【目的】比较单链构象多态性分析(SSCP)和异源双链分析(HA)在基因突变筛查中的意义。【方法】在帕金森病人和正常对照中,同时应用这两种实验方法筛查α-共核蛋白基因3、4外显子突变,电泳结果由测序考证。【结果】对照组第29号标本3号外...【目的】比较单链构象多态性分析(SSCP)和异源双链分析(HA)在基因突变筛查中的意义。【方法】在帕金森病人和正常对照中,同时应用这两种实验方法筛查α-共核蛋白基因3、4外显子突变,电泳结果由测序考证。【结果】对照组第29号标本3号外显子 SSCP 存在单链泳动异常。但 HA 检测无双链泳动异常,测序证实无基因突变。其它标本 SSCP 和 HA 检测均未发现异常泳动。【结论】将单链构象分析和异源双链分析相结合应用于基因突变筛查,既不用增加试剂、设备和技术难度,又可以优势互补,利于提高检测的准确性。展开更多
内脂素(Visfatin)是脂肪细胞因子家族的新成员,主要由内脏脂肪组织产生.研究表明内脂素具有类胰岛素样作用.在检测固始鸡-安卡鸡资源群体3代(亲本,F1,F2)964只鸡Visfatin基因9bp插入/缺失(9 bp 'TAACCTGTG' insertion-deletion...内脂素(Visfatin)是脂肪细胞因子家族的新成员,主要由内脏脂肪组织产生.研究表明内脂素具有类胰岛素样作用.在检测固始鸡-安卡鸡资源群体3代(亲本,F1,F2)964只鸡Visfatin基因9bp插入/缺失(9 bp 'TAACCTGTG' insertion-deletion)多态的过程中,发现其杂合子的变性和非变性聚丙烯酰胺胶上除2条同源双链DNA(282bp和273bp)外有2条未知条带(命名为A和B).A,B条带经回收、二次PCR、再次聚丙烯酰胺凝胶电泳及DNA测序表明:Visfatin基因第10内含子中9bp insertion-deletion突变杂合子的PCR产物中,本身包含2种同源双链DNA片段和2种异源双链DNA片段,不需要经过额外的变性、退火处理,其PCR产物可以直接进行突变检测,在229个杂合突变中异源双链DNA的检出率为100%.因此,通过异源双链DNA这一标示物作为基因分型时的依照或者参考,建立适当的异源双链DNA分析法可进行基因中几个核苷酸插入/缺失多态的检测.展开更多
Background: A dominant T- cell clone can be detected by polymerase chain reaction (PCR) in 40- 90% of cutaneous samples from patients with cutaneous T- cell lymphoma (CTCL). Materials and methods: From 1996 to 2003 we...Background: A dominant T- cell clone can be detected by polymerase chain reaction (PCR) in 40- 90% of cutaneous samples from patients with cutaneous T- cell lymphoma (CTCL). Materials and methods: From 1996 to 2003 we analysed 547 cutaneous biopsies performed to exclude CTCL (mycosis fungoides, MF/Sé zary syndrome, SS). The final diagnosis was benign inflammatory disease (BID) in 353 samples (64.5% )- and CTCL in 194 (35.5% ). T- cell receptor (TCR)- γ gene rearrangement was studied by using a multiplex PCR/heteroduplex (HD) analysis. The PCR results were correlated with the clinical picture, the histological pattern and the presence of T- cell lineage antigen loss, using univariate and multivariate logistic regression analyses. Objective: To determine the sensitivity and specificity of the multiplex PCR/HD analysis and to identify which are the clinical, histopathological or immunophenotypical features significantly associated with a positive T- cell clonality. Results: A clonality was demonstrated in 83.5% of CTCL and in 2.3% of BID (P < 0.001). A significantly higher percentage of clonal cases was associated with the cutaneous T- score (71.4% in T1, 76.1% in T2 and 100% in nodular and erythrodermic MF samples) and with the presence of a T- cell lineage antigen loss (93.9% vs. 77.4% ). Moreover, clonality was closely related to an increase in the histopathological score (51.3% in the samples with a score < 5, compared with 92% in the lesions with ≥ 5). No significant difference in the percentage of clonal cases was found between T1/T2 and T3/T4 lesions with a histopathological score ≥ 5. The multivariate logistic regression showed that the density and extent of the cell infiltrate, the degree of epidermotropism and the presence of cytological atypia share an independent predictive value for clonality in T1/T2 samples, even if the highest odds ratios (3.6) were associated with the density of the cell infiltrate. The disease course of T1/T2 patients was analysed according to the PCR findings. All the PCR- negative patients showed a long- standing stable disease course; on the other hand, a disease progression occurred in 12/87 (13.8% ) positive patients. Conclusions: The multiplex PCR/HD analysis is associated with a high diagnostic accuracy (92.7% ) in CTCL patients. The finding of a clonal T- cell rearrangement is more closely associated with the histological pattern (in particular with the density and extent of the cell infiltrate) rather than with the MF cutaneous T- score or immunophenotype.展开更多
文摘【目的】比较单链构象多态性分析(SSCP)和异源双链分析(HA)在基因突变筛查中的意义。【方法】在帕金森病人和正常对照中,同时应用这两种实验方法筛查α-共核蛋白基因3、4外显子突变,电泳结果由测序考证。【结果】对照组第29号标本3号外显子 SSCP 存在单链泳动异常。但 HA 检测无双链泳动异常,测序证实无基因突变。其它标本 SSCP 和 HA 检测均未发现异常泳动。【结论】将单链构象分析和异源双链分析相结合应用于基因突变筛查,既不用增加试剂、设备和技术难度,又可以优势互补,利于提高检测的准确性。
文摘内脂素(Visfatin)是脂肪细胞因子家族的新成员,主要由内脏脂肪组织产生.研究表明内脂素具有类胰岛素样作用.在检测固始鸡-安卡鸡资源群体3代(亲本,F1,F2)964只鸡Visfatin基因9bp插入/缺失(9 bp 'TAACCTGTG' insertion-deletion)多态的过程中,发现其杂合子的变性和非变性聚丙烯酰胺胶上除2条同源双链DNA(282bp和273bp)外有2条未知条带(命名为A和B).A,B条带经回收、二次PCR、再次聚丙烯酰胺凝胶电泳及DNA测序表明:Visfatin基因第10内含子中9bp insertion-deletion突变杂合子的PCR产物中,本身包含2种同源双链DNA片段和2种异源双链DNA片段,不需要经过额外的变性、退火处理,其PCR产物可以直接进行突变检测,在229个杂合突变中异源双链DNA的检出率为100%.因此,通过异源双链DNA这一标示物作为基因分型时的依照或者参考,建立适当的异源双链DNA分析法可进行基因中几个核苷酸插入/缺失多态的检测.
文摘Background: A dominant T- cell clone can be detected by polymerase chain reaction (PCR) in 40- 90% of cutaneous samples from patients with cutaneous T- cell lymphoma (CTCL). Materials and methods: From 1996 to 2003 we analysed 547 cutaneous biopsies performed to exclude CTCL (mycosis fungoides, MF/Sé zary syndrome, SS). The final diagnosis was benign inflammatory disease (BID) in 353 samples (64.5% )- and CTCL in 194 (35.5% ). T- cell receptor (TCR)- γ gene rearrangement was studied by using a multiplex PCR/heteroduplex (HD) analysis. The PCR results were correlated with the clinical picture, the histological pattern and the presence of T- cell lineage antigen loss, using univariate and multivariate logistic regression analyses. Objective: To determine the sensitivity and specificity of the multiplex PCR/HD analysis and to identify which are the clinical, histopathological or immunophenotypical features significantly associated with a positive T- cell clonality. Results: A clonality was demonstrated in 83.5% of CTCL and in 2.3% of BID (P < 0.001). A significantly higher percentage of clonal cases was associated with the cutaneous T- score (71.4% in T1, 76.1% in T2 and 100% in nodular and erythrodermic MF samples) and with the presence of a T- cell lineage antigen loss (93.9% vs. 77.4% ). Moreover, clonality was closely related to an increase in the histopathological score (51.3% in the samples with a score < 5, compared with 92% in the lesions with ≥ 5). No significant difference in the percentage of clonal cases was found between T1/T2 and T3/T4 lesions with a histopathological score ≥ 5. The multivariate logistic regression showed that the density and extent of the cell infiltrate, the degree of epidermotropism and the presence of cytological atypia share an independent predictive value for clonality in T1/T2 samples, even if the highest odds ratios (3.6) were associated with the density of the cell infiltrate. The disease course of T1/T2 patients was analysed according to the PCR findings. All the PCR- negative patients showed a long- standing stable disease course; on the other hand, a disease progression occurred in 12/87 (13.8% ) positive patients. Conclusions: The multiplex PCR/HD analysis is associated with a high diagnostic accuracy (92.7% ) in CTCL patients. The finding of a clonal T- cell rearrangement is more closely associated with the histological pattern (in particular with the density and extent of the cell infiltrate) rather than with the MF cutaneous T- score or immunophenotype.
