| 周翠兰,陈婕,许云思,付乙人,彭翠英.限制性内切酶酶切后去磷酸化联合蓝白斑筛选检测KRAS基因第12位稀有突变.[J].中南医学科学杂志.,2024,(1):26-30. |
| 限制性内切酶酶切后去磷酸化联合蓝白斑筛选检测KRAS基因第12位稀有突变 |
| Detection of KRAS rare mutation of codon 12 blue/white colony screening coupled with dephosphorylation of enzymatic products |
| 投稿时间：2023-02-27 修订日期：2023-12-18 |
| DOI：10.15972/j.cnki.43-1509/r.2024.01.006 |
| 中文关键词: 限制性内切酶酶切KRAS基因 蓝白斑技术 去磷酸化 稀有突变 [ |
| 英文关键词:restriction endonuclease digestion KRAS gene blue-white screening dephosphorylation rare mutation |
| 基金项目:湖南省自然基金(2020JJ4536,2021JJ30598);南华大学大学生创新课题(210XCX533,S202110555302);南华大学博士科研启动金(2018XQD12) |
| 作者 | 单位 | E-mail | | 周翠兰 | 南华大学衡阳医学院 基础医学院应用解剖学与生殖医学研究所,湖南衡阳 421001
南华大学衡阳医学院 生态健康与人类重要疾病防控湖南省高校重点实验室,湖南衡阳 421001 | e-mail为406272538@qq.com,e-mail为pengcuiying2004@126.com | | 陈婕 | 南华大学衡阳医学院 基础医学院应用解剖学与生殖医学研究所,湖南衡阳 421001
南华大学衡阳医学院 生态健康与人类重要疾病防控湖南省高校重点实验室,湖南衡阳 421001 | | | 许云思 | 南华大学衡阳医学院 基础医学院应用解剖学与生殖医学研究所,湖南衡阳 421001
南华大学衡阳医学院 生态健康与人类重要疾病防控湖南省高校重点实验室,湖南衡阳 421001 | | | 付乙人 | 南华大学衡阳医学院 基础医学院应用解剖学与生殖医学研究所,湖南衡阳 421001
南华大学衡阳医学院 生态健康与人类重要疾病防控湖南省高校重点实验室,湖南衡阳 421001 | | | 彭翠英 | 南华大学衡阳医学院 基础医学院应用解剖学与生殖医学研究所,湖南衡阳 421001
南华大学衡阳医学院 生物毒理与生态修复衡阳市重点实验室,湖南衡阳 421001
南华大学衡阳医学院 有色金属矿区耕地重金属污染生态阻抗技术研究所衡阳市重点实验室,湖南衡阳 421001 | e-mail为406272538@qq.com,e-mail为pengcuiying2004@126.com |
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| 中文摘要: |
| 目的研究限制性内切酶酶切后去磷酸化联合蓝白斑筛选检测KRAS基因第12位密码子稀有突变的可行性。 方法将采用限制性内切酶酶切后去磷酸化联合蓝白斑筛选检测方法作为实验组,限制性内切酶酶切联合蓝白斑筛选检测方法作为对照组。对照组将KRAS基因第12位密码子突变型(MUT)/野生型(WT)(MUT/WT 12)质粒按0∶1、1∶300、1∶1 000、1∶3 000的比例混合,限制性内切酶酶切PCR产物,酶切后产物进行蓝白斑筛选。实验组将MUT/WT 12质粒按0∶1、1∶3 000、1∶10 000、1∶30 000的比例混合,在对照组方法的基础上酶切产物去磷酸化后再蓝白斑筛选。比较两组蓝白斑克隆数。限制性内切酶酶切鉴定实验组MUT/WT 12为1∶30 000的阳性克隆,一代测序阳性克隆验证插入片段的序列。采用限制性内切酶酶切后去磷酸化联合蓝白斑筛选验证26例肺癌病例游离DNA的KRAS基因第12位稀有突变。 结果对照组1∶3 000的培养皿上白色克隆17个,蓝色克隆2 329个,白色/蓝色克隆为1/137；而实验组1∶30 000的培养皿上白色克隆23个,蓝色克隆394个,白色/蓝色克隆为1/17,1∶30 000实验组白色/蓝色克隆比例明显高于1∶30 00对照组。实验组1∶30 000培养血上5个阳性克隆酶切鉴定出3个阳性,其插入片段为KRAS第12位突变片段。限制性内切酶酶切后去磷酸化联合蓝白斑筛选26例肺癌病例可检测出2例为KRAS基因第12位密码子突变。 结论采用限制性内切酶酶切后去磷酸化联合蓝白斑筛选可检测出1∶30 000 KRAS基因稀有突变。 |
| 英文摘要: |
| AimTo study the feasibility of detecting rare mutations in the 12th codon of the KRAS gene through restriction endonuclease digestion followed by dephosphorylation combined with blue and white spot screening. MethodsThe experimental group employed restriction enzyme digestion,dephosphorylation, combined with blue-white screening, while the control group utilized restriction enzyme digestion followed by blue-white screening. In the control group, plasmids containing KRAS gene codon 12 mutant (MUT) and wild type (WT) (MUT/WT 12) sequences were mixed at ratios of 0∶1,1∶300,1∶1 000, or 1∶3 000, followed by restriction enzyme digestion and blue-white screening. In the experimental group, plasmids containing MUT/WT 12 were mixed at ratios of 0∶1, 1∶3 000,1∶10 000, or 1∶30 000. In addition to the methods used in the control group, the digested products in the experimental group were subjected to dephosphorylation before blue-white screening. The number of blue-white clones was compared between the two groups. The experimental group was identified as positive clones with a MUT/WT 12 ratio of 1∶30 000, and the sequences of the inserted fragments were verified through first-generation sequencing. Restriction enzyme digestion combined with dephosphorylation and blue-white screening were used to validate 12th codon mutations in rare KRAS gene in 26 cases of lung cancer. ResultsIn the control group with a 1∶3 000 ratio, there were 17 white clones and 2 329 blue clones, resulting in a white-to-blue clone ratio of 1∶137. In the experimental group with a 1∶30 000 ratio, there were 23 white clones and 394 blue clones, resulting in a white-to-blue clone ratio of 1∶17. The white/blue clone ratio in the 1∶30 000 experimental group was significantly higher than that in the 1∶3 000 control group.Three positive clones were identified by enzymatic digestion of five positive clones on a 1∶30 000 culture dish in the experimental group. Restriction enzyme digestion combined with dephosphorylation and blue-white screening successfully identified 12th codon mutations in KRAS gene in 2 out of 26 cases of lung cancer. ConclusionThe use of restriction enzyme digestion combined with dephosphorylation and blue-white screening allows for the detection sensitivity of rare KRAS gene mutations as 1∶30 000. |
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