HydroLink gel electrophoresis (HLGE). III. High DNA loading capacity and recovery of dsDNA

Novel polymers have been prepared for high performance electrophoretic separations of double-stranded DNA (dsDNA). These materials are part of a family of HydroLink high performance electrophoresis polymers. A comparison of the resolving capabilities of dsDNA HydroLink gels to agarose and polyacrylamide separations has been described in an accompanying paper. In this study, we demonstrate that dsDNA HydroLink gels possess ten times the loading capacity of comparable polyacrylamide or agarose gels without compromise to resolution or biological integrity of the separated DNA. A simplified procedure for recovery of separated components is also described.     

HydroLink gel electrophoresis (HLGE). II. Applications of a new polymer matrix to dsDNA analysis

HydroLink materials represent a novel family of gels composed of unique polymer matrices. The applications of HydroLink to molecular biology and, specifically, to DNA technology have been carefully investigated. Our results indicate that the HydroLink matrix developed for double-stranded DNA (dsDNA) is an excellent tool for electrophoretic separations in fixed electric fields. Excellent linear resolution from 100 to 5000 base pairs is easily achieved with good resolution albeit non-linear from 6000 to 23000 base pairs. The broad range of separation in addition to increased mechanical strength of dsDNA HydroLink represents a distinct advantage over other matrices currently used in DNA electrophoretic analysis.     

Hybridization of DNA to RNA in methylmercuric hydroxide agarose gels

We describe a method for hybridization of cDNA probes to RNA directly in agarose gels which provides a practical alternative to methods involving transfer of the RNA out of the gel. Total cellular RNA is subjected to electrophoresis in agarose gels containing methylmercuric hydroxide as the denaturing agent. After removal of the methylmercuric hydroxide, the gel is dried and 32P-labeled DNA probes are hybridized to the immobilized RNA. This method is more economical in time and expense than methods involving transfer of the RNA out of the gel, while maintaining a level of sensitivity comparable to other procedures.     

High capacity gel preparative electrophoresis for purification of fragments of genomic DNA

We have designed a high-capacity gel electrophoretic device for the purification of large amounts of restriction endonuclease fragments of genomic DNA. This device exploits the high resolution of gel electrophoresis in conjunction with an electronic system permitting discontinuous sample elution over a large gel surface area. This feature preserves resolution and greatly increases capacity and yield. The resulting DNA fractions may be used in restriction endonuclease, ligation, transfection, and transformation reactions without further extensive manipulation. Furthermore, DNA fragments of a broad size range are recovered with high efficiency from the gel.     

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.     

Southern and Northern analysis

This review on Southern and Northern analysis, rather than providing step-by-step protocols, focuses on a critical evaluation of the existing experimental methods and modifications thereof. Principal parameters influencing electrophoresis of DNA/RNA in agarose gels are outlined and at the same time alterations in these parameters for optimal resolution of DNA of varying length are discussed. Further, methods for evaluating the quality of DNA/RNA size separation in agarose gels are described. Since efficient transfer of DNA/RNA from the gel onto a membrane support is critical in both methods, several experimental approaches for transfer are compared. Also discussed in this review are alternative methods for radioactive and non-radioactive labelling of DNA probes. Finally, detailed protocols are provided for an effective hybridization of Southern and Northern blots.     

Enhanced resolution of random amplified polymorphic DNA genotyping of Pseudomonas aeruginosa

AIMS: To develop a rapid, sensitive and reproducible screening test for the detection of nosocomial spreading of Pseudomonas aeruginosa. METHODS AND RESULTS: Ps. aeruginosa genomic DNA extraction, RAPD-PCR, electrophoresis on acrylamide gel and silver staining were performed by using standardized reagents and conditions. The results were compared with the agarose gel electrophoresis followed by ethidium bromide staining. CONCLUSIONS: The coupling of acrylamide gel electrophoresis and silver staining gave about 80% more DNA bands than the traditional method, allowing a finer discrimination among different Ps. aeruginosa strains. SIGNIFICANCE AND IMPACT OF THE STUDY: By enhancing the resolution of the electrophoretic separation and the sensitivity of the staining, random amplification could be easily applied to the surveillance and prevention of nosocomial infections by clinical microbiology laboratories.     

Sensitivity of Prestaining RNA with Ethidium Bromide Before Electrophoresis and Performance of Subsequent Northern Blots Using Heterologous DNA Probes

Adding ethidium bromide (EtBr) at low concentrations to RNA samples before running formaldehyde–agarose gels affords the advantages of checking RNA integrity and evaluating the quality of size-separation at any time during electrophoresis or immediately after either electrophoresis or blotted the separated RNA onto the membrane without significantly compromising mobility, transfer, or hybridization. In this study, we systematically examined the factors that affect the sensitivity of RNA prestaining by heating RNA samples that include EtBr before electrophoresis under different denaturation conditions. We also examined the efficiency of the hybridization of EtBr-prestained RNA with heterologous DNA probes. The results showed that the fluorescent intensity of EtBr-prestained RNA was affected not only by the EtBr concentration as previously reported but also by the RNA amount, denaturation time, and denaturation temperature. Prior staining of RNA with 40 μg/mL EtBr significantly decreased the efficiency of Northern blot hybridization with heterologous DNA probes. We propose that to best combine staining sensitivity and the efficiency of Northern blot hybridization with heterologous DNA probes, the concentration of EtBr used to prestain RNA should not exceed 30 μg/mL. The efficiency of the hybridization of EtBr-prestained RNA was affected not only by factors that affect staining sensitivity but also by the type of probe used.     

Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA.

Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels. The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes. Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size. Comparison of the Downs strain with other H. capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility. The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.     

Single universal primer multiplex ligation-dependent probe amplification with sequencing gel electrophoresis analysis

In this study, a novel single universal primer multiplex ligation-dependent probe amplification (SUP-MLPA) technique that uses only one universal primer to perform multiplex polymerase chain reaction (PCR) was developed. Two reversely complementary common sequences were designed on the 5′ or 3′ end of the ligation probes (LPs), which allowed the ligation products to be amplified through only a single universal primer (SUP). SUP-MLPA products were analyzed on sequencing gel electrophoresis with extraordinary resolution. This method avoided the high expenses associated with capillary electrophoresis, which was the commonly used detection instrument. In comparison with conventional multiplex PCR, which suffers from low sensitivity, nonspecificity, and amplification disparity, SUP-MLPA had higher specificity and sensitivity and a low detection limit of 0.1 ng for detecting single crop species when screening the presence of genetically modified crops. We also studied the effect of different lengths of stuffer sequences on the probes for the first time. Through comparing the results of quantitative PCR, the LPs with different stuffer sequences did not affect the ligation efficiency, which further increased the multiplicity of this assay. The improved SUP–MLPA and sequencing gel electrophoresis method will be useful for food and animal feed identification, bacterial detection, and verification of genetic modification status of crops.     

Optimized conditions for pulsed field gel electrophoretic separations of DNA.

Quantitative measurement of DNA migration in gel electrophoresis requires precisely controlled homogeneous electric fields. A new electrophoresis system has allowed us to explore several parameters governing DNA migration during homogeneous field pulsed field gel (PFG) electrophoresis. Migration was measured at different switch times, temperatures, agarose concentrations, and voltage gradients. Conditions which increase DNA velocities permit separation over a wider size range, but reduce resolution. We have also varied the angle between the alternating electric fields. Reorientation angles between 105 degrees and 165 degrees give equivalent resolution, despite significant differences in DNA velocity. Separation of DNA fragments from 50 to greater than 7000 kilobases (Kb) can easily be optimized for speed and resolution based on conditions we describe.     

Detection of sickle-cell mutation by electrophoresis of partial RNA:DNA hybrids following solution hybridization

We have developed a method in which partially single-stranded (ss) DNA molecules containing a defined region of duplex RNA:DNA are electrophoretically separated in agarose gels. The partial hybrids are formed by solution hybridization with a uniform length RNA probe complementary to part of the DNA sequence of interest. Following hybridization, the RNA/DNA mixture is fractionated by agarose gel electrophoresis at high temperature to minimize intrastrand base pairing which causes mobility heterogeneity. Not requiring the steps of DNA transfer from the gel to a solid support and subsequent probing, pre-electrophoretic hybridization allows the direct identification of single-copy fragments. Conditions for the detection of single-copy genes in human DNA digested with specific restriction endonucleases were developed and applied to the diagnosis of sickle-cell disease. This method should be applicable for the analysis of DNAs of high complexity where the presence of DNA polymorphisms and interspersed repeated DNA sequences often make impossible the creation of complete RNA:DNA hybrids.     

Resolution of DNA molecules greater than 5 megabases by contour-clamped homogeneous electric fields.

Excellent resolution of chromosomal DNA molecules from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe has been obtained using alternating contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The largest of these molecules is greater than 5 Mb in size and is resolved after 130 hours in a 0.6% agarose gel at a field strength of 1.3 V/cm and a switching interval of 1 hour. Separation of concatamers of phage lambda DNA reveals four regions of resolution in alternating CHEF gel electrophoresis. There are two regions of good resolution in which mobility approximates a linear function of molecular weight. These are separated by a region of lower resolution and bounded at high molecular weights by a region of little or no resolution. The four regions are of practical and possibly theoretical importance.     

Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family.

A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows one to estimate the number of hybridizing genes contained in each large DNA fragment. We have also developed a novel method to increase the separation, resolution and hybridization signal in the second dimension by condensing the bands from the first dimension into spots. As an example, we have applied these techniques to determine the organization of the murine T-cell receptor gamma locus. The murine gamma gene family was found to be contained on two DNA fragments encompassing 195 kilobases of DNA. The two-dimensional gel electrophoresis method is particularly useful in the analysis of the organization of multigenic families where single copy probes are not readily available, and should extend the potential usefulness of field inversion gel electrophoresis in gene mapping.     

Homogeneous versus heterogeneous probes for microbial ecological microarrays

Microbial ecological microarrays have been developed for investigating the composition and functions of microorganism communities in environmental niches. These arrays include microbial identification microarrays, which use oligonucleotides, gene fragments or microbial genomes as probes. In this article, the advantages and disadvantages of each type of probe are reviewed. Oligonucleotide probes are currently useful for probing uncultivated bacteria that are not amenable to gene fragment probing, whereas the functional gene fragments amplified randomly from microbial genomes require phylogenetic and hierarchical categorization before use as microbial identification probes, despite their high resolution for both specificity and sensitivity. Until more bacteria are sequenced and gene fragment probes are thoroughly validated, heterogeneous bacterial genome probes will provide a simple, sensitive and quantitative tool for exploring the ecosystem structure.     

Definition of the limits of the Wilms tumor locus on human chromosome 11p13

In a previous report, we described a contiguous restriction map of chromosome band 11p13 that localized the Wilms tumor locus to a small group of NotI fragments. In an effort to identify and isolate the 11p13-associated sporadic Wilms tumor locus, we developed a panel of NotI fragment-specific DNA probes. These probes were selected from genomic libraries constructed using the Chinese hamster ovary-human somatic cell hybrid carrying only human 11p. The libraries were prepared from NotI-digested DNA after size selection by pulsed-field gel electrophoresis. The selected NotI fragments had been previously targeted on the basis of deletion mapping as having a high probability of containing the Wilms tumor locus. We used these newly identified 11p13-specific probes to improve the resolution of the restriction map spanning the Wilms tumor locus. The locus has been defined by a homozygous deletion in a sporadic Wilms tumor. Using these probes, the region of homozygous deletion in this tumor and presumably all or part of the Wilms tumor gene have been confined to two small SfiI fragments spanning less than 350 kb.     

Fluorescent single-stranded DNA binding protein as a probe for sensitive, real-time assays of helicase activity

The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases.     

Ultraviolet nicking of large DNA molecules from pulsed-field gels for southern transfer and hybridization.

Large DNA molecules separated in pulsed-field gels are not efficiently transferred from the gel for Southern hybridization. Various procedures for fragmenting the DNA prior to transfer are in use, but quantitative details that permit reproducible application have not been reported. We have determined the optimum level of energy for uv nicking of large DNA needed to promote efficient Southern transfer and detection by hybridization. To ensure consistent results we have used a uv oven equipped with a detector that measures only 200-400 nm wavelengths, and we report the total energy delivered. Using uv nicking and the transfer techniques described, we can obtain hybridization signals overnight with single-copy DNA probes on Southern blots of large DNA fragments separated by pulsed-field gel electrophoresis.     

DNA microarray probe preparation by gel isolation nested PCR

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.