Non-gridded library: a new approach for BAC (bacterial artificial chromosome) exploitation in hexaploid wheat (Triticum aestivum).
The feasibility of exploiting non-gridded bacterial artificial chromosome (BAC) libraries and some major factors affecting the efficiency of handling such libraries were studied in hexaploid wheat. Even for a bacterial culture containing only 55% recombinants, some 2000 BAC clones with inserts ranging from 45 to 245 kb could be pooled. The pooled BAC clones could be amplified by culturing for up to 6 h without losing any target clones. These results imply that even for hexaploid wheat, which has an extremely large genome, some 250 pools are sufficient for a BAC library that should satisfy many research objectives. This non-gridded strategy would dramatically reduce the cost and make robotic equipment non-essential in exploiting BAC technology. To construct a representative library and to minimise clone competition, thawing and re-freezing ligation mixtures and bacterial cultures should be avoided in BAC library construction and application.
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CSIRO Plant Industry, 306 Carmody Road, St Lucia, Queensland 4067, Australia.
Weining
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Sharp
P J
PJ
Liu
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D015139:Blotting, Southern; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases / Q000378:metabolism; D015342:DNA Probes / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D005615:Freezing; D005822:Genetic Vectors / Q000235:genetics; D018745:Genome, Plant; D015698:Genomic Library; D005990:Glycerol; D016133:Polymerase Chain Reaction; D011123:Polyploidy; D013997:Time Factors; D014169:Transformation, Bacterial; D014908:Triticum / Q000235:genetics*
D015342:DNA Probes; D004274:DNA, Recombinant; D011088:DNA Ligases; D005990:Glycerol