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A physical map of a BAC clone contig covering the entire autosome insertion between ovine MHC Class IIa and IIb.

Li G, Liu K, Jiao S, Liu H, Blair HT, Zhang P, Cui X, Tan P, Gao J, Ma RZ - BMC Genomics (2012)

Bottom Line: A total of 368 positive BAC clones were identified and 108 of the effective clones were ordered into an overlapping BAC contig to cover the consensus region between ovine MHC class IIa and IIb.The map confirmed the bovine sequence assembly for the same homologous region.The entire ovine MHC region is now fully covered by a continuous BAC clone contig.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life Sciences, Shihezi University, Xinjiang 832003, China.

ABSTRACT

Background: The ovine Major Histocompatibility Complex (MHC) harbors genes involved in overall resistance/susceptibility of the host to infectious diseases. Compared to human and mouse, the ovine MHC is interrupted by a large piece of autosome insertion via a hypothetical chromosome inversion that constitutes ~25% of ovine chromosome 20. The evolutionary consequence of such an inversion and an insertion (inversion/insertion) in relation to MHC function remains unknown. We previously constructed a BAC clone physical map for the ovine MHC exclusive of the insertion region. Here we report the construction of a high-density physical map covering the autosome insertion in order to address the question of what the inversion/insertion had to do with ruminants during the MHC evolution.

Results: A total of 119 pairs of comparative bovine oligo primers were utilized to screen an ovine BAC library for positive clones and the orders and overlapping relationships of the identified clones were determined by DNA fingerprinting, BAC-end sequencing, and sequence-specific PCR. A total of 368 positive BAC clones were identified and 108 of the effective clones were ordered into an overlapping BAC contig to cover the consensus region between ovine MHC class IIa and IIb. Therefore, a continuous physical map covering the entire ovine autosome inversion/insertion region was successfully constructed. The map confirmed the bovine sequence assembly for the same homologous region. The DNA sequences of 185 BAC-ends have been deposited into NCBI database with the access numbers HR309252 through HR309068, corresponding to dbGSS ID 30164010 through 30163826.

Conclusions: We have constructed a high-density BAC clone physical map for the ovine autosome inversion/insertion between the MHC class IIa and IIb. The entire ovine MHC region is now fully covered by a continuous BAC clone contig. The physical map we generated will facilitate MHC functional studies in the ovine, as well as the comparative MHC evolution in ruminants.

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PCR verification of the overlapping relationship between pairs of overlapping BAC clones. Pairs of overlapped BAC clones were PCR amplified using a primer pair designed based on the BAC-end sequence. The markers above the black lines define the primer pairs and the ones below the lines are numbers of positive clones used as PCR templates.
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Figure 3: PCR verification of the overlapping relationship between pairs of overlapping BAC clones. Pairs of overlapped BAC clones were PCR amplified using a primer pair designed based on the BAC-end sequence. The markers above the black lines define the primer pairs and the ones below the lines are numbers of positive clones used as PCR templates.

Mentions: For additional cross-verification of the BAC clone orders, a total of 108 pairs of BAC-end oligo primers were designed for amplification by PCR of the common loci in two overlapping BACs (Figure3). Verification PCR confirmed the results of DNA fingerprinting at a high level of accuracy. Out of the 108 primer pairs used, 103 produced the specific PCR products with the expected size, the overall success rate reached 95% (Additional file 1: Table S1). An overlapping relationship between two BACs was further verified if the common target loci were detected from both BACs in the overlapped region. A total of five pairs of oligo primers failed to generate the specific PCR band, or failed to produce the PCR fragment at the expected size.


A physical map of a BAC clone contig covering the entire autosome insertion between ovine MHC Class IIa and IIb.

Li G, Liu K, Jiao S, Liu H, Blair HT, Zhang P, Cui X, Tan P, Gao J, Ma RZ - BMC Genomics (2012)

PCR verification of the overlapping relationship between pairs of overlapping BAC clones. Pairs of overlapped BAC clones were PCR amplified using a primer pair designed based on the BAC-end sequence. The markers above the black lines define the primer pairs and the ones below the lines are numbers of positive clones used as PCR templates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475007&req=5

Figure 3: PCR verification of the overlapping relationship between pairs of overlapping BAC clones. Pairs of overlapped BAC clones were PCR amplified using a primer pair designed based on the BAC-end sequence. The markers above the black lines define the primer pairs and the ones below the lines are numbers of positive clones used as PCR templates.
Mentions: For additional cross-verification of the BAC clone orders, a total of 108 pairs of BAC-end oligo primers were designed for amplification by PCR of the common loci in two overlapping BACs (Figure3). Verification PCR confirmed the results of DNA fingerprinting at a high level of accuracy. Out of the 108 primer pairs used, 103 produced the specific PCR products with the expected size, the overall success rate reached 95% (Additional file 1: Table S1). An overlapping relationship between two BACs was further verified if the common target loci were detected from both BACs in the overlapped region. A total of five pairs of oligo primers failed to generate the specific PCR band, or failed to produce the PCR fragment at the expected size.

Bottom Line: A total of 368 positive BAC clones were identified and 108 of the effective clones were ordered into an overlapping BAC contig to cover the consensus region between ovine MHC class IIa and IIb.The map confirmed the bovine sequence assembly for the same homologous region.The entire ovine MHC region is now fully covered by a continuous BAC clone contig.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life Sciences, Shihezi University, Xinjiang 832003, China.

ABSTRACT

Background: The ovine Major Histocompatibility Complex (MHC) harbors genes involved in overall resistance/susceptibility of the host to infectious diseases. Compared to human and mouse, the ovine MHC is interrupted by a large piece of autosome insertion via a hypothetical chromosome inversion that constitutes ~25% of ovine chromosome 20. The evolutionary consequence of such an inversion and an insertion (inversion/insertion) in relation to MHC function remains unknown. We previously constructed a BAC clone physical map for the ovine MHC exclusive of the insertion region. Here we report the construction of a high-density physical map covering the autosome insertion in order to address the question of what the inversion/insertion had to do with ruminants during the MHC evolution.

Results: A total of 119 pairs of comparative bovine oligo primers were utilized to screen an ovine BAC library for positive clones and the orders and overlapping relationships of the identified clones were determined by DNA fingerprinting, BAC-end sequencing, and sequence-specific PCR. A total of 368 positive BAC clones were identified and 108 of the effective clones were ordered into an overlapping BAC contig to cover the consensus region between ovine MHC class IIa and IIb. Therefore, a continuous physical map covering the entire ovine autosome inversion/insertion region was successfully constructed. The map confirmed the bovine sequence assembly for the same homologous region. The DNA sequences of 185 BAC-ends have been deposited into NCBI database with the access numbers HR309252 through HR309068, corresponding to dbGSS ID 30164010 through 30163826.

Conclusions: We have constructed a high-density BAC clone physical map for the ovine autosome inversion/insertion between the MHC class IIa and IIb. The entire ovine MHC region is now fully covered by a continuous BAC clone contig. The physical map we generated will facilitate MHC functional studies in the ovine, as well as the comparative MHC evolution in ruminants.

Show MeSH
Related in: MedlinePlus