Current methods for single nucleotide polymorphism （SNP） analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification （MASA） method fo...Current methods for single nucleotide polymorphism （SNP） analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification （MASA） method for rapid SNP analysis. The method is a marriage of two technologies： MASA primers for target DNA and a double-stranded DNA-selective fluorescent dye, SYBR Green I. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experimental results showed that the different genotypes can be clearly discriminated by the assay. The real-time fluorescence MASA method will have an enormous potential for fast and reliable SNP analysis due to its simplicity and low cost.展开更多
A new approach combined the specificity of allele-specific amplification （ASA） with the sensitivity of electrochemiluminescence （ECL） assay for single nucleotide polymorphism （SNP） analysis was proposed. Briefly...A new approach combined the specificity of allele-specific amplification （ASA） with the sensitivity of electrochemiluminescence （ECL） assay for single nucleotide polymorphism （SNP） analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru（bpy）3 ^2＋（TBR）-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA-ECL assay. The method is useful in SNP analysis due to its sensitivity,safety, and simplicity.展开更多
HLA-A＊02 is the most prevalent and polymorphic major histocompatibility complex （MHC） allele family in humans. Functional differences have been revealed among subtypes, demanding further subtyping of HLA-A＊02 in b...HLA-A＊02 is the most prevalent and polymorphic major histocompatibility complex （MHC） allele family in humans. Functional differences have been revealed among subtypes, demanding further subtyping of HLA-A＊02 in basic and clinical settings. However, the fast growing polymorphisms render traditional primeror probe-based typing methods impractical and result in increasing ambiguities in direct sequence-based typing. In this study, we combined group-specific amplification and mono-allelic sequencing to design and validate a simple scheme for the complete screening and accurate subtyping of all 540 reported HLA-A＊02 alleles. This scheme could be performed in routine labs to facilitate studies with an interest in HLA-A＊02.展开更多
将荧光定量PCR技术与等位基因特异性扩增(Allele specific amplification,ASA)方法相结合,发展了一种可以快速检测基因点突变的实时荧光等位基因特异性扩增(Real-time ASA)方法.将该法用于检测K-ras癌基因第12位密码子发生的点突变,分...将荧光定量PCR技术与等位基因特异性扩增(Allele specific amplification,ASA)方法相结合,发展了一种可以快速检测基因点突变的实时荧光等位基因特异性扩增(Real-time ASA)方法.将该法用于检测K-ras癌基因第12位密码子发生的点突变,分别采用针对其不同点突变方式(GAT,GTT,CGT)设计的突变型引物对待测样品进行ASA,只有突变型样品能被顺利扩增出双链DNA产物,该产物才能与双链DNA染料SYBR GreenⅠ结合,发出荧光信号从而被检测到.用该法检测31例结肠癌组织中的K-ras癌基因点突变,其中有15例样品检出为突变型.Real-time ASA法可检测到样品中含量为1/1000的突变型基因,具有灵敏、快速、简便、安全、高通量和低成本等优点,可望用于大量临床样本的点突变筛查.展开更多
针对传统电泳检测方法存在操作复杂、费时等缺点,提出一种用于检测K-ras癌基因点突变的实时荧光等位基因特异性扩增(Allele specific amplification,ASA)方法。该法采用突变型引物对结肠癌基因组中的K-ras基因进行等位基因特异性扩增,...针对传统电泳检测方法存在操作复杂、费时等缺点,提出一种用于检测K-ras癌基因点突变的实时荧光等位基因特异性扩增(Allele specific amplification,ASA)方法。该法采用突变型引物对结肠癌基因组中的K-ras基因进行等位基因特异性扩增,只有突变型样品能被顺利扩增出双链DNA产物,该产物能与双链DNA染料SYBR GreenⅠ结合,产生荧光信号从而被检测到。通过对荧光域值和溶解曲线分析来区分不同的基因突变类型。该法可以检测到野生型DNA中含量为1/1 000的突变型DNA,整个检测时间小于1 h。我们用该法检测31例结肠癌样品中K-ras基因密码子12发生的点突变,其中有15例检出为阳性。此外,还采用等位基因特异性扩增结合电泳分析对样品进行了检测,并对两种方法进行了比较。结果显示:实时荧光等位基因特异性扩增方法具有操作简便、快速、检测成本低等优点,为临床诊断基因突变引起的疾病提供了一种可行的手段。展开更多
目的将新近建立的单管双向等位基因专一性扩增(single-tube bi-directional allele specific amplification,SB-ASA)方法用于分析近交系小鼠基因组中的单核苷酸多态性(SNP)。方法以5个近交系小鼠为研究对象,采用SB-ASA方法对其16个SNP...目的将新近建立的单管双向等位基因专一性扩增(single-tube bi-directional allele specific amplification,SB-ASA)方法用于分析近交系小鼠基因组中的单核苷酸多态性(SNP)。方法以5个近交系小鼠为研究对象,采用SB-ASA方法对其16个SNP位点进行检测,并通过双盲实验和测序验证该方法的可靠性;且考察了该方法中PCR反应各成分及扩增条件对结果的影响。结果16个SNP位点,SB-ASA都成功地对5个品系小鼠进行了分型,与测序结果完全一致;双盲实验结果显示通过3个SNP位点即可鉴别5个品系。结论SB-ASA方法可用于近交系小鼠SNP的遗传检测,可望作为一种新的分子生物学遗传检测方法推广应用。展开更多
基金This research is supported by the National Natural Science Foundation of China(60378043,30470494)the Natural Science Foundation of Guangdong Province(015012,04010394).
文摘Current methods for single nucleotide polymorphism （SNP） analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification （MASA） method for rapid SNP analysis. The method is a marriage of two technologies： MASA primers for target DNA and a double-stranded DNA-selective fluorescent dye, SYBR Green I. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experimental results showed that the different genotypes can be clearly discriminated by the assay. The real-time fluorescence MASA method will have an enormous potential for fast and reliable SNP analysis due to its simplicity and low cost.
基金the National Natural Science Foundation of China （Nos. 30600128, 30670507,30470494） the Natural Science Foundation of Guangdong Province （No. 015012）.
文摘A new approach combined the specificity of allele-specific amplification （ASA） with the sensitivity of electrochemiluminescence （ECL） assay for single nucleotide polymorphism （SNP） analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru（bpy）3 ^2＋（TBR）-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA-ECL assay. The method is useful in SNP analysis due to its sensitivity,safety, and simplicity.
基金The authors would like to thank Jueqin Yang for assistance with sample preparation. The authors would also like to thank the Fred Hutchinson Cancer Research Center IHWG Cell and Gene Bank for providing reference genomic DNA samples. This work was supported through grants from the National Natural Science Foundation of China （NSF-30830093） and the National Key Program （973） for Basic Research of China （2009CB522409） to HJ. Supplementary Information accompanies the paper on Cellular ＆ Molecular Immunology website.
文摘HLA-A＊02 is the most prevalent and polymorphic major histocompatibility complex （MHC） allele family in humans. Functional differences have been revealed among subtypes, demanding further subtyping of HLA-A＊02 in basic and clinical settings. However, the fast growing polymorphisms render traditional primeror probe-based typing methods impractical and result in increasing ambiguities in direct sequence-based typing. In this study, we combined group-specific amplification and mono-allelic sequencing to design and validate a simple scheme for the complete screening and accurate subtyping of all 540 reported HLA-A＊02 alleles. This scheme could be performed in routine labs to facilitate studies with an interest in HLA-A＊02.
基金supported by the National Natural Science Foundation of China(81071790,30600128,81102159)The Key Project of Chinese Ministry of Education(211131)+2 种基金The Natural Science Foundation of Guangdong Province(7005825,S2011040003622)The Science and Technology Project of Guangzhou Nansha District(RG201001003)The Foundation for Distinguished Young Talents in Higher Education of Guangdong(2009)
文摘针对传统电泳检测方法存在操作复杂、费时等缺点,提出一种用于检测K-ras癌基因点突变的实时荧光等位基因特异性扩增(Allele specific amplification,ASA)方法。该法采用突变型引物对结肠癌基因组中的K-ras基因进行等位基因特异性扩增,只有突变型样品能被顺利扩增出双链DNA产物,该产物能与双链DNA染料SYBR GreenⅠ结合,产生荧光信号从而被检测到。通过对荧光域值和溶解曲线分析来区分不同的基因突变类型。该法可以检测到野生型DNA中含量为1/1 000的突变型DNA,整个检测时间小于1 h。我们用该法检测31例结肠癌样品中K-ras基因密码子12发生的点突变,其中有15例检出为阳性。此外,还采用等位基因特异性扩增结合电泳分析对样品进行了检测,并对两种方法进行了比较。结果显示:实时荧光等位基因特异性扩增方法具有操作简便、快速、检测成本低等优点,为临床诊断基因突变引起的疾病提供了一种可行的手段。
文摘目的将新近建立的单管双向等位基因专一性扩增(single-tube bi-directional allele specific amplification,SB-ASA)方法用于分析近交系小鼠基因组中的单核苷酸多态性(SNP)。方法以5个近交系小鼠为研究对象,采用SB-ASA方法对其16个SNP位点进行检测,并通过双盲实验和测序验证该方法的可靠性;且考察了该方法中PCR反应各成分及扩增条件对结果的影响。结果16个SNP位点,SB-ASA都成功地对5个品系小鼠进行了分型,与测序结果完全一致;双盲实验结果显示通过3个SNP位点即可鉴别5个品系。结论SB-ASA方法可用于近交系小鼠SNP的遗传检测,可望作为一种新的分子生物学遗传检测方法推广应用。
