Solid-phase method for the purification of DNA sequencing reactions.
Solid-phase method for the purification of DNA sequencing reactions.
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DOI：
10.1021/ac00046a004
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发表时间：
1992-11
影响因子：
7.4
通讯作者：
X. Tong;L. Smith
中科院分区：
文献类型：
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作者：
X. Tong;L. Smith
已经开发了用于纯化酶促测序反应中产生的单链DNA分子的固相方法。合成在内部位置含有生物素部分的引物寡核苷酸。该引物用于酶促延伸反应，所得产物与链霉亲和素包被的磁珠结合。污染物质如蛋白质、盐、模板DNA和未掺入或降解的脱氧和双脱氧核苷酸三磷酸可以通过在用固定磁体将它们固定在样品管中之后洗涤珠来去除。通过在10 mM EDTA、95%甲酰胺中加热，在90 ℃下上样染料，将所得纯单链DNA片段从固体支持物上除去，然后可以直接上样到聚丙烯酰胺凝胶上用于序列分析。该方法用于研究各种污染物对DNA序列数据的影响。
A solid-phase method for the purification of the single-stranded DNA molecules produced in enzymatic sequencing reactions has been developed. A primer oligonucleotide is synthesized containing a biotin moiety at an internal position. This primer is utilized in enzymatic extension reactions, and the resulting products are bound to streptavidin-coated magnetic beads. Contaminating species such as protein, salts, template DNA, and unincorporated or degraded deoxy and dideoxy nucleotide triphosphates may be removed by washing the beads after immobilizing them in the sample tube with a fixed magnet. The resulting pure single-stranded DNA fragments are removed from the solid support by heating in 10 mM EDTA, 95% formamide, loading dye at 90 degrees C, and may then be directly loaded onto a polyacrylamide gel for sequence analysis. This method was used to investigate the effect of various contaminants upon DNA sequence data.