两步磁珠法提取血浆游离DNA的评价及临床应用
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摘要
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。
Objective To evaluate the two-step magnetic bead method for extracting plasma free DNA,and to preliminarily explore the significance of plasma free DNA quantification in experimental diagnosis of lung cancer patients. Methods Standard DNA including127 bp,208 bp and Quick-load~@100 bp DNA-ladder were spiked into healthy volunteer blood. Then the standard DNA was extracted by two-step magnetic beads method and spin columns method. The quality of purified DNA was examined by Qubit,and the size of purified DNA was analyzed on 1. 5% agarose gels and Agilent 2100 chip analysis. Then plasma cf-DNA of 15 lung cancer patients was measured. The plasma cf-DNA was extracted by two-step magnetic bead method and spin columns method respectively. After purification,the quality and the size of cf-DNA were examined as mentioned above. Results The re-purification of standard DNA( 127 bp and208 bp) by two-step magnetic beads method showed positive correlation with the concentration of spiked standard DNA( r~2= 0. 985 7,P <0. 001). The results showed that the size of DNA extracted by the first isolation step of two-step magnetic bead method was ≥300 bp,and DNA extracted by the second isolation step was ≤300 bp. The size of DNA extracted by spin columns method was ≤1 500 bp.The plasma cf-DNA concentration of lung cancer patients extracted by two-step magnetic bead method was positively correlated with the stage of lung cancer( r~2= 0. 866 4,P = 0. 004 9),while there was no significant correlation between the concentration of cf-DNA extracted by spin columns method and the stage of lung cancer( r~2= 0. 500 9,P = 0. 214 5). Conclusion Two-step magnetic bead method can extract different size fragments of DNA standard. Cf-DNA( less than or equal to 300 bp) from lung cancer patient plasma is effectively enriched by this method,and the concentration of these cf-DNA is positively correlated with the stage of lung cancer.
Objective To evaluate the two-step magnetic bead method for extracting plasma free DNA,and to preliminarily explore the significance of plasma free DNA quantification in experimental diagnosis of lung cancer patients. Methods Standard DNA including127 bp,208 bp and Quick-load~@100 bp DNA-ladder were spiked into healthy volunteer blood. Then the standard DNA was extracted by two-step magnetic beads method and spin columns method. The quality of purified DNA was examined by Qubit,and the size of purified DNA was analyzed on 1. 5% agarose gels and Agilent 2100 chip analysis. Then plasma cf-DNA of 15 lung cancer patients was measured. The plasma cf-DNA was extracted by two-step magnetic bead method and spin columns method respectively. After purification,the quality and the size of cf-DNA were examined as mentioned above. Results The re-purification of standard DNA( 127 bp and208 bp) by two-step magnetic beads method showed positive correlation with the concentration of spiked standard DNA( r~2= 0. 985 7,P <0. 001). The results showed that the size of DNA extracted by the first isolation step of two-step magnetic bead method was ≥300 bp,and DNA extracted by the second isolation step was ≤300 bp. The size of DNA extracted by spin columns method was ≤1 500 bp.The plasma cf-DNA concentration of lung cancer patients extracted by two-step magnetic bead method was positively correlated with the stage of lung cancer( r~2= 0. 866 4,P = 0. 004 9),while there was no significant correlation between the concentration of cf-DNA extracted by spin columns method and the stage of lung cancer( r~2= 0. 500 9,P = 0. 214 5). Conclusion Two-step magnetic bead method can extract different size fragments of DNA standard. Cf-DNA( less than or equal to 300 bp) from lung cancer patient plasma is effectively enriched by this method,and the concentration of these cf-DNA is positively correlated with the stage of lung cancer.
引文
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[2]张世强,王保庆,王海清,等.非小细胞肺癌患者血清游离DNA定量检测的临床意义[J].山西医科大学学报,2014,45(10):944-946.
[3]Traver S,Assou S,Scalici E,et al.Cell-free nucleic acids as non-invasive biomarkers of gynecological cancers,ovarian,endometrial and obstetric disorders and fetal aneuploidy[J].Hum Reprod Update,2014,20(6):905-923.
[4]Chan KC,Jiang P,Zheng YW,et al.Cancer genome scanning in plasma:detection of tumor-associated copy number aberrations,single-nucleotide variants,and tumoral heterogeneity by massively parallel sequencing[J].Clin Chem,2013,59(1):211-224.
[5]Lim JH,Lee BY,Kim JW,et al.Evaluation of extraction methods for methylated cell-free fetal DNA from maternal plasma[J].J Assist Reprod Genet,2018,35(4):637-641.
[6]Qin Lan,Wang Jing,Tian Xia,et al.Detection and Quantification of Mosaic Mutations in Disease Genes by Next-Generation Sequencing[J].J Mol Diagn,2016,18(3):446-453.
[7]Biancon G,Gimondi S,Vendramin A,et al.Noninvasive Molecular Monitoring in Multiple Myeloma Patients Using Cell-Free Tumor DNA:A Pilot Study[J].J Mol Diagn,2018,20(6):859-870.
[8]He WS,Bishop KS.The potential use of cell-free-circulatingtumor DNA as a biomarker for prostate cancer[J].Expert Rev Mol Diagn,2016,16(8):839-852.
[9]Ettinger DS,Wood DE,Hughes M,et al.Non-small cell lung cancer,Version 6.2015[J].J Natl Compr Canc Netw,2015,13(5):515-524.
[10]Cicchillitti L,Corrado G,De AM,et al.Circulating cell-free DNA content as blood based biomarker in endometrial cancer[J].Oncotarget,2017,8(70):115230-115243.
[11]Mouliere F,El Messaoudi S,Pang D,et al.Multi-marker analysis of circulating cell-free DNA toward personalized medicine for colorectal cancer[J].Mol Oncol,2014,8(5):927-941.
[12]严子禾,潘世扬,陈丹,等.四种血浆游离DNA提取方法的比较[J].临床检验杂志,2006,24(5):363-365.