Construction of a highly enriched marsupial Y chromosome-specific BAC sub-library using isolated Y chromosomes |
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Authors: | N Sankovic M L Delbridge F Grützner M A Ferguson-Smith P C M O’Brien J A Marshall Graves |
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Institution: | (1) Comparative Genomics Group, Research School of Biological Sciences, Australian National University, Canberra, ACT, 2601, Australia;(2) Department of Zoology, University of Melbourne, Parkville, VIC, 3010, Australia;(3) School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, 5005, Australia;(4) Department of Veterinary Medicine, Cambridge University, Cambridge, CB3 0ES, UK |
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Abstract: | The Y chromosome is perhaps the most interesting element of the mammalian genome but comparative analysis of the Y chromosome
has been impeded by the difficulty of assembling a shotgun sequence of the Y. BAC-based sequencing has been successful for
the human and chimpanzee Y but is difficult to do efficiently for an atypical mammalian model species (Skaletsky et al.
2003, Kuroki et al.
2006). We show how Y-specific sub-libraries can be efficiently constructed using DNA amplified from microdissected or flow-sorted
Y chromosomes. A Bacterial Artificial Chromosome (BAC) library was constructed from the model marsupial, the tammar wallaby
(Macropus eugenii). We screened this library for Y chromosome-derived BAC clones using DNA from both a microdissected Y chromosome and a flow-sorted
Y chromosome in order to create a Y chromosome-specific sub-library. We expected that the tammar wallaby Y chromosome should
detect ∼100 clones from the 2.2 times redundant library. The microdissected Y DNA detected 85 clones, 82% of which mapped
to the Y chromosome and the flow-sorted Y DNA detected 71 clones, 48% of which mapped to the Y chromosome. Overall, this represented
a ∼330-fold enrichment for Y chromosome clones. This presents an ideal method for the creation of highly enriched chromosome-specific
sub-libraries suitable for BAC-based sequencing of the Y chromosome of any mammalian species. |
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Keywords: | BAC library Macropus eugenii tammar wallaby Y chromosome |
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