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Biomedical subjects

E Sverdlov

Publications and source records attributed to E Sverdlov.

9 recordsLinked to original sources
PubMed2000

Contiguous strings of strongly binding short oligonucleotides as a useful tool for completing sequencing experiments.

Large-scale DNA sequencing is currently based mostly on the shotgun approach. Although widely used, it is not free of shortcomings: the total length of randomly sequenced subclones appear to be five to eight times more than the total DNA length; such a high statistical redundancy of random sequences cannot guarantee the absence of unsequenced gaps. We calculated that these problems could be alleviated if the average length of the subcloned random fragments were increased. In this paper we present the primer walking approach based on the use of contiguous modified hexamer strings strongly binding to complementary templates as segmented primers. The approach exhibits the following characteristic features: 1) Single-stranded templates appear to have advantage over double-stranded ones. 2) With single-stranded templates, an overall sequencing success rate is about 80%. 3) A relatively small library of 1400 hexanucleotides selected according to the most frequent occurrence in the human genome is sufficient for sequencing all clones. The approach allows one to reduce the number of templates necessary for the shotgun strategy and also to decrease the number of gaps between sequenced contigs. This should be kept in mind when considering the ways for enhancement of the present strategy of large-scale sequencing.

DNA↗
PubMed1999

PCR based targeted genomic and cDNA differential display.

We previously described a targeted genomic differential display method (TGDD: Broude NE, Chandra A, Smith CL. Differential display of genomic subsets containing specific interspersed repeats. Proc. Natl. Acad. Sci. USA 1997;94:4548-53). In that method, presently characterized as method I, targeting was accomplished by capturing DNA fragments containing specific a sequence by hybridization with complementary single-stranded DNA. The captured fragments were amplified by PCR. Here, we describe method II where targeting is accomplished by PCR using primers specific to the target sequence. Method II takes advantage of PCR suppression to eliminate fragments not containing the target sequence (Siebert PDA, Chenchik A, Kellogg DE, Lukyanov KA and Lukyanov SA. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 1995;23:1087-1088). Targeting focuses analysis on and around interesting areas and additionally serves to reduce the complexity of the amplified subset. These approaches are useful to amplify genome subsets containing a variety of targets including various conserved sequences coding for cis-acting elements or protein motifs.

Alleles↗
PubMed1997

Positioning of 72 potentially full size LTRs of human endogenous retroviruses HERV-K on the human chromosome 19 map. Occurrences of the LTRs in human gene sites.

Seventy-two near full size long terminal repeats (LTRs) of human endogenous retrovirus of K-family (HERV-K) have been precisely located on the metric map of human chromosome 19. The LTR-related sequences were identified and assigned to cosmids by hybridization with two independent chromosome 19 specific cDNA clones corresponding to different parts of U3 region of LTR of HERV-K. The presence of full-size LTR sequences in a cosmid was further verified by PCR assay with a pair of primers complementary to the termini of the LTR. Coincidences of the LTR and the known genes positions are discussed.

Base Sequence↗
PubMed1996

Parallel Processing in Genome Mapping and Sequencing

Conventional genome mapping and sequencing involves the analysis and processing of individual samples and pieces of experimental data. Although these methods work, it is quite clear that more efficient and less expensive methods are needed. Our top down physical mapping experiments have focused on the parallel processing of information from multiple samples at one time. This approach has aided the construction of genomic restriction maps and allowed us to assess the degree of large-scale conservation across wide regions of the human genome. The principles of parallel processing were applied in top down experiments that ordered an overlapping cosmid library from the 14-Mb Schizosaccharomyces pombe genome. This approach produced an eight-fold increase in efficiency in clone ordering over similar efforts. Recently, we have developed an enhanced sequencing by hybridization protocol that allows DNA sequence information to be collected on a large number of samples at once. Our current research focuses on applying parallel processing principles to make genome-wide comparisons between pairs of samples for analyzing disease states.

Journal Article↗
PubMed1996

Oligonucleotides containing 2-aminoadenine and 5-methylcytosine are more effective as primers for PCR amplification than their nonmodified counterparts.

Oligonucleotides containing the modified bases 5-methylcytosine and 2-aminoadenine in place of cytosine and adenine, respectively, have higher than normal affinity for complementary sequences The strong binding oligonucleotides (SBO) are much better than their normal counterparts in PCR amplification: they yield significantly more product per cycle, allow amplification at annealing temperatures as high as 72 degrees C and, unlike their normal counterparts, allow efficient priming from within a palindromic sequence. We propose that such strong binding oligonucleotides will be valuable in numerous PCR applications, including: (i) minimization of the frequency of mutants among PCR products; (ii) when only short specific primers can be designed based on available sequence information; (iii) when the material available for the analysis is limited in quantity; and (iv) when primer binding is blocked by DNA secondary structure involving a primer binding site, or chain extension is impeded by secondary structure in downstream sequences.

2-Aminopurine↗
PubMed1996

Factors affecting the priming efficiency of short contiguous oligonucleotide strings in the primer walking strategy of DNA sequencing.

We use modified oligonucleotides with enhanced strength of complementary DNA binding for primer walking DNA sequencing with strings of short contiguous oligonucleotides as primers. Such an approach allows us to reduce the probability of primer failures due to unstable binding of oligos with templates. In this paper the factors affecting the priming efficiency of segmented primers (strings composed of several short oligonucleotides contiguously juxtaposed on the template) used for DNA sequencing were investigated. Modified oligonucleotides were used to discriminate the effects caused by intrinsic properties of the oligonucleotides and by template features. It was shown that the most crucial factor is the stability of the duplex formed by the template with the 3'-outermost oligonucleotide of a string. The data were obtained with a model M13 template and in the process of sequencing the region flanking a long terminal repeat of human endogenous retrovirus HERVK mapped on chromosome 19. The sequencing was done by primer walking with strings of contiguous modified hexanucleotides. The effects revealed should be taken into consideration when choosing oligonucleotide units of segmented primers and for construction of minimised libraries composed of short unit oligonucleotides.

Bacteriophage M13↗
PubMed1995

An arrayed library enriched in hncDNA corresponding to transcribed sequences of human chromosome 19: preparation and analysis.

A simple technique for preparation of libraries of the human chromosome specific transcribed sequences is developed. It uses hnRNA from human-rodent hybrid cell lines containing particular human chromosomes or their fragments and includes three stages: (i) reverse transcription of the hnRNA with Alu-specific primers directing the transcription beyond the Alu-repeats to flanking non-repetitive sequences of the chromosome; (ii) nested primer PCR strategy with specifically designed primers; (iii) direct selective cloning of the second-stage nested primer PCR products. An arrayed hncDNA library was prepared from a hybrid cell line containing chromosome 19 and fragments of 22 and X chromosomes. The library contains around 98% of human-specific transcribed sequences. Sequences of 52 human-specific, according to PCR analysis, clones differed from each other and had no close analogs in the EMBL Data Bank. Of 17 clones assigned to certain human chromosomes, 9 belonged to chromosome 19, 5 to chromosome 22 and 3 to chromosome X. Some of the human specific clones contained repetitive elements scattered over different human chromosomes. Clones from hncDNA libraries are useful as STSs/ESTs, as probes for detecting full-size genes in genomic libraries, for RFLP analysis and for identification of chromosome specific cDNAs.

Animals↗
PubMed1977

Expression of esterases during ontogenesis of the flour beetle Tribolium castaneum (Tenebrionidae; Coleoptera).

Two electrophoretically fast-migrating, nonspecific esterases were detected in two strains of the flour beetle Tribolium castaneum and designated F (fast) and S (slow) according to their relative migration distances. Both isozymes are associated with the alimentary canal and display ontogenetic changes. Their activity is very low in the egg stage, increases in the larva, and declines dramatically in the pharate pupa and pupa. The overall activity in the pupal stage is low, yet increases gradually throughout this period. In the adult, the activity of the esterases rises sharply. The larval and adult F and S isozymes were found to hydrolyze alpha- and beta-naphthylacetate and alpha-naphthylpropionate with almost equal capacity. alpha-Naphthyl laurate was cleaved by the F enzyme of both larvae and adults. The F and S were insensitive to inhibitors of arylesterases and cholinesterases and were markedly inhibited by the organophosphate di-isopropylphosphorofluoridate (DFP) and could be classified as carboxylesterases. Differential sensitivities of larval and adult esterases to urea and heat treatment as well as to DFP may indicate the expression of different genes during metamorphosis.

Animals↗