CAJ | 학술논문

背景:dystrophin基因是人类常见的X染色体连锁隐性遗传的神经肌肉系统疾病,dystrophin基因缺失集中在两个热点区域即外显子2～20和44～53,其主要缺失可以通过对一系列外显子的检测来发现.然而对dystrophin基因缺失的18个常见易缺失外显子片段的系统检测却鲜有报道.目的:拟对dystrophin基因18个常见易缺失外显子片段进行克隆、鉴定.方法:以人类基因组DNA为模板,应用18对引物对dystrophin基因常见易缺失外显子片段进行PCR扩增.将扩增产物与pGEM-TEasy载体连接,转化E.coli JM109感受态细胞.通过平板培养,挑选阳性克隆.提取重组质粒,Notl酶切,获得完整的探针片段,并作测序鉴定.通过核酸序列数据库相似性检索工具验证序列的来源及其与GeneBank收录序列的相似性.结果与结论:PCR扩增出18个片段,与dystrophin基因预期扩增片段大小相一致.重组克隆质粒的酶切产物与PCR产物大小相近,与预期相一致.经测序获得18个克隆片段全序列,其核苷酸数量与预期基本一致,序列相似性检索分析证实了这些克隆片段与GeneBank收录的dystrophin基因片段具有极高的同源性.克隆产物确为dystrophin基因常见易缺失外显子片段.
배경:dystrophin기인시인류상견적X염색체련쇄은성유전적신경기육계통질병,dystrophin기인결실집중재량개열점구역즉외현자2～20화44～53,기주요결실가이통과대일계렬외현자적검측래발현.연이대dystrophin기인결실적18개상견역결실외현자편단적계통검측각선유보도.목적:의대dystrophin기인18개상견역결실외현자편단진행극륭、감정.방법:이인류기인조DNA위모판,응용18대인물대dystrophin기인상견역결실외현자편단진행PCR확증.장확증산물여pGEM-TEasy재체련접,전화E.coli JM109감수태세포.통과평판배양,도선양성극륭.제취중조질립,Notl매절,획득완정적탐침편단,병작측서감정.통과핵산서렬수거고상사성검색공구험증서렬적래원급기여GeneBank수록서렬적상사성.결과여결론:PCR확증출18개편단,여dystrophin기인예기확증편단대소상일치.중조극륭질립적매절산물여PCR산물대소상근,여예기상일치.경측서획득18개극륭편단전서렬,기핵감산수량여예기기본일치,서렬상사성검색분석증실료저사극륭편단여GeneBank수록적dystrophin기인편단구유겁고적동원성.극륭산물학위dystrophin기인상견역결실외현자편단.
BACKGROUND: Dystrophin gene is X-linkage recessive heredity nerve-muscle system disease. Dystrophin gene deletions cluster in two hotspot regions, comprising exons 2-20 and 44-53. The majority of deletions can be detected by examining only a subset of exons. However, little is known regarding systematic detection of 18 common deletion exons of dystrophin gene.OBJECTIVE: To obtain and identify the cloning of 18 deletion-prone exons of dystrophin gene.METHODS: A total of 18 fragments of dystrophin gene were obtained through polymerase chain reaction (PCR) amplification with human genomic DNA as template and 18 pairs of primers respectively. The fragments were connected with pGEM-T Easy vector.The recombinants were transformed into E.coli JM109 competent cells, followed by planted on Luria-Bertani (LB)/ampicillin(Amp)/isopropylthio-β-D-galactoside(IPTG)/X-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) plates and cultured.Positive transformants were selected with blue/white color screening, and the recombinant plasmids DNA was extracted and digested with restriction enzyme Not I. DNA sequences of the fragments were analyzed. Nucleotide analyses were performed through the National Center for Biotechnology Information (NCBI) Basic Local Alighment Search Tool (BLAST) against GenBank.RESULTS AND CONCLUSION: Size of the18 fragments by PCR amplification was in accordance with anticipation. Size of the fragments of recombinant cloning by Not I digestion was in accordance with that of PCR and expectation. Sequence size of the 18cloned fragments was in accordance with expectation. The cloned fragments have high homology with dystrophin gene through NCBI BLAST against GenBank. These cloned fragments were the main deletion-prone exons of dystrophin gene.