. The chapter discusses several procedures, such as isolation of fragment, fragment self-ligation, sonication, and others. Regarding size selection for DNA purification, two factors determine the minimum size to be selected. First, the insert to be sequenced should be at least as long as the maximum, which can be read from a normal sequence gel run. Second, as the subfragments have ends generated at random and not at specific primary sequence sites as with restriction enzymes, it is not possible to detect the junctions of religated noncontiguous fragments simply by inspection. M13 is the vector of choice for dideoxy sequencing for two main reasons. First, M13 bacteriophages are packaged in single strands of DNA, which are extruded from infected Escherichia coli cells into the surrounding culture medium. This means that considerable quantities of single-stranded template DNA can be easily produced. Second, the M13 mp vectors have a quick color assay to identify bacterial cells infected with phage containing an insert.