一种新的小分子DNA纯化方法——石英棉纯化法

建立了小分子DNA的高效分离纯化方法,适合于分离几十到300个核苷酸组成的基因,在此基础上,建立更大的DNA片段的分离纯化方法。对不同大小的DNA片段经琼脂糖凝胶电泳后,采用石英棉分离纯化法纯化DNA。结果显示,石英棉的分离效果最好,对于200个碱基以下的DNA的回收率约高达90%以上,对于300个碱基的DNA回收率为约85~90,该项技术纯化的DNA的酶切、酶连效率与试剂盒法纯化的DNA的酶切、酶连效率相同。该分离纯化DNA技术明显优于试剂盒方法。     

Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene-specific probes

It has been shown that minor differences, such as single-base-pair substitutions between otherwise identical DNA fragments can result in altered melting behavior detectable by denaturing gradient gel electrophoresis (DGGE). Sequence variations in only a small DNA region within one locus can be detected using the previously described procedures. We have developed a method for the efficient Southern transfer of genomic DNA fragments from the denaturing gradient gels in order to be able to analyze larger regions in several loci for variation. The gels were made using polyacrylamide containing 2% low-geling-temperature agarose (LGT). The polyacrylamide gel (PAG) was crosslinked with a reversible crosslinker, and after electrophoresis the crosslinks were cleaved, the structure of the gel being maintained by the agarose. After this treatment of the denaturing gels, more than 90% of the DNA fragments could be transferred to nylon membranes by alkaline transfer, while electroblotting transferred only 10% of the DNA. Hybridization with gene-specific probes was then performed. We have used this technique to identify an RFLP in the COL1A2 gene in a human genomic DNA sample. The transfer technique described should make the use of DGGE more widely applicable since the genomic DNA fragments separated on one gel can be screened with several different probes, both cDNA and genomic probes.     

Electroelution of DNA and protein from polyacrylamide and agarose gels

An electroelution method is described for the recovery of DNA and protein from agarose or polyacrylamide gels. The samples to be electroeluted are compartmentalized in a modified microcentrifuge tube fitted with dialysis membranes. This procedure is simple, rapid, inexpensive and efficient. Within 30 min to 2 hrs, the recovery of the sample is nearly quantitative. DNA fragments recovered can be directly subjected to DNA sequence analysis or enzymatic reactions after ethanol precipitation. Proteins can also be recovered after separation by acrylamide gel in the presence or absence of detergents and be ready for further analysis.     

Simultaneous electroelution and concentration of DNA fragments from agarose gels

A rapid and inexpensive method for the electroelution of DNA fragments from agarose gels is described. DNA fragments were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Selected DNA fragments were placed into electroeluter tubes capped with dialysis membrane and electroeluted into a small volume of buffer using a conventional horizontal gel electrophoresis apparatus. The method successfully eluted and concentrated DNA fragments with molecular weights ranging from 2.7 to 13.9 MDa in 3 h.     

Separation and recovery of DNA fragments by electrophoresis through a thermoreversible hydrogel composed of poly(ethylene oxide) and poly(propylene oxide)

We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology.     

从非变性聚丙烯酰胺凝胶回收纯化DNA样本

介绍一种从非变性聚丙烯酰胺凝胶中回收和纯化目标DNA片段的方法,经比较回收纯化前后PCR产物的电泳结果,表明该方法具有简单快捷和效率高的优点。     

Preparation of GDP-D-mannose specifically labeled with tritium in the D-mannosyl moiety

A method, called “bidirectional transfer”, has been described for the transfer of DNA and RNA from agarose or polyacrylamide gels onto diazobenzyloxymethyl (DBM)-paper or nitrocellulose filters. The gels were sandwiched between either two nitrocellulose filters or two diazobenzyloxymethyl-papers. Next, the nucleic acids were allowed to diffuse out of the gels onto the filters. In this way, duplicate blots were obtained from a single gel. The bidirectional transfer of DNA or RNA from 0.5 to 1% agarose gels was complete and nearly quantitative after 1 h of transfer. DNA fragments from 5% polyacrylamide gels were efficiently blotted after 36 h onto nitrocellulose filters using bidirectional transfer. The fragments were transferred with good resolution and were shown to be efficient substrates for homologous [³²P]DNA probes.     

Improved resolution of DNA fragments in polysaccharide-supplemented agarose gels

The electrophoretic separation of nucleic acids, including small DNA fragments in the range 50-1000 bp, is presently carried out in polyacrylamide gels or in gels containing high concentrations of agarose. We have developed an alternative gel matrix composition which is inexpensive, nontoxic, easy to prepare, and highly transparent to visible and uv light. The composition combines a soluble nonionic polysaccharide such as hydroxyethylcellulose, methylcellulose, or galactomannan with a minimum but sufficient concentration of agarose to form a gel which immobilizes the "liquid phase sieve." These mixtures do not replace polyacrylamide for resolving fragments smaller than approximately 75 nucleotides. However, the new gels show DNA fragment resolution (band separation versus distance traveled) and optical clarity superior to those of conventional agarose.     

平末端DNA随机连接构建重组质粒

目的:拼接DNA片段并克隆。方法:用T4DNA连接酶将DNA片段以平末端随机连接,随后用限制性内切酶切割,琼脂糖电泳分离酶切产物,挑选特定片段纯化回收,与线性化的载体质粒连接,转化大肠杆菌感受态细胞。结果:通过以上步骤,成功拼接了不同DNA片段,构建了含有目的拼接片段的重组质粒。结论:该方法简便、易行、可靠,可作为拼接、克隆DNA的备选方案,在分子生物学研究和基因工程中应用。     

从琼脂糖凝胶中高效回收DNA技术的探讨

用两只离心管制成的凝胶过滤装置,从电泳后的琼脂糖凝胶中回收DNA片段的简易方法。它依次包括以下步骤：凝胶过滤装置的制作、凝胶切割、凝胶低温冷冻、低温高速离心、ddH20洗胶、DNA纯化和回收效果检测等。用此方法回收的DNA片段产率高、质量纯,可直接用于分子生物学实验的后续操作,如载体连接、PCR模板获得、DNA探针制备、基因测序等。其优点是：DNA片段的回收率高（90％以上）,质量好;操作简便,耗时短;回收装置简单,成本低廉,可进行商品化开发。     

A simple and efficient method for the separation and detection of small DNA fragments by electrophoresis in formamide containing agarose gels and Southern blotting to DBM-paper.

We show in this report that DNA fragments smaller than 300 bp are separated with high resolution by electrophoresis in concentrated (up to 7%) agarose gels containing 50% formamide. The separated DNA fragments can subsequently be quantitatively transferred to DBM-paper [Alwine, J.C. et al., Proc. Natl. Acad. Sci. USA (1977) 74, 5350-54] using the Southern technique [Southern, E.M., J. Mol. Biol. (1975) 98, 503-517], while preserving the sharpness of the original gel pattern. Since thin (0.2-0.4 mm) and thick (up to 5 mm) agarose slab gels can be easily handled in vertical or horizontal apparatus, this method should prove to be a very useful extention of the Southern technique, applicable to a variety of analytical and preparative purposes.     

A simple method for filter hybridization of labeled restriction fragments excised from gels

Labeled DNA restriction fragments excised from agarose or bisacrylylcystamine-acrylamide gels can be used for hybridization to nitrocellulose-bound DNA without eliminating the gel matrix. A gel slice containing the labeled fragment is excised, dissolved by heating at 105 degrees C (in the presence of beta-mercaptoethanol for bisacrylylcystamine-acrylamide gels), and added to the hybridization mixture. The presence of agarose or polyacrylamide in the solution does not inhibit hybridization. The method is simple, rapid, and allows complete recovery of the probe.     

Rapid and quantitative recovery of DNA fragments from gels by displacement electrophoresis (isotachophoresis)

The use of displacement electrophoresis (synonymous to isotachophoresis, steady-state stacking, and moving boundary electrophoresis) for recovery of DNA fragments from agarose and polyacrylamide gels is described. Complete recovery of DNA molecules ranging from oligonucleotides to 20 000-basepairs-long fragments was achieved. The DNA is recovered in a small volume (0.1-0.3 ml) and can be used directly in enzyme-mediated cleavage and ligation reactions. The recovered DNA contained no inhibitory contaminants as revealed by ligation or restriction enzyme cleavage.     

Electroeluting DNA Fragments

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.     

A rapid and efficient procedure for the purification of DNA from agarose gels

DNA fragments electrophoresed through a horizontal agarose slab gel can be recovered by inserting strips of filter paper backed by dialysis membrane into slits cut in the gel in front of the DNA bands and continuing electrophoresis until the DNA is collected in the paper. Elution of the DNA from the filter paper is then achieved by low-speed centrifugation. Recovery well above 70% is routinely obtained with this technique and the DNA recovered is biologically active and can be recleaved, ligated, labeled in vitro by nick translation and hybridized to RNA.     

A method for the recovery of DNA from agarose gels.

We describe a quick and versatile method for the isolation of DNA from agarose gels. The DNA is electrophoresed into a trough containing hydroxyapatite, where it is bound. The hydroxyapatite is taken out and the DNA eluted with phosphate buffer. By putting the hydroxyapatite on a small column of Sephadex G50, elution and subsequent removal of phosphate can be performed in one step. The DNA recovered can be used equally well in enzymatic incubations as DNA not purified through agarose gel electrophoresis. Several applications of this technique are described.     

Transverse agarose pore gradient gel electrophoresis of DNA.

Transverse agarose pore gradient gels were prepared on GelBond in the concentration range of nominally 0.2-1.5% SeaKem GTG agarose, using density stabilization by glycerol and incorporation of a dye to define the gel concentration at each point on the pore gradient gel. The distribution of the dye was evaluated by photography, video-acquisition and digitization of the gradient mixture and by densitometry of the gel. The gel was applied to the electrophoresis of a 1-kb standard ladder of DNA fragments, using standard submarine apparatus. The method extends to agarose gel electrophoresis the benefits of semi-automated analysis of 'Ferguson curves' described in application to polyacrylamide gel by Wheeler et al. (J. Biochem. Biophys. Methods 24, 171-180).     

Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose

A method for efficient electrophoretic transfer of DNA fragments from polyacrylamide gels to nitrocellulose sheets was developed. Hybridization to these fragments can be performed by standard techniques. The method is also applicable to agarose gels, allowing this transfer method to be used for DNA ranging from 40 to at least 23,000 bp.     

Electrophoretic mobility of high-molecular-weight, double-stranded DNA on agarose gels.

A new theoretical model for the migration of high-molecular-weight, double-stranded DNA on agarose gels is presented. This leads to the prediction that under certain conditions of electrophoresis, a linear relationship will exist between the molecular weight of a DNA molecule, raised to the (-2/3) power, and its electrophoretic mobility. Agarose gel electrophoresis of the fragments of bacteriophage lambda DNA produced by several restriction endonucleases confirms this relationship, and establishes some of the limits on its linearity. For this work, a polyacrylamide slab gel apparatus was modified for use with agarose gels. This apparatus has several advantages over others commercially available for agarose gel electrophoresis, including the abilities to run a larger number of samples at one time, to use lower-concentration gels, and to maintain better temperature stability across the width of the gel. The validation of the relationship developed here between molecular weight and electrophoretic mobility should make this a useful method for determining the molecular weights of DNA fragments.     

Large-scale purification of plasmid insert DNA sequences using low-percentage agarose exclusion chromatography

Linear double-stranded DNA fragments ranging from 0.14 to 8.4 kbp have been fractionated on low-percentage agarose exclusion gels. Both Ultragel A2 (2% agarose) and Bio-Gel A150m (1% agarose) exclude DNA fragments greater than 900 bp, while the exclusion limit of Bio-Gel A50m (2% agarose) is about 350 bp. All gels result in moderate resolution of DNA fragments smaller than the exclusion limits; we generally observe nearly complete one-step separation of fragments that differ in size by a factor of 2. On the basis of these results, we have used these exclusion gels to routinely purify greater than 0.4 mg of plasmid insert DNA sequences in one step and over 2.5 mg with a single column, demonstrating that these gel matrices can be ideally suited for repeated rapid large-scale purification of plasmid inserts. In addition, this knowledge allows for a more rational design of plasmids in those cases where large-scale use of the insert DNA is required.