Serial cloner cohesive ends1/7/2024 1999), double-stranded cDNA is synthesized from poly (A) + mRNA by priming first-strand cDNA synthesis with a biotinylated oligo (dT) 18 primer. In the original SAGE procedure ( Velculescu et al. Genomic variation and, if genome information is available, provide immediate species identity. 2002) in that it produces large numbers of positionally defined 21-bp tag sequences that can be used to examine intraspecific It is similar to long serial analysis of gene expression (long SAGE Velculescu 2001 Saha et al. Genomes by use of genomic signature tags (GSTs), which like AFLP-related methods does not rely on a priori knowledge of the In this paper, we describe a new higher-throughput, direct sequence-based approach for characterizing prokaryotic or eukaryotic Is labor intensive and, in most cases, requires further PCR amplification or cloning of the eluted DNAs. Usually, individualįragments are extracted from the gels and the corresponding sequences determined by direct DNA sequencing however, this approach A drawback to these techniques is how to further analyze novel bands. The DNA fingerprints are then visualized by means of autoradiography, phosphor-imaging,įluorescence, or other labeling methods. Landmark genome scanning (RLGS)-are generally based on some combination of restriction digestion of genomic DNA, PCR amplification,Īnd gel electrophoretic separation. Polymorphism (T-RFLP), denaturing gradient gel electrophoresis (DGGE), amplified rDNA restriction analysis, (ARDRA), and restriction These fingerprinting techniques-such as amplified fragment length polymorphism (AFLP), terminal restriction fragment length 2000 Kozdrój and van Elsas 2001 Torsvik and Øvreås 2002). Thompson.]Ī variety of DNA-based fingerprinting techniques now exist to characterize and compare whole genomes of prokaryotes and eukaryotes,Įither as independent organisms or as members of communities ( Schloter et al. [The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: W. Relative to the archetype CO92 strain, including deletion of a 57-kb region of the chromosome known to be an unstable pathogenicity The GST profile predicts that this strain has several changes Of the 4.7-Mb Yersinia pestis EV766 genome using BamHI as the fragmenting enzyme and NlaIII as the tagging enzyme validated the precision of our approach. To provide additional nucleotide information or used as probes to identify specific clones in metagenomic libraries. To be long enough for use as oligonucleotide primers to amplify adjacent segments of the DNA, which can then be sequenced The tag sequences and abundances are used to create a high-resolution GST sequence profile of the genomic DNA. These tags are PCR-amplified, purified, concatenated, and then cloned and sequenced. Site for MmeI, a type IIS restriction enzyme, is then used to release 21-bp tags from fixed positions in the DNA relative to the sites An oligonucleotide adaptor containing a recognition The DNA is initially fragmented with a type II restriction enzyme. coli, and those more experienced researchers a broad-ranging, proven array of successful techniques.Genomic signature tags (GSTs) are the products of a method we have developed for identifying and quantitatively analyzing coli Plasmid Vectors offers those new to the field a basic guide to the use of plasmid vectors in the cloning host E. Each fully tested protocol is described in step-by-step detail by an established expert in the field and includes an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, tips on troubleshooting and avoiding known pitfalls and, where needed, a discussion of the interpretation and use of the results.Ĭomprehensive and highly practical, E. Also presented are methods for common downstream applications, such as mutagenesis, expression of recombinant proteins and RNA transcripts, and uses of reporter genes. They also include protocols for the construction and screening of libraries, as well as specific techniques for specialized cloning vehicles, such as cosmids, bacterial artificial chromosomes, l vectors, and phagemids. The authors describe readily reproducible methods for cloning DNA into plasmid vectors, transforming plasmids into E. coli Plasmid Vectors, experienced bench researchers describe their proven techniques for the manipulation of recombinant plasmids utilizing this popular bacterial host. coli, constitutes one of the fundamental methodologies of molecular biotechnology. Manipulation of recombinant DNA, which is almost exclusively performed using the host E.
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