ABSTRACT
Quantitation of DNA bands separated in polyacrylamide or agarose gels was tested under a variety of conditions to examine key factors contributing to the ability to obtain quantitative data. Variations tested included comparison of different fluorescent stains (ethidium bromide and GelStar stain), and variations of other parameters relating to the electrophoretic separation, e.g. gel and sample geometry, mode of staining, and mode of excitation of stains. The results showed that linear results were seen for a similar 20-30-fold range of DNA concentrations with both stains tested and that it is critical to standardize separations to obtain consistent results. Variations in separation and detection could lead to relatively large changes in fluorescent signals seen for similar amounts of DNA.
Subject(s)
DNA/analysis , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Image Processing, Computer-Assisted , DNA/isolation & purification , Ethidium , Fluorescent Dyes , Reproducibility of ResultsABSTRACT
GelStar nucleic acid gel stain can be used for sensitive fluorescent detection of both double-stranded (ds) and single-stranded (ss) DNAs, oligonucleotides and RNA in gels. The stain can be added to agarose gels at casting for immediate imaging after electrophoresis or can be used after electrophoresis with both agarose and acrylamide gels. GelStar stain is highly fluorescent only when bound to nucleic acids thus giving superior signal-to-noise ratios and obviating the need to destain the gel. The detection limits of GelStar strain are 20 pg for dsDNA, 25 pg for ssDNA and 10 ng for native or glyoxal-treated RNA.
Subject(s)
Coloring Agents , Nucleic Acids/analysis , Biotechnology , DNA Mutational Analysis , Electrophoresis, Agar Gel , Ethidium , Evaluation Studies as Topic , Fluorescent Dyes , Gels , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Sensitivity and Specificity , Tandem Repeat SequencesABSTRACT
Short tandem repeats (STRs) are traditionally analyzed on large polyacrylamide electrophoresis gels. We demonstrate in this study that a small (10-cm-long, 1-mm-thick) agarose gel is sufficient for analysis of multiplexed samples for several commonly used STR loci. A system was developed using a high-resolution agarose, MetaPhor. Within an hour of electrophoresis, sufficient resolution was obtained to allow discrimination of triple-multiplexed STR loci. We show that this agarose is capable of resolving di- as well as tetranucleotide ladders. Using PCR conditions similar to those routinely used with sensitive detection systems, we found that direct staining of gels with SYBR Green I stain was comparable with silver staining, autoradiography or fluorescently tagged primers for sample detection and was considerably easier. This procedure significantly simplifies STR analysis and is much faster than many standard protocols.
Subject(s)
DNA/isolation & purification , Electrophoresis, Agar Gel/methods , Repetitive Sequences, Nucleic Acid , Sepharose , Genetic Linkage , Humans , Pedigree , Polymerase Chain ReactionABSTRACT
Properties of agarose potentially relevant to PFGE (pulsed-field gel electrophoresis) are reviewed, and some new information is presented. Agarose polymers appear to have molecular weights in the range of 100,000 to 200,000 Da, but this is not tightly related to the effective gel strength. Agarose has some residual charge, and hence exhibits electroendosmosis (EEO). It is possible to markedly increase the speed of separation of DNA molecules by using agarose of low EEO, especially in low ionic strength, non-borate buffers. This increase is especially noticeable in the relatively long experiments required for separation of large DNAs. It is also possible to increase the range of separation in a single run by use of step gradients of agarose concentration, which allows visualization of yeast chromosomes and lambda-phage restriction fragments in the same lane. Because of the strong influence of concentration on separation, it may be useful for investigators to control water content and related variables. Our lack of knowledge of the detailed microstructure of gels may be barrier to complete understanding of PFGE.
Subject(s)
Electrophoresis, Gel, Pulsed-Field , Sepharose/chemistry , Chemical Phenomena , Chemistry, Physical , Molecular Structure , Polymers/chemistryABSTRACT
This report describes the use of a new type of agarose (FastLane agarose) for faster separation of DNA by agarose gel electrophoresis. DNA molecules separated in this agarose exhibited electrophoretic mobilities up to 30% higher than similar separations in standard analytical grade agarose. DNA molecules of all sizes examined showed higher mobilities in FastLane agarose. The mobility increase was predominantly due to the low electroendosmosis of FastLane agarose and was most pronounced in pulsed field gel electrophoresis separations. The magnitude of mobility increase varied depending on the conditions used for electrophoresis.
Subject(s)
Buffers , DNA/isolation & purification , Electrophoresis, Gel, Pulsed-Field/methods , Sepharose , Boric Acids , DNA, Fungal/isolation & purification , Edetic Acid , Saccharomyces cerevisiae , TromethamineABSTRACT
Large DNA fragments (greater than or equal to 1 kb), separated in low melting temperature SeaPlaque GTG agarose gels, can be enzymatically processed directly in the presence of this agarose (in-gel). Time saving protocols are discussed for in-gel processing of large DNA fragments in the presence of remelted SeaPlaque GTG agarose, including cloning into pUC18, nick translation, random priming and restriction digestion. These in-gel molecular biology techniques are as efficient as those using DNA recovered from agarose. The effects of UV irradiation, Mg2+ concentration and agarose concentration on selected in-gel protocols are also discussed.
