PCR-based Method for Screening Libraries

نویسنده

  • David I. Israel
چکیده

One of the fundamental techniques of molecular biology is the isolation of a rare clone from a complex mixture of DNA sequences contained within a library. The isolation of cDNA and genomic clones from highly complex libraries is often an early step in studies aimed at understanding basic biological processes as well as in applied biological research. In a typical genomic library with an average insert size of 20,000 bp from an organism with a haploid genome size of 2 x 10 bp, the occurrence of a single-copy gene will be approximately 1/10. Likewise, for cDNA clones within a highly complex library derived from a tissue or cell line that expresses many different genes, a particular clone may occur with a similarly low frequency. Screening of libraries of high complexity by techniques such as filter hybridization (Benton and Davis 1977) or expression cloning (Wong et al. 1985) is therefore a labor-intensive and time-consuming process because of the large number of clones that must be screened to obtain the clone of interest. PCR results in the amplification of a given nucleic acid sequence by many orders of magnitude (Saiki et al. 1985). When applied to the screening of highly complex DNA libraries contained within either bacteriophage or plasmid vectors, PCR offers the opportunity to identify rare DNA sequences in complex mixtures of molecular clones by increasing the abundance of a particular sequence, thereby allowing the easy identification of a particular clone in a portion of the library. This is accomplished by subdividing the original library into pools of decreased complexity and screening each pool or groups of pools for a given DNA sequence (Fig 1). A pool that contains the desired clone is subsequently subdivided into smaller pools, each of which is screened using the same PCR protocol that was used for the primary screen. After several cycles of subdividing and screening, the initially rare clone is greatly enriched and can be easily obtained as a pure clone (Israel 1993). A method for screening highly complex DNA libraries using PCR is described in this chapter. This method allows a library of high complexity to be screened in a short time and provides an alternative to more traditional and time-consuming screening methods that entail plaque or colony hybridization, or methods that require the expression of a functional gene product. The main advantages of this screening technique are its speed, sensitivity, and ease. Some providers of molecular biology reagents, such as MRC geneservice, have PCR-ready library pools available for screening. In addition, the method as described below requires more than one oligonucleotide to anneal correctly to the template DNA and/or PCR product, thereby providing a high degree of stringency for a true positive signal. 24

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تاریخ انتشار 2004