Showing papers in "Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society in 1990"

Modifications to improve the effectiveness of restriction fragment length polymorphism typing.

Abstract: A streamlined and effective method for RFLP analysis of DNA has been developed. Southern transfers are accomplished by alkali blotting DNA onto positively charged nylon membranes. The prehybridization step has been eliminated. The hybridization solution is composed of three cost-effective reagents: 7% SDS, 10% PEG, and phosphate buffer. By using probes that hybridize to variable number of tandem repeat loci, and RFLP analysis of one to two micrograms of genomic DNA can be achieved within five working days under normal working hours. With longer autoradiographic exposures, as little as 20-100 ng of human genomic DNA is sufficient for analysis.

Identification of haptoglobin alpha-2FF variants in mid-trimester maternal serum as potential markers for Down syndrome.

Abstract: Exploratory protein analysis in mid-trimester maternal sera by 2-dimensional electrophoresis and image analysis was performed to determine the differences between mothers carrying fetuses with Down syndrome (DS) and control mothers. Nine haptoglobin alpha-1 and alpha-2 (Hp1 and Hp2) variants were detected. Three apparent isoforms of the Hp2FF protein having the same relative charge but different Mr's were detected, with the two smaller variants--Hp2FF (-0.7k) and Hp2FF (-1.3k)--believed to be first reported here. Ninety-two percent of the cases (n = 83) had the Hp2FF (-0.7k) protein, and 61% had Hp2FF (-1.3k). The geometric mean concentration of Hp2FF (Mr = 18.0 kd) was found to be significantly increased (P less than 0.01) in mid-trimester sera from cases (n = 27) compared to controls (n = 56). When the concentrations of the three Hp2FF isoforms were added, the cases had larger sums on average than controls (P less than 0.01).

Increased migration rate observed in DNA from evidentiary material precludes the use of sample mixing to resolve forensic cases of identity.

Abstract: The analysis of restriction fragment length polymorphisms in forensic DNA samples can be used to determine whether any two or more samples have the same biological origin. However, sometimes DNA recovered from evidentiary material, such as blood or semen stains, migrates at a different rate than an exemplar sample. This difference in migration, while maintaining the same overall pattern, produces a shift in the position of the bands. To verify that a shift in migration has occurred between evidence and exemplar samples, we have utilized two DNA probes that recognize DNA fragments that do not vary in size between individuals (monomorphic). The results obtained with this type of internal control show that differences in migration rate between exemplar and evidentiary samples can be recognized and accounted for and do not affect the ability to decide whether two patterns match. A common practice in many analytical tests, to show identity between two samples, is to test the properties of the samples individually and mixed. However, this approach is not applicable to all forensic DNA identity tests. In many cases, DNA from forensic samples may be irreversibly modified and this can alter the migration rate of the DNA samples. Thus, in a mixture of DNA from exemplar and evidence, the same polymorphic DNA fragments may not comigrate and produce a composite pattern which could lead to false exclusions.

Detection and recovery of proteins from gels following zinc chloride staining.

Abstract: This report describes a method for detecting proteins in SDS polyacrylamide gels using ZnCl2 and their recovery using passive elution. Washing unfixed gels in a dilute solution of ZnCl2 produces two desirable effects. First, it makes the proteins easily visible as clear zones in a white background, and second, it prevents loss of proteins due to diffusion into the wash solution. Compared to other commonly used methods of protein detection such as Coomassie, KCl, copper or silver staining, the zinc stain offers some distinct advantages. Zinc staining can be completed in 15 min for most applications, making it much faster than Coomassie or most silver stains. The zinc stain is more sensitive than Coomassie, KCl or copper stain. Since no harsh chemical conditions are used in the zinc staining procedure, recovery of proteins from the gel is facilitated. More than 90% of selected proteins were recovered from 2-D gels by simple elution from the gel pieces with a buffer containing 10 mM EDTA.

Agarose gel electrophoresis of linear genomic DNA in the presence of ethidium bromide: band shifting and implications for forensic identity testing.

Abstract: We demonstrate that agarose gel electrophoresis of linear duplex DNA in the presence of ethidium bromide has a marked effect on the mobility of genomic DNA fragments detected by Southern hybridization. Mobility shifts of greater than 6% of the actual molecular weight were detected when different amounts of the same DNA sample were analyzed in 1.0% agarose gels containing 0.5 micrograms ml-1 ethidium bromide. For forensic applications, shifts of this magnitude could complicate the task of comparing restriction fragment length polymorphism profiles and introduce a degree of uncertainty to allele frequency population databases.

Denaturing gradient gel electrophoretic analysis of the human cHa-ras1 proto-oncogene.

Abstract: Activation of ras proto-oncogenes frequently involves point mutations at different sites in the gene. Here we describe the application of denaturing gradient gel electrophoresis to identify and characterize such mutations in the cHa-ras1 proto-oncogene. We calculated a melting map of the cHa-ras1 gene and designed optimal conditions to separate mutant from wild type sequences in the first exon. As an example we examined the T24 guanosine to thymidine point mutation in the first exon which has been found in T24 bladder carcinoma cells. Denaturing gradient gel analysis of both homoduplex as well as heteroduplex molecules resulted in separation of the wild type sequence from the mutant sequence. On the basis of the melting map we present a general scheme for screening the cHa-ras1 proto-oncogene sequence for the occurrence of point mutations.

Affinity electrophoresis in agarose gels. Theory and some applications.

Abstract: Affinity electrophoresis is the electrophoresis of components which interact during the electrophoresis procedure. Among the numerous modifications of the basic principle, affinity electrophoresis in agarose gels combined with subsequent immunochemical detection (crossed affinity immunoelectrophoresis) has emerged as a useful means of characterizing biospecific macromolecular interactions. Demonstration of ligand-binding proteins, enumeration of binding sites, evaluation of microheterogeneous forms, and estimation of binding constants can be achieved with this procedure even when analyzing small amounts of unpurified material. With an emphasis on interactions between lectins and glycoproteins as model systems, the principles and applicability of crossed affinity immunoelectrophoresis are exemplified. This includes examples of modifications developed for screening for lectins in plant extracts, for estimation of the binding capacity of affinity matrices, and for the calculation of dissociation constants.

Acid depurination after field inversion agarose gel electrophoresis reduces transfer of large DNA molecules

Abstract: Field-inversion gel electrophoresis (FIGE) is an economical method for the resolution of DNA fragments 20 to 1000 kilobase pairs (kbp) in length, sizes beyond the resolving capabilities of normal agarose gel electrophoresis. A theoretical limitation to FIGE is the subsequent transfer of large DNA molecules to membrane supports for hybridization. After normal electrophoresis, uniform and rapid capillary transfer of DNA fragments from agarose gels has been previously reported to be facilitated by brief depurination of DNA with 0.25 N HCl. However, after FIGE we have found that brief treatment of large (greater than 200 kbp) linear DNA fragments with 0.25 N HCl reduces the extent of subsequent transfer by capillary methods. After FIGE and transfer, ethidium bromide staining of DNA suggests that acid treatment causes trapping of DNA within the agarose matrix.

Gel and buffer effects on the migration of DNA molecules in agarose.

Abstract: The migration rate of linear and supercoiled DNA molecules in four brands of agarose from two different lots was compared using three buffer systems. Differences in DNA mobility were observed between brands and lots of agarose, in standard as well as in pulsed-field gel electrophoresis. A buffer-related effect on the migration of DNA molecules in agarose gels was also observed. Our results underline the need for an actual lot analysis of agarose, this extraction product varying from one preparation to another in several aspects.

Serum autoantibodies in type 1 (insulin-dependent) diabetes mellitus: identification of reactivity against a 29 kd pancreas-specific protein.

Abstract: Serum samples from patients with type 1 (insulin-dependent) diabetes mellitus and controls were incubated with two-dimensional Western immunoblots of pancreas and other tissues. Two out of 26 (8%) of the diabetics and 0 out of 45 of the controls demonstrated reactivity against four pancreas-specific proteins with identical molecular weights of 29,000 daltons and different isoelectric points ranging from pH 7.0-8.0. It is concluded that 29 Kd autoantibody reactivity is not a major marker for type 1 diabetes, but may help identify a subgroup of type 1 diabetics.

Visualization of alkaline phosphatase-labelled antibodies on immunoblots by means of formazan staining using indoxyl phosphate and thiazolyl blue

Abstract: Alkaline phosphatase-conjugated secondary antibodies on blotting membranes were visualized by means of a modified formazan staining method (Hodson & Skillen 1988). It is based on the standard 5-bromo-4-chloro-3-indolyl phosphate/Nitroblue tetrazolium method, but employs different buffer conditions and thiazolyl blue instead of Nitroblue. It gave a sensitivity of 1 ng albumin in a 12 mm2 dot and less background staining on dot blots than the standard method. The new protocol provides an advantage with regard to ease of staining and preparation of reagents while it equals the former method with respect to sensitivity.

Development of a high-performance electrophoretic light scattering apparatus for mobility determination of particles with their Stokes' radii of several nanometers.

Abstract: A sophisticated electrophoretic light scattering apparatus was developed. It allows reliable measurements of electrophoretic mobilities of charged macromolecules, such as proteins, nucleic acids and synthetic polyelectrolytes which have been out of the range of the apparatuses of this type aimed at relatively large particles such as microspheres and biological cells. The present apparatus features the monitoring of the electroosmotic flow profile and the determination of the sign of the electrophoretic mobility of an object as well as its high-performance based on the improvement in various facets of acquisition and handling of the signals and data.