Abstract
We are developing a novel strategy for efficient and specific isolation of cDNA clones encoded by a particular genomic region. This sandwich-selection method consists of: 1. Hybridization of single-stranded (ss) circular cDNA molecules to tagged in vitro synthesized RNA from the genomic region; 2. Retention of “genomic RNA” - cDNA hybrids on an avidin-matrix through a biotinylated RNA complementary to the tag; and 3. Elution of specific cDNAs from the avidin-matrix with ribonuclease A. The selected ss cDNAs are directly used for electroporation of competent E. coli cells without requiring any amplification or cloning step. Here, we report the construction of appropriate vectors and results from model experiments. This protocol has been applied for specifically selecting NRL (neural retina leucine zipper) cDNA from a mixture of cDNA clones using a sub-library from the NRL genomic region. The control experiments have allowed us to optimize various steps in the procedure and provided valuable data regarding the yield and specificity of selection process. The strategy is now being used for the isolation of cDNAs from large regions of genomic DNA cloned in cosmids or yeast artificial chromosome (YAC) vectors and from microdissected and flow-sorted chromosomal DNA. We expect that a high degree of specificity and efficiency will permit wider application of this cDNA selection strategy.
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Yan, D., Swaroop, A. (1994). A Sandwich-Hybridization Method for Specific and Efficient Selection of cDNA Clones from Genomic Regions. In: Hochgeschwender, U., Gardiner, K. (eds) Identification of Transcribed Sequences. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-2562-2_10
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DOI: https://doi.org/10.1007/978-1-4615-2562-2_10
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