We established a quick and reliable method for recovering cell-free seminal DNA （cfsDNA）, by using the binding-washing-elution procedure on the DNA purification column. Low variations （below 15%） among the triplic...We established a quick and reliable method for recovering cell-free seminal DNA （cfsDNA）, by using the binding-washing-elution procedure on the DNA purification column. Low variations （below 15%） among the triplicate values of cfsDNA quantity verified the reproducibility of our cfsDNA recovery method. Similar cfsDNA yield and size distribution between seminal plasma acquired by filtration and centrifugation confirmed the presence of cfsDNA. To investigate the general characterization of cfsDNA, the quantitation and size distribution of cfsDNA from normozoospermic and azoospermic semen were analyzed by real-time PCR and electrophoresis, respectively. CfsDNA concentration in semen with normozoospermia （n = 11） was 1.34 ± 0.65 μg ·mL^-1, whereas a higher cfsDNA concentration was observed in azoospermia （2.56 ± 1.43 μg ·mL^-1, n = 9）. The continuous distribution of DNA fragments ranging from -1 kb to 15 kb and a spectrum of multiples of 180-bp fragments were observed in each normozoospermic and azoospermic sample. Distinct characteristic DNA ladder fragmentations in some azoospermic samples implicated that cfsDNA originate partly from apoptotic cells. CfsDNAs of 36 selected azoospermic patients with known information of Y chromosome microdeletion were subjected to the same microdeletion analysis by multiplex PCR and PCR amplification of sY114 （1 450 bp）. All multiplex PCR reactions with cfsDNA amplified successfully and provided the same result as leukocyte DNA. PCR amplification of sY114 gave a 1 450-bp amplicon as expected. Our data suggested the potential use of cfsDNA in search of biomarker or diagnostic procedures.展开更多
Synovial sarcoma(SS) is genetically characterized by chromosomal translocation, which generates SYT-SSX fusion transcripts. Although SS can occur in any body part, primary gastric SS is substantially rare. Here we des...Synovial sarcoma(SS) is genetically characterized by chromosomal translocation, which generates SYT-SSX fusion transcripts. Although SS can occur in any body part, primary gastric SS is substantially rare. Here we describe a detection of the fusion gene sequence of gastric SS in plasma cell-free DNA(cf DNA). A gastric submucosal tumor was detected in the stomach of a 27-year-old woman and diagnosed as SS. Candidate intronic primers were designed to detect the intronic fusion breakpoint and this fusion sequence was confirmed in intron 10 of SYT and intron 5 of SSX2 by genomic polymerase chain reaction(PCR) and direct sequencing. A locked nucleic acid(LNA) probe specificto the fusion sequence was designed for detecting the fusion sequence in plasma and the fusion sequence was detected in preoperative plasma cfD NA, while not detected in postoperative plasma cfD NA. This technique will be useful for monitoring translocation-derived diseases such as SS.展开更多
目的探讨孕妇血浆游离DNA定量检测在唐氏综合征筛查中的价值。方法采用实时定量荧光PCR检测孕中期孕妇,唐氏综合征高危孕妇的血浆中3-磷酸甘油醛脱氢酶基因(GAPDH)及男性特有Y性别决定区(sex determ in ingregion Y,S R Y)基因。结果孕...目的探讨孕妇血浆游离DNA定量检测在唐氏综合征筛查中的价值。方法采用实时定量荧光PCR检测孕中期孕妇,唐氏综合征高危孕妇的血浆中3-磷酸甘油醛脱氢酶基因(GAPDH)及男性特有Y性别决定区(sex determ in ingregion Y,S R Y)基因。结果孕中期、唐氏综合征高危孕妇GAPDH拷贝数均值分别为(4.28±1.36)×103copy/m l,(3.08±1.67)×104copy/m l;孕中期、唐氏综合征高危孕妇的SRY拷贝数均值分别为174.02±98.56 copy/m l、1065.01±588.72 copy/m l。孕中期和唐氏高危孕妇血浆游离DNA含量有显著性差异。结论孕妇血浆游离DNA的定量分析在唐氏综合征筛查中有重要的价值。展开更多
目的对两步磁珠法提取血浆游离DNA的方法进行评价,初步探讨血浆游离DNA定量对于肺癌患者实验诊断的意义。方法在健康志愿者血浆中掺入DNA标准品127 bp,208 bp和Quick-load~@100 bp DNA ladder(由100-1 500 bp DNA片段组成),采用两步磁...目的对两步磁珠法提取血浆游离DNA的方法进行评价,初步探讨血浆游离DNA定量对于肺癌患者实验诊断的意义。方法在健康志愿者血浆中掺入DNA标准品127 bp,208 bp和Quick-load~@100 bp DNA ladder(由100-1 500 bp DNA片段组成),采用两步磁珠法和分离柱提取法提取血浆中掺入的DNA标准品;用Qubit测定DNA浓度,安捷伦2100生物芯片分析仪和琼脂糖凝胶电泳检测分析DNA片段大小分布。用两步磁珠法和分离柱提取法分别提取15例肺癌患者的血浆cfDNA,利用安捷伦2100生物芯片分析仪和琼脂糖凝胶电泳比较两种方法提取的cf-DNA片段大小分布,分析cf-DNA浓度与肺癌分期的相关性。结果两步磁珠法第二步分离的DNA浓度与掺入标准品DNA(127 bp,208 bp)片段浓度呈正相关(r^2=0. 985 7,P <0. 001);两步磁珠法提取的Quick-load~@100 bp DNA ladder第一步分离的DNA片段≥300 bp,第二步分离的DNA片段≤300 bp,分离柱提取法重提取的Quick-load~@DNA ladder≤1 500 bp。两步磁珠法第二步分离的肺癌患者血浆cf-DNA浓度与肺癌分期呈正相关(r^2=0. 866 4,P=0. 004 9)。分离柱提取法提取的cf-DNA浓度与肺癌分期无显著相关性(r^2=0. 500 9,P=0. 214 5)。结论两步磁珠法可分别提取不同大小片段的DNA标准品,同时有效提取肺癌患者血浆中小片段游离DNA,且其浓度与肺癌分期呈正相关。展开更多
基金Acknowledgment The investigation was supported by grants from the National Natural Science Foundation of China （No. 30801144）, by the Specialized Research Fund for the Doctoral Program of Higher Education （No. 200804871092） and by the National Key Technology Research and Development Program for the 10th Five- Year Plan, China （No. 2004BA720A33-01）.
文摘We established a quick and reliable method for recovering cell-free seminal DNA （cfsDNA）, by using the binding-washing-elution procedure on the DNA purification column. Low variations （below 15%） among the triplicate values of cfsDNA quantity verified the reproducibility of our cfsDNA recovery method. Similar cfsDNA yield and size distribution between seminal plasma acquired by filtration and centrifugation confirmed the presence of cfsDNA. To investigate the general characterization of cfsDNA, the quantitation and size distribution of cfsDNA from normozoospermic and azoospermic semen were analyzed by real-time PCR and electrophoresis, respectively. CfsDNA concentration in semen with normozoospermia （n = 11） was 1.34 ± 0.65 μg ·mL^-1, whereas a higher cfsDNA concentration was observed in azoospermia （2.56 ± 1.43 μg ·mL^-1, n = 9）. The continuous distribution of DNA fragments ranging from -1 kb to 15 kb and a spectrum of multiples of 180-bp fragments were observed in each normozoospermic and azoospermic sample. Distinct characteristic DNA ladder fragmentations in some azoospermic samples implicated that cfsDNA originate partly from apoptotic cells. CfsDNAs of 36 selected azoospermic patients with known information of Y chromosome microdeletion were subjected to the same microdeletion analysis by multiplex PCR and PCR amplification of sY114 （1 450 bp）. All multiplex PCR reactions with cfsDNA amplified successfully and provided the same result as leukocyte DNA. PCR amplification of sY114 gave a 1 450-bp amplicon as expected. Our data suggested the potential use of cfsDNA in search of biomarker or diagnostic procedures.
文摘Synovial sarcoma(SS) is genetically characterized by chromosomal translocation, which generates SYT-SSX fusion transcripts. Although SS can occur in any body part, primary gastric SS is substantially rare. Here we describe a detection of the fusion gene sequence of gastric SS in plasma cell-free DNA(cf DNA). A gastric submucosal tumor was detected in the stomach of a 27-year-old woman and diagnosed as SS. Candidate intronic primers were designed to detect the intronic fusion breakpoint and this fusion sequence was confirmed in intron 10 of SYT and intron 5 of SSX2 by genomic polymerase chain reaction(PCR) and direct sequencing. A locked nucleic acid(LNA) probe specificto the fusion sequence was designed for detecting the fusion sequence in plasma and the fusion sequence was detected in preoperative plasma cfD NA, while not detected in postoperative plasma cfD NA. This technique will be useful for monitoring translocation-derived diseases such as SS.
