Rapid isolation of terminal sequences from cloned plant DNA fragments |
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Authors: | Xianghuai Lu Diane M Ruezinsky James J Giovannoni |
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Institution: | (1) Crop Biotechnology Center, Department of Horticultural Sciences, Texas A&M University, 77843-2133 College Station, TX, USA |
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Abstract: | The isolation of DNA clone termini is an important step in the development of DNA contigs utilized for a range of applications,
including physical mapping, genetic map-based cloning, insertion mutagenesis cloning, and isolation of complete gene sequences.
We describe a rapid PCR-based method for the isolation of vector-insert junctions, or insert terminal sequences, of cloned
plant DNA fragments. PCR amplification is performed using a vector-specific primer and a nonspecific primer, originally designed
for use in animal systems, containing degenerative bases that we have shown can also anneal to plant insert DNA. Using this
method we have successfully isolated end-terminal sequences from plant genomic clones harbored in YAC, BAC, and bacteriophage
λ vectors. Termini of genomic clones from both tomato andArabidopsis were isolated demonstrating the utility of this technique among a range of plant species. |
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Keywords: | map-based cloning physical mapping contigs insert termini |
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