CN109554480A - 一种快速检测遗传性皮肤弹性基因的方法 - Google Patents
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Abstract
本发明为一种快速检测遗传性皮肤弹性基因的方法,涉及分子生物学领域。本发明涉及检测三个基因的三个SNP位点,提供待检测位点的检测用特异性引物及其反应条件(详见表1~3)。本发明采用高分辨率熔解曲线分析技术(High Resolution Melting,HRM)以分析待检测样本的各SNP位点基因型。为辅助分析待检测样品的基因型,本发明还将提供各SNP位点对应标准基因型模板(含野生型、突变杂合型及突变型)。
Description
技术领域
本发明为一种快速检测遗传性皮肤弹性基因的方法,涉及分子生物学领域,尤其针对美肤行业肤质评测。
背景技术
目前,常用的基因检测方法为测序法、基因芯片法、飞行质谱法,这些方法都需要在完成PCR扩增后进行后续实验分析,实验周期相对较长,适用于中、高通量的SNP检测。使用支持高分辨率熔解曲线分析技术(High Resolution Melting,HRM)的仪器仅需要一次PCR扩增后即可根据扩增产物直接进行分析报告得出样品的基因型,极大的缩短了实验时长。
HRM是一种基于单核苷酸熔解温度不同而形成不同形态熔解曲线的基因分析新技术,具有极高的敏感性,可以检测出单个碱基的差异,并且成本低、通量高、速度快、结果准确、不受检测位点的局限,实现了真正的闭管操作。
HRM无需使用序列特异性探针,而是利用一种饱和染料对PCR反应产物进行分析。基本原理:双链核苷酸(double strand DNA,dsDNA)的热稳定性受其长度和碱基组成的影响,序列变化会导致升温过程中dsDNA解链行为的改变。因为所用的荧光染料只能嵌入并结合到dsDNA上,因此利用实时PCR技术,通过实时检测dsDNA熔解过程中荧光信号值的变化,就能以生成不同形状熔解曲线的方式将PCR产物中存在的差异直观地展示出来。同时,借助于专业性的分析软件就可以对测试群体实现基于不同形状熔解曲线的基因分型或归类。
皮肤由表皮、真皮、皮下组织三层构成,表皮处于皮肤的最外层,覆盖全身,仅有0.2毫米厚,可防止外界异物入侵,有过滤吸收紫外线、锁水、分裂增生细胞等作用;真皮主要由胶原纤维及弹性纤维所构成,胶原纤维和弹性纤维交叉形成一张弹性网,如同弹簧一样,保持肌肤的弹性和张力。皮肤弹性不仅受到年龄、紫外线和自由基损伤等的影响,导致胶原纤维和弹性纤维受损、硬化、断裂,导致真皮层网状结构疏松,开成凹陷,在皮肤表面出现皱纹和松弛。同时,还会受到基因表达的调控,如:基质金属蛋白酶(MMPs)家族是一类依赖金属锌离子的蛋白水解酶家族,能有效地降解细胞外基质,一种MMP可降解多种细胞外基质成份,而某一种细胞外基质成分又可被多种MMP降解,但不同酶的降解效率可不同。MMP1又称间质性胶原酶,属基质金属蛋白酶家族,其主要作用底物为纤维性胶原,可降解细胞外基质中的胶原纤维和明胶,MMP-1 是少数可以降解I型以及III型胶原的酶之一;MMP1将胶原断开后,明胶酶 MMP9与断开的胶原结合,进一步将其降解成更小的分子,同时网织层的IV、 V、VII、X、XI型胶原、蛋白聚糖的核心蛋白、明胶、纤维粘连蛋白、层粘连蛋白、弹性蛋白等,都是MMP-9的作用底物,MMP9将进一步破坏皮肤的网织层,使皮肤丧失弹性。
matrix metallopeptidase 1/3/9(MMP-1/3/9),该基因编码基质金属蛋白酶(MMPs)的肽酶M10家族的成员。该家族中的蛋白质参与正常生理过程中细胞外基质的分解,例如胚胎发育,繁殖和组织重塑,以及疾病过程,例如关节炎和转移。对编码的前原蛋白进行蛋白水解加工以产生成熟蛋白酶。这种分泌的蛋白酶可以分解间质胶原蛋白,包括I,II和III型。MMP-1是少数可以降解 I型以及III型胶原的酶之一,位于MMP-1基因启动子区域-1607bp的1G/2G 的单核苷酸多态性影响MMP-1的转录子水平,MMP-1启动子-1607位点的2GSNP提供了一个ETS结合位点,和-1602位点的AP-1结合位点共同作用,促进MMP-1的转录,因此,2G与1G相比,MMP-1的转录活性提高。与MMP-1的启动子区域的SNP位点相同,在MMP-3及MMP-9的启动子区域也存在有影响表达量的SNP位点。
发明内容
为了加快检测速度、简化检测过程,本发明选用了HRM技术对与皮肤抗痘能力相关的基因突变位点进行基因型分析。为实现这一检测我们提供了一系列用于HRM试验的特异性扩增引物及对应的标准对照品。
本发明涉及检测三个基因共三个SNP位点,提供待检测位点的检测用特异性引物及其使用LightCycler 480High Resolution Melting Master试剂盒特异性扩增DNA片段的反应体系和条件(详见表1)。本发明采用高分辨率熔解曲线分析技术(High ResolutionMelting,HRM)以分析待检测样本的各SNP位点基因型。为辅助分析待检测样品的基因型,本发明还将提供各SNP位点对应标准基因型模板(含野生型、突变杂合型及突变型)。
表1.检测用引物及其条件
本发明通过以下技术方案实现(以其中某一特定位点:rs1799750为例):
1)设计扩增含rs1799750在内的过氧化氢酶特定基因片段的1组引物;
2)将样品总DNA的浓度调至20ng/μL、制备一组含三种不同基因型的标准品;
3)使用1)的引物和2)的模板及标准品,使用HRM试剂盒并根据试剂盒说明对过氧化氢酶基因片段的扩增;
4)使用LightCycler 480 software软件对试验数据进行基因分型分析,确定样品的基因型。
所述标准品的制备:
a.通过PCR扩增筛选得到野生型及突变型基因(若突变型基因不易筛选可进行化学合成法);
b.将筛选所得的基因(野生型及突变型)连接到克隆常用质粒中,将连接后质粒进行转化扩增,最后回收连接成功的质粒(并将相应菌落进行菌种保存)
c.将连接有两种基因型的质粒进行定量稀释处理,同时取两种不同质粒1∶ 1混合即可得到突变杂合型模板。将这三种质粒进行定量处理,并取其中一部分稀释为工作浓度在后续实验中作为标准品DNA使用。
所述试剂盒的反应体系及其反应条件:
表2.PCR扩增反应体系
表3.PCR扩增反应条件
所述检测结果解读:判断样品的基因型,其特质在于不同基因型的dsDNA 在解链时的温度有细微的差别,从而导致其溶解曲线在突变点位有不同的曲线形状。以添加的三种不同基因型标准品为参照即可得出样品的基因型。
附图说明
图1、2为三种不同基因型的标准品在突变位点的溶解曲线线型(被检测样品线型与其中某一条重合)
图3为实例中的检测结果
具体实施方式
现结合实施例,进一步说明使用本发明的引物、标准品结合HRM试剂盒检测样品基因型的良好效果。本发明使用LightCycler 480 High Resolution Melting Master试剂盒及LightCycler 480仪器进行试验,但并不限制使用其他公司的仪器及试剂盒。
以下操作均已rs1799750位点的检测过程为例。
某位客户在刮取口腔上皮细胞之后,实验员进行核酸提取,将提取出的基因组DNA进行质量评估(检测其浓度及纯度)。根据所测得的浓度结果对样本进行稀释(稀释至工作浓度20ng/μL)。
标准模板的制备:
1)将筛选所得的基因(野生型及突变型)连接到克隆常用质粒中,将连接后质粒进行转化扩增,最后回收连接成功的质粒(并将相应菌落进行菌种保存);
2)将连接有两种基因型的质粒进行定量稀释处理,同时取两种不同质粒1∶1 混合即可得到突变杂合型模板。将这三种质粒进行定量处理,并取其中一部分稀释为工作浓度在后续实验中作为标准品DNA使用。
1.反应溶液配制
1)根据表2中提供的反应体系避光配制除模板DNA外的公共体系(反应数量为样本数X+标准品数量3),为避免多次移液操作造成损耗,应多配0.5个或数个反应的量;
2)将公共体系按照每孔18μl分装置各孔;
3)分装完毕,将板离心数秒钟;
4)根据按规划每孔加入2μl对应编号的DNA模板
2.上机检测
1)根据表3提供的条件设定反应温度和时间
2)反应结束LightCycler 480 software软件参照标准品分析确定各样本的基因型,同时,扩增后的样本仍可进行测序分析以验证实验结果的真实性。
Claims (5)
1.一种快速检测遗传性皮肤弹性基因的方法,其特征在于包括以下步骤:
1)设计3对特异性引物,并确定各引物对的扩增条件;
2)制备3组含三种不同基因型的标准品;
3)将待检测样品总DNA的浓度稀释至20ng/μL作为模板;
4)使用上述步骤中的引物及模板,借助HRM技术对各基因片段进行特异性扩增并测定其溶解曲线;
5)根据4)中的实验结果,依据与标准品的线型关系判断样品的基因型。
2.如专利要求1所述的3对引物,其特征为下表所示:
3.如权利要求1所述的3组标准品制备,其特征在于:
1)通过PCR扩增筛选得到野生型及突变型基因(若突变型基因不易筛选可进行化学合成法);
2)将筛选所得的基因(野生型及突变型)连接到克隆常用质粒中,将连接后质粒进行转化扩增,最后回收连接成功的质粒(并将相应菌落进行菌种保存);
3)将连接有两种基因型的质粒进行定量稀释处理,同时取两种不同质粒1∶1混合即可得到突变杂合型模板。将这三种质粒进行定量处理,并取其中一部分稀释为工作浓度在后续实验中作为标准品DNA使用。
4.如专利要求1所述的特异性扩增体系与条件,其特质如下:
扩增体系:
扩增条件:
5.如权利要求1中所述根据试验结果判断样品的基因型,其特征在于:使用LightCycler 480 software软件对样品的突变位点进行溶解曲线进行线型分析,与三种标准品进行对比分析样品的基因型。同时,扩增后的样本仍可进行测序分析以验证实验结果的真实性。
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US20150359483A1 (en) * | 2013-09-13 | 2015-12-17 | Genocosmetics Lab Sl | Methods and systems for improving perceived age based on phenotypic and genetic features of the skin |
CN105886645A (zh) * | 2016-05-27 | 2016-08-24 | 福建爱我健康生物科技有限公司 | 一种用于评估皮肤抗皱情况的引物和探针及试剂盒 |
CN106498071A (zh) * | 2016-11-14 | 2017-03-15 | 天津市康婷生物工程有限公司 | 从基因水平检测和评估皮肤肤质的体系 |
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US20150359483A1 (en) * | 2013-09-13 | 2015-12-17 | Genocosmetics Lab Sl | Methods and systems for improving perceived age based on phenotypic and genetic features of the skin |
CN105886645A (zh) * | 2016-05-27 | 2016-08-24 | 福建爱我健康生物科技有限公司 | 一种用于评估皮肤抗皱情况的引物和探针及试剂盒 |
CN106498071A (zh) * | 2016-11-14 | 2017-03-15 | 天津市康婷生物工程有限公司 | 从基因水平检测和评估皮肤肤质的体系 |
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