Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences · 2008
[Construction and screening of genomic library from Raji cells].
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Gao Jian-ming, Li Xiao-ling, Li Gui-yuan
Аннотация
OBJECTIVE: To construct the genomic library of Raji cells and screen it by EBV DNA probe.
METHODS: High molecular weight genomic DNA of Raji cells was digested by restriction enzyme BamHI. DNA fragments ranging from 9 to 23 kb were recovered by agarose gel electrophoresis, which were ligated with Lambda DASH II vector BamHI arms pre-treated with calf intestine alkaline phosphatase (CIAP). Ligated DNA was packed in vitro using Gigapack III gold packaging extract. The library was plated on XL1-blue MRA (P2) host strain.Titering and screening of the Raji genomic library were performed.
RESULTS: The primary titer of the Raji genomic library was 1.8 x 10(5) pfu/mL, while that of the amplified library was 2.8 x 10(8) pfu/mL. Plaques (1 x 10(5)) were screened with (32)P-labeled EBV DNA probe(EBV genome 5-3271), 4 positive clones were obtained, and 1 of the 4 positive clones was picked out randomly for the second round of plaque screening. All the phage plaques were positive. DNA of the positive clone was extracted and was digested with BamHI. The length of the inserted fragment was 8.5 kb. Sequencing and BLAST analysis revealed that the inserted fragments consisted of the BamHI-W fragment at one end and clone RP11-665A22 on chromosome 15 at the other end.
CONCLUSION: The successfully established genomic library of Raji cells will provide a basis for cloning the sequences of the EBV junction sites and interpreting the mechanism of oncogenesis of EBV integration.
METHODS: High molecular weight genomic DNA of Raji cells was digested by restriction enzyme BamHI. DNA fragments ranging from 9 to 23 kb were recovered by agarose gel electrophoresis, which were ligated with Lambda DASH II vector BamHI arms pre-treated with calf intestine alkaline phosphatase (CIAP). Ligated DNA was packed in vitro using Gigapack III gold packaging extract. The library was plated on XL1-blue MRA (P2) host strain.Titering and screening of the Raji genomic library were performed.
RESULTS: The primary titer of the Raji genomic library was 1.8 x 10(5) pfu/mL, while that of the amplified library was 2.8 x 10(8) pfu/mL. Plaques (1 x 10(5)) were screened with (32)P-labeled EBV DNA probe(EBV genome 5-3271), 4 positive clones were obtained, and 1 of the 4 positive clones was picked out randomly for the second round of plaque screening. All the phage plaques were positive. DNA of the positive clone was extracted and was digested with BamHI. The length of the inserted fragment was 8.5 kb. Sequencing and BLAST analysis revealed that the inserted fragments consisted of the BamHI-W fragment at one end and clone RP11-665A22 on chromosome 15 at the other end.
CONCLUSION: The successfully established genomic library of Raji cells will provide a basis for cloning the sequences of the EBV junction sites and interpreting the mechanism of oncogenesis of EBV integration.