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Biomedical subjects

L S Lerman

Publications and source records attributed to L S Lerman.

At least 19 recordsLinked to original sources
PubMed1991

The effects of lithium, rubidium, cesium and magnesium ions on the close packing of persistence-length DNA fragments.

By application of scaled particle theory to persistence-length DNA fragments in sedimentation-equilibrium at speeds high enough to maintain close packing, the range of interhelical electrostatic repulsion was evaluated with LiCl, RbCl, CsCl, and MgCl2 as supporting electrolytes. Analysis of the data in terms of the Zimm cluster function confirmed that the net interaction between helices is purely repulsive in all cases. At constant ionic strength the electrostatic radius of the rod-like DNA decreases as the counterion changes from Li+ to Rb+ to Cs+. In contrast to univalent counterions, electrostatic radius increases with Mg2+ concentration, except at very low (mM) MgCl2 concentrations. All solutions undergo a reversible transition to a turbid, optically anisotropic phase at a slightly salt-dependent, critical DNA concentration, as observed previously for NaDNA.

Animals↗
PubMed1990

Comprehensive detection of single base changes in human genomic DNA using denaturing gradient gel electrophoresis and a GC clamp.

We present a simple, efficient extension of denaturing gradient gel electrophoresis that allows the detection of nearly any sequence change in a defined fragment of DNA. The fragment can be obtained either by means of the polymerase chain reaction or by restriction digestion of genomic DNA. With restriction fragments of genomic DNA, sequence information is not required, and covalent modifications in genomic DNA that are lost in a PCR, such as methylation, are detectable. We describe how a GC clamp (an arbitrary, G+C-rich sequence of 30 to 60 bp) can be attached to a selected restriction fragment present in a digest of genomic DNA. The GC clamp alters the melting properties of the fragment; this change greatly increases the fraction of possible mutations that is detectable. In a 272-bp HaeIII fragment from the human beta-globin gene, we were able to detect 13 of 13 mutations tested in human genomic DNA. Four additional mutations in cloned plasmids were analyzed. The data agree with a simple theoretical model for DGGE, which predicts how two fragments, differing at a single (specified) base pair, are resolved in a gradient gel as a function of running time for the gel. The calculation assists in the design of probes and gel conditions that aid in the detection of sequence changes.

Base Composition↗
PubMed1989

Relaxation effects in the gel electrophoresis of DNA in intermittent fields.

The electrophoretic mobility of restriction fragments of lambda DNA in agarose gels declines if the field is intermittent rather than continuous, with a greater effect on the longer fragments. The changes are compatible with the assumption of two exponential relaxation processes for field-dependent configurational changes, one when the field is turned on and another when it terminates. The length dependence at the extrapolated limit of mobility for short pulses with long intervals corresponds closely to the simple inverse proportionality to length expected from theoretical considerations when the molecular configuration is not affected by the electric field. Simple intermittent fields would allow separation of longer molecules than can ordinarily be resolved. The relaxation times for both the change in conformation imposed by the field and the return to field-free conformation vary as approximately the second power of the length of the molecule, independent of the salt concentration or field strength and varying only slightly with gel density. These relations are not in good agreement with properties expected from reputation theory, and they suggest that a different mechanism must be invoked for the electrophoretic migration of long DNA molecules at ordinary values of field strength.

Bacteriophage lambda↗
PubMed1989

Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes.

Denaturing gradient gel electrophoresis (DGGE) can be used to distinguish two DNA molecules that differ by as little as a single-base substitution. This method detects approximately 50% of all possible single-base changes in DNA fragments ranging from 50 to approximately 1000 base pairs. To increase the number of single-base changes that can be distinguished by DGGE, we used the polymerase chain reaction to attach a 40-base-pair G + C-rich sequence, designated a GC-clamp, to one end of amplified DNA fragments that encompass regions of the mouse and human beta-globin genes. We show that this GC-clamp allows the detection of mutations, including the hemoglobin sickle (HbS) and hemoglobin C (HbC) mutations within the human beta-globin gene, that were previously indistinguishable by DGGE. In addition to providing an easy way to attach a GC-clamp to genomic DNA fragments, the polymerase chain reaction technique greatly increases the sensitivity of DGGE. With this approach, DNA fragments derived from less than 5 ng of human genomic DNA can be detected by ethidium bromide staining of the gel, obviating the need for radioactive probes. These improvements extend the applicability of DGGE for the detection of polymorphisms and mutations in genomic and cloned DNA.

Animals↗
PubMed1985

A general method for saturation mutagenesis of cloned DNA fragments.

A new procedure for generating and isolating random single-base substitutions in cloned DNA fragments is presented. The mutations are generated by treatment of single-stranded DNA with various chemicals, followed by the synthesis of the complementary strand with reverse transcriptase. Misincorporation frequently occurs when the enzyme encounters a damaged base in the mutagenized template DNA. The resulting duplex DNA fragments containing random single-base substitutions are cloned, amplified as a population, and isolated from wild-type DNA by preparative denaturing gradient gel electrophoresis. The physical separation of mutant DNA fragments makes it possible to isolate and characterize large numbers of site-directed single-base substitutions in the absence of a phenotypic selection. This procedure should be generally applicable to the fine-structure genetic analysis of regulatory and protein-coding sequences.

Animals↗
PubMed1985

Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis.

The melting behavior of a DNA fragment carrying the mouse beta maj-globin promoter was investigated as a means of establishing procedures for separating DNA fragments differing by any single base substitution using the denaturing gradient gel electrophoresis procedure of Fischer and Lerman (1,2). We find that attachment of a 300 base pair GC-rich DNA sequence, termed a GC-clamp, to a 135 bp DNA fragment carrying the mouse beta-globin promoter significantly alters the pattern of DNA melting within the promoter. When the promoter is attached to the clamp, the promoter sequences melt without undergoing strand dissociation. The calculated distribution of melting domains within the promoter differs markedly according to the relative orientation of the clamp and promoter sequences. We find that the behavior of DNA fragments containing the promoter and clamp sequences on denaturing gradient polyacrylamide gels is in close agreement with the theoretical melting calculations. These studies provide the basis for critical evaluation of the parameters for DNA melting calculations, and they establish conditions for determining whether all single base substitutions within the promoter can be separated on denaturing gradient gels.

Animals↗
PubMed1985

Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis.

Duplex DNA fragments differing by single base substitutions can be separated by electrophoresis in denaturing gradient polyacrylamide gels, but only substitutions in a restricted part of the molecule lead to a separation (1). In an effort to circumvent this problem, we demonstrated that the melting properties and electrophoretic behavior of a 135 base pair DNA fragment containing a beta-globin promoter are changed by attaching a GC-rich sequence, called a 'GC-clamp' (2). We predicted that these changes should make it possible to resolve most, if not all, single base substitutions within fragments attached to the clamp. To test this possibility we examined the effect of several different single base substitutions on the electrophoretic behavior of the beta-globin promoter fragment in denaturing gradient gels. We find that the GC-clamp allows the separation of fragments containing substitutions throughout the promoter fragment. Many of these substitutions do not lead to a separation when the fragment is not attached to the clamp. Theoretical calculations and analysis of a large number of different mutations indicate that approximately 95% of all possible single base substitutions should be separable when attached to a GC-clamp.

Animals↗
PubMed1979

Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis.

When double helical DNA is exposed to conditions favoring partial melting in polyacrylamide gels, its electrophoretic mobility undergoes a sharp cooperative transition, resulting in a large reduction in mobility. In the present experiments, where the transition is effected at a uniform temperature of 60 degrees C in a concentration gradient of a urea-formamide mixture, each Eco RI fragment of lambda or E. coli DNA exhibits the mobility transition at a characteristic concentration of the denaturant. The sudden retardation of fragments moving toward higher denaturant concentration in the gradient results in a pattern of sharpened zones in order depending upon nucleotide sequence, rather than size, and only very slightly dependent upon the time after the last fragment has been retarded. When combined with length-dependent electrophoresis in agarose in the perpendicular direction, this system provides a two-dimensional separation of fragments. The resolving power of the system is demonstrated by the clear resolution of over 250 fragments of the Eco RI digest of E. coli DNA. Corresponding fragments from an isogenic lambda lysogen of E. coli are found in the same positions, and additional fragments unique to the lysogen are evident.

Coliphages↗