Construction of a highly enriched marsupial Y chromosome-specific BAC sub-library using isolated Y chromosomes.
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 approximately 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 approximately 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.
Sankovic
N
N
Comparative Genomics Group, Research School of Biological Sciences, Australian National University, Canberra, ACT 2601, Australia.
Delbridge
M L
ML
Grützner
F
F
Ferguson-Smith
M A
MA
O'Brien
P C M
PC
Marshall Graves
J A
JA
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial; D015723:Gene Library; D017404:In Situ Hybridization, Fluorescence; D007614:Macropodidae / Q000235:genetics*; D008297:Male; D014998:Y Chromosome