**2. BACs used for gene mapping and sequencing of target regions**

Prior to the availability of BAC libraries for marsupials and monotremes, gene mapping by fluorescence in situ hybridization (FISH) was an arduous task, which relied on the isolation of the gene of interest from a lambda phage genomic library. The construction of BAC libraries for the species listed above has facilitated the mapping of many marsupial and monotreme genes by FISH. Initially, PCR products were used to screen these BAC libraries for genes of interest but more recently overgo probes (overlapping oligonucleotides) have proven to be the method of choice, permitting the isolation of many genes from one screening, thereby facilitating the rapid construction of gene maps. Likewise, before the availability of genome sequence, isolating and sequencing BACs containing genes of interest proved a very useful method for obtaining sequence from particular regions of interest. In some cases, even after whole genome sequencing had been performed, it proved necessary to take this targeted approach. These mapped or sequenced BACs have led to a number of important findings, with examples of those having had a significant impact on previously held theories reviewed here. Examples include the determination of the origins of monotreme and marsupial sex chromosomes, the evolution of regions imprinted in eutherian mammals, the unique arrangement of the Major Histocompatibility Complex (MHC) in the tammar wallaby and the evolution of the - and -globin gene clusters. BACs have played a vital role in many more studies using gene mapping and/or target region sequencing than can be included in detail in this review and hence, other studies that have utilized BACs for these purposes are listed in Table 3. This is not an exhaustive list but an indication of the breadth of studies in which BACs have played a role.


Table 3. Studies in marsupial and monotreme comparative genomics that relied on BAC clones.
