DNA sequencing provides the most complete characterization of recombinant plasmid DNAs. Using primers targeting the plasmid backbone and/or the insert sequence, the identity and order of nucleotide bases for any given DNA can be determined. In the context of cloning, sequencing allows users to confirm the DNA sequence of the insert, insert orientation, and to examine the junctions between the plasmid and insert DNA. The size of a DNA insert will dictate how many and which primers are necessary to determine the complete sequence. In cases where the complete insert sequence must be determined, it is advised to use primers targeting both the plasmid DNA, ~100-150 bases outside the insert site, and the insert DNA. Primers should be designed to give at least 2x coverage of the entire insert sequence for reliable characterization.
Today, the Sanger sequencing technique is the most commonly-used method for determining DNA sequences of recombinant plasmids. Sanger sequencing involves the use of a DNA polymerase, a primer, unlabeled deoxynucleotide triphosphates (dNTPs), and fluorescently labeled dideoxynucleotide triphosphates (ddNTPs), where each base is labeled with a unique fluorophore. Incorporation of a ddNTP into the newly synthesized strand prevents the addition of subsequent nucleotides, stopping the further elongation of the DNA molecule and resulting in a DNA product with a fluorescently labeled ddNTP at the end of the strand. The sequence of nucleotides can then be determined by separating the DNA products by size.
Library Preparation for Next Generation Sequencing
While Sanger sequencing remains the standard for analysis of recombinant DNAs, next generation sequencing (NGS) technologies such as Illumina®, Ion TorrentTM, SOLiDTM, 454TM, and Pacific BiosciencesTM provide platforms for high-throughput, low cost sequence analysis. NEBNext® reagents are a series of highly pure reagents that facilitate library preparation of DNA for multiple NGS platforms, as well as for, ChIP, RNA and Small RNA.