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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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Representative gel images on initial PCR screening of an ovine BAC library using comparative primers from the bovine sequences. Approximately 190,500 random BAC clones were organized into pooled super DNA plates of rows, columns, and plates to facilitate PCR screening. Location of a target positive BAC clone in the library was determined usually by two runs of PCRs, one for “plate” and the other for “row + column”. The procedure eliminated the need for hybridization-based screening with radioactive 32P labeling. Gel images of PCR screen band on (A): Row pool of P098 BAC plate using the primer pair S036; (B): Row pool of P056 BAC plate using the primer pair S109; (C): Row N of P083 BAC plate; (D): Row F of P162 BAC plate. M: DL2000. Sample: PCR Products. A ~ P: Number of Row. 1 ~ 16: Number of Column (only partial shown here). P: Positive control (The amplified PCR products using the sheep genome DNA as templates).
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Figure 1: Representative gel images on initial PCR screening of an ovine BAC library using comparative primers from the bovine sequences. Approximately 190,500 random BAC clones were organized into pooled super DNA plates of rows, columns, and plates to facilitate PCR screening. Location of a target positive BAC clone in the library was determined usually by two runs of PCRs, one for “plate” and the other for “row + column”. The procedure eliminated the need for hybridization-based screening with radioactive 32P labeling. Gel images of PCR screen band on (A): Row pool of P098 BAC plate using the primer pair S036; (B): Row pool of P056 BAC plate using the primer pair S109; (C): Row N of P083 BAC plate; (D): Row F of P162 BAC plate. M: DL2000. Sample: PCR Products. A ~ P: Number of Row. 1 ~ 16: Number of Column (only partial shown here). P: Positive control (The amplified PCR products using the sheep genome DNA as templates).

Mentions: Organization of ~190,500 random ovine BAC clones into three dimensional super DNA pool of rows (n = 16), columns (n = 24), and plates (n = 496) significantly increased the efficiency of PCR screening of the sheep BAC library (Figure1). The whole BAC library of 8.4× genome equivalents was screened through with a maximum of 536 (=496 + 16 + 24) PCR reactions, and a positive BAC clone could be frequently identified by as few as 136 (=96 + 16 + 24) PCR reactions using the super pool DNA as templates. In addition, PCR-based BAC clone screening also helped to eliminate the need for hybridization-based screening using radioactive 32P labeling.


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)

Representative gel images on initial PCR screening of an ovine BAC library using comparative primers from the bovine sequences. Approximately 190,500 random BAC clones were organized into pooled super DNA plates of rows, columns, and plates to facilitate PCR screening. Location of a target positive BAC clone in the library was determined usually by two runs of PCRs, one for “plate” and the other for “row + column”. The procedure eliminated the need for hybridization-based screening with radioactive 32P labeling. Gel images of PCR screen band on (A): Row pool of P098 BAC plate using the primer pair S036; (B): Row pool of P056 BAC plate using the primer pair S109; (C): Row N of P083 BAC plate; (D): Row F of P162 BAC plate. M: DL2000. Sample: PCR Products. A ~ P: Number of Row. 1 ~ 16: Number of Column (only partial shown here). P: Positive control (The amplified PCR products using the sheep genome DNA as templates).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Representative gel images on initial PCR screening of an ovine BAC library using comparative primers from the bovine sequences. Approximately 190,500 random BAC clones were organized into pooled super DNA plates of rows, columns, and plates to facilitate PCR screening. Location of a target positive BAC clone in the library was determined usually by two runs of PCRs, one for “plate” and the other for “row + column”. The procedure eliminated the need for hybridization-based screening with radioactive 32P labeling. Gel images of PCR screen band on (A): Row pool of P098 BAC plate using the primer pair S036; (B): Row pool of P056 BAC plate using the primer pair S109; (C): Row N of P083 BAC plate; (D): Row F of P162 BAC plate. M: DL2000. Sample: PCR Products. A ~ P: Number of Row. 1 ~ 16: Number of Column (only partial shown here). P: Positive control (The amplified PCR products using the sheep genome DNA as templates).
Mentions: Organization of ~190,500 random ovine BAC clones into three dimensional super DNA pool of rows (n = 16), columns (n = 24), and plates (n = 496) significantly increased the efficiency of PCR screening of the sheep BAC library (Figure1). The whole BAC library of 8.4× genome equivalents was screened through with a maximum of 536 (=496 + 16 + 24) PCR reactions, and a positive BAC clone could be frequently identified by as few as 136 (=96 + 16 + 24) PCR reactions using the super pool DNA as templates. In addition, PCR-based BAC clone screening also helped to eliminate the need for hybridization-based screening using radioactive 32P labeling.

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