Rapid transfer of DNA from agarose gels to nylon membranes.

The unique properties of nylon membranes allow for dramatic improvement in the capillary transfer of DNA restriction fragments from agarose gels (Southern blotting). By using 0.4 M NaOH as the transfer solvent following a short pre-treatment of the gel in acid, DNA is depurinated during transfer. Fragments of all sizes are eluted and retained quantitatively by the membrane; furthermore, the alkaline solvent induces covalent fixation of DNA to the membrane. The saving in time and materials afforded by this simple modification is accompanied by a marked improvement in resolution and a ten-fold increase in sensitivity of subsequent hybridization analyses. In addition, we have found that nylon membrane completely retains native (and denatured) DNA in transfer solvents of low ionic strength (including distilled water), although quantitative elution of DNA from the gel is limited to fragments smaller than 4 Kb. This property can be utilized in the direct electrophoretic transfer of native restriction fragments from polyacrylamide gels. Exposure of DNA to ultraviolet light, either in the gel or following transfer to nylon membrane, reduces its ability to hybridize.     

Semi-dry electroblotting of DNA

A procedure is described for the rapid transfer of DNA from agarose gels to nylon membranes using the semi-dry electroblotting technique. A Hind III digest of lambda DNA which was separated in a 1% agarose gel containing Tris, Borate, and EDTA (pH 8.0) was employed for the electrotransfer experiments. Transfer efficiency was determined by staining the DNA on the nylon membranes with a colloidal iron reagent. Current densities of 3-5 mA/sq. cm of gel permitted the transfer of high (23 kb) and low (0.3 kb) molecular weight fragments within 15 min. However, efficient transfer required a high ionic strength buffer that would prevent uneven dehydration of the agarose gel. Critical parameters for the transfer of nucleic acids with the semi-dry technique are discussed.     

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.     

Native genomic blotting: a novel approach to mapping DNase I hypersensitive sites and protein-DNA interactions at high resolution

We have developed a new high resolution method for screening 400-600 base pairs of DNA in chromatin for DNase I hypersensitive sites and protein-DNA interactions. By separating the DNA isolated from nuclease-digested nuclei in small, native polyacrylamide gels prior to electroblotting onto nylon membranes, we increased the resolution by greater than 3-fold as compared with the traditional approach whereby the nuclease-digested DNA is fractionated electrophoretically in agarose gels (11). In addition, our native genomic blotting method has the advantage of combining the ability of the traditional agarose approach to detect DNase I hypersensitive sites, with the genomic sequencing method (2), where individual protein-DNA contacts can be observed. Native genomic blotting therefore permits for the first time the display of DNase I hypersensitive sites and protein-DNA interactions at high resolution on the same autoradiograph. This method allows us to investigate a new level of chromatin structure and to therefore obtain better insight into levels of gene structure, organization and gene regulation.     

Rapid alkaline blot-transfer of viral dsRNAs

The double-stranded genomic RNAs of reovirus and bluetongue virus can be transferred very efficiently from either sodium dodecyl sulfate-polyacrylamide gels or NuSieve agarose gels onto several nylon membranes. After a brief acid depurination treatment, viral dsRNAs from the gels are transferred at room temperature using 0.2 N NaOH as the transfer medium. Four blots can be obtained within 1 h and each blot contains 15-20% of the input RNA sample. These blots can be used immediately without baking in vacuo. Less than 5% of the "fixed" dsRNAs are removed after repeated washings of the membrane blots. As little as 10 pg of the genomic dsRNA segment can be detected in this alkaline Northern blot. A 20- to 50-fold increase in resolution and sensitivity over traditional Northern blots is routinely achieved. These alkaline blots can be reused 6-10 times after appropriate strip washing and proper handling.     

Orientation of DNA molecules in agarose gels by pulsed electric fields

The electric birefringence of DNA restriction fragments of three different sizes, 622, 1426, and 2936 base pairs, imbedded in agarose gels of different concentrations, was measured. The birefringence relaxation times observed in the gels are equal to the values observed in free solution, if the median pore diameter of the gel is larger than the effective hydrodynamic length of the DNA molecule in solution. However, if the median pore diameter is smaller than the apparent hydrodynamic length, the birefringence relaxation times increase markedly, becoming equal to the values expected for the birefringence relaxation of fully stretched DNA molecules. This apparent elongation indicates that end-on migration, or reptation is a likely mechanism for the electrophoresis of large DNA molecules in agarose gels. The relaxation times of the stretched DNA molecules scale with molecular weight (or contour length) as N2.8, in reasonable agreement with reptation theories.     

A procedure for DNA and RNA transfer to membrane filters avoiding weight-induced gel flattening.

We describe a technique of rapid (within 1-2 h) transfer of DNA and RNA from agarose gels to nitrocellulose or nylon membrane filters. It is characterized by nearly complete elimination of mechanical action on the gel (a thin layer of liquid is placed over the gel and, filtering through the gel into a stack of paper towels beneath, it transfers nucleic acids onto the filter under the gel). This "descending" transfer, as opposed to the widely used "ascending" Southern transfer, reduces the transfer time (to about 1 h) with equal or higher quality of the hybridization signal. The comparison of transfer kinetics by the both methods shows that (a) the Southern transfer of large size DNA fragments proceeds quicker than it has been thought so far and is almost complete within 4 h; (b) the descending transfer has an advantage over the ascending one in the rate of transfer (1-2 h) and its efficiency; and (c) the time of transfer may become a critical parameter upon using a filter with an apparently low retention capacity (Hybond N, Amersham) that is manifested by a decreased signal at longer than optimal transfer times.     

Efficient electroblotting of human very-low-density lipoproteins and low-density lipoproteins from composite gradient gels to nylon membranes

Rinsing of gradient composite acrylamide/agarose gels with 1% Triton X-100 permits the efficient electrotransfer of very-low-density and low-density lipoproteins from the gels and does not appear to interfere with subsequent capture of the lipoproteins by charged nylon membranes. Overall efficiency of the transfer/capture process can approach 95% and does not appear to be significantly affected by total lipoprotein concentrations up to 5000 mg/dl. Direct immunoquantification of transferred apolipoproteins on the membrane is feasible as well. The nylon membranes used, however, must be pretested to ensure capture efficiency.     

Orientation of DNA Molecules in Agarose Gels By Pulsed Electric Fields

Abstract

The electric birefringence of DNA restriction fragments of three different sizes, 622,1426, and 2936 base pairs, imbedded in agarose gels of different concentrations, was measured. The birefringence relaxation times observed in the gels are equal to the values observed in free solution, if the median pore diameter of the gel is larger than the effective hydrodynamic length of the DNA molecule in solution. However, if the median pore diameter is smaller than the apparent hydrodynamic length, the birefringence relaxation times increase markedly, becoming equal to the values expected for the birefringence relaxation of fully stretched DNA molecules. This apparent elongation indicates that end-on migration, or reptation is a likely mechanism for the electrophoresis of large DNA molecules in agarose gels. The relaxation times of the stretched DNA molecules scale with molecular weight (or contour length) as N^2.8, in reasonable agreement with reptation theories.     

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.     

Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.

We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.     

Random amplified polymorphic DNA analysis of DNA extracted from Trichuris trichiura (Linnaeus, 1771) eggs and its prospective application to paleoparasitological studies

Random amplified polymorphic DNA analysis was applied to DNAs extracted from Trichuris trichiura eggs recovered from human fecal samples. Four out of 6 primers tested displayed 18 distinct and well defined polymorphic patterns, ranging from 650 to 3200 base pairs. These results, upon retrieval and DNA sequencing of some of these bands from agarose gels, might help in establishing. T. trichiura specific genetic markers, not available yet, and an important step to design primers to be used in molecular diagnosis approaches.     

Alkaline transfer of DNA to plastic membrane

DNA forms a stable complex with Gene Screen Plus membrane at alkaline pH. Based on this, a method of alkaline transfer of DNA from agarose gel to Gene Screen Plus membrane was elaborated. The procedure entails the use of 0.4 M NaOH for both, the DNA denaturation and DNA transfer steps. The alkaline transfer offers a higher hybridization efficiency and simplifies the transfer procedure as compared with the standard method of DNA transfer at neutral pH. In addition, it can be used to remove RNA contamination from the transferred DNA.     

蜘蛛HMW-gDNA的电洗脱纯化提取

该文研究了蜘蛛大分子量基因组DNA（HMW-gDNA）的提取以及一种高效电洗脱纯化装置的构建。以蜘蛛胸部肌肉组织为原料,通过自改良CTAB法提取蜘蛛HMW-gDNA,利用透析膜和2 mL离心管构建一种新的HMW-gDNA快速凝胶回收装置,并对蜘蛛HMW-gDNA进行电洗脱分离回收。结果显示,改良CTAB法可高效提取蜘蛛HMW-gDNA（>48.5 kb）,且通过透析膜的截留作用,对普通琼脂糖凝胶中目的HMW-gDNA进行快速电洗脱分离,其回收率超过75%,OD₂₆₀/OD₂₈₀处于1.8～2.0之间,对HMW-gDNA完整性无影响。综合结果表明, 改良CTAB法可用于蜘蛛HMW-gDNA的提取,此电洗脱纯化装置可从普通琼脂糖中高效回收HMW-gDNA,是一种低成本、简捷、高效且实用性强的凝胶回收方法。     

从琼脂糖电泳凝胶中回收DNA的几种简便方法

介绍两类从普通琼脂糖电泳凝胶中回收ＤＮＡ的简便、快捷、高效且廉价的方法．第一类为电泳洗脱法．方法ａ：利用１．５ｍＬ微量离心管、ｌｍＬ吸头、尼龙网膜和透析膜做成的一个小装置,快速有效回ＤＮＡ,最终回收率为７０％左右．方法ｂ：不用ＤＥＡＥ－纤维素膜,而用透析膜在凝胶中作出横隔挡在ＤＮＡ条带前,最终回收率为５０％左右;第二类为冰冻融解法,最终回收率也在５０％左右．如果联合使用冰冻融解法和电泳洗脱法,回收率可进一步提高至９０％．     

One-hour downward alkaline capillary transfer for blotting of DNA and RNA.

The downward alkaline capillary transfer of DNA and RNA from agarose gel to a hybridization membrane was performed using a transfer solution containing 3 M NaCl and 8 mM NaOH. Under mild alkaline conditions, DNA and RNA were completely eluted from the agarose gel and bound to a hybridization membrane within 1 h. On the basis of this new method of transfer a blotting protocol, downward alkaline blotting, was elaborated. It provides a fast and efficient alternative to commonly used Southern and Northern blotting protocols. The downward alkaline blotting of DNA and RNA can be completed in 2.5 and 1.5 h, respectively, and can be used with both plastic and nitrocellulose membranes. In addition, the downward alkaline blotting protocol allows for a hybridization efficiency of DNA and RNA higher than that of the standard blotting protocols performed at neutral pH.     

Dictyostelium rDNA consists of non-chromosomal palindromic dimers containing 5S and 36S coding regions

Restriction enzyme mapping of snap-back DNA has been used to show that the ribosomal DNA (rRNA genes and spacers) from Dictyostelium discoideum exists as 88 kb (kb=1,000 base pairs) linear palindromic dimers. Analysis of the mobility of total cell DNA in 0.15% agarose gels indicates that the majority of the rDNA is not covalently attached to chromosomal DNA.     

Quantitation of single- and double-strand DNA breaks in vitro and in vivo

This communication describes a rapid and convenient procedure for quantitation of strand breaks in bacterial DNA, both in vitro and in vivo, using agarose gel electrophoresis. The electrophoretic determination of single strand breaks is carried out in alkaline medium, followed by renaturation of the gel and intercalation of the fluorescent dye, ethidium bromide. Double-strand breaks are determined by electrophoresis in neutral medium containing the dye. The distribution of DNA fragment sizes, the determination of the number-average molecular weight, the quantitation of the average number of DNA breaks per molecule, and the ratio between the single- and double-strand breaks are evaluated from microdensitometric scanning of the gels. The application of this analysis to damage caused by a combination of ascorbate and copper is demonstrated.     

Express hybridization of molecular colonies with fluorescent probes

DNA colonies formed during PCR in a polyacrylamide gel and RNA colonies grown in an agarose gel containing Qbeta replicase can be identified using the procedure of transfer of molecular colonies onto a nylon membrane followed by membrane hybridization with fluorescent oligonucleotide probes. The suggested improvements significantly simplify and accelerate the procedure. By the example of a chimeric AML1-ETO sequence, a marker of frequently occurring leukemia, the express hybridization method was shown to allow the rapid identification of single molecules and the determination of titers of DNA and RNA targets. Hybridization with a mixture of two oligonucleotide probes labeled with different fluorophores complementary to components of the chimeric molecule ensures the identification of molecular colonies containing both parts of the chimeric sequence and improves the specificity of diagnostics.     

RNA molecular weight determination by agarose gel electrophoresis using formaldehyde as denaturant: comparison of RNA and DNA molecular weight markers

Reliable molecular weight measurements of RNA molecules as large as 4.0 X 10(6) dalton can be made on agarose gels containing 2.2 M formaldehyde as denaturant (Lehrach et al., 1977). Both eucaryotic and procaryotic ribosomal RNAs have generally been used as molecular weight markers. However, Maniatis et al. (1982) have suggested the use of restriction fragments of DNA as convenient molecular weight markers for RNA samples run in formaldehyde/agarose gels. This communication compares RNA and DNA molecular weight markers run under identical conditions.