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L Ulanovsky

Publications and source records attributed to L Ulanovsky.

6 recordsLinked to original sources
PubMed1999

DENS (differential extension with nucleotide subsets): application to the sequencing of human genomic DNA and cDNA.

Here we describe further development of our method of DNA sequencing by Differential Extension with Nucleotide Subsets (DENS) and its application to the sequencing of human genomic DNA and full-insert cDNA. Essentially, DENS is primer walking without custom primer synthesis; instead, DENS uses a presynthesized library of octamer primers degenerate in two positions (4,096 tubes/sequences for a complete library). DENS converts an octamer selected from this library into a long primer on the template, at the intended site only. This is done using a two-step procedure which starts with a limited extension of the octamer (at 20 degrees C) in the presence of only two of the four possible dNTPs. The primer is extended by five bases or more at the intended priming site, which is deliberately selected to maximize the extension length (as are the two-dNTP set and the primer itself). The subsequent termination reaction at 60 degrees C then accepts the primer extended at the intended site, but not at alternative sites, where the initial extension (if any) is generally much shorter. This paper presents a set of rules for selection of DENS priming sites. We also compare different ways of template preparation for DENS sequencing. The data were obtained from primer walking on three human genomic DNA subclones of 3 to 4 kbp and four cDNA clones containing inserts of 1.9, 2.3, 3.8, and 4.9 kbp. Full-length sequences were obtained from both strands of each subclone by automated dye-terminator fluorescent DNA sequencing using DENS with degenerate octamer primers. We compared the following types of DNA templates: single-stranded and double-stranded phagemid DNA, double-stranded PCR products, asymmetric PCR products, and single-stranded DNA produced by digestion with Lambda Exonuclease of double-stranded PCR product phosphorylated at one end (Exo-PCR). While all of the preps were found to work, the best results were obtained with Exo-PCR and phagemid single-stranded DNA. Exo-PCR directly from overnight bacterial culture with no plasmid prep of any kind yielded templates for DENS as good as Exo-PCR from purified DNA. We found that the Tm of the differentially extended octamers is an important factor in the success of DENS. Clustering of successful reactions was clearly distinguished in the Tm range of 50-66 degrees C, with success rates of 70% for Exo-PCR and 65% for ss phagemid templates.

Base Sequence↗
PubMed1994

DNA sequencing: modular primers for automated walking.

Here we describe our progress in the development of the technology of DNA sequencing by primer walking based on the modular primer approach, which eliminates the primer synthesis bottleneck. Modular primers are assembled from 5-mers, 6-mers, or 7-mers selected from a presynthesized library of as few as 1000 oligonucleotides. This technology is expected to reduce the time per walk by a factor of 20 to 50, and the cost of DNA sequencing by a factor of 5 to 15. Both time and expense will be saved not only on the primer synthesis per se but, more importantly, as a result of the closed-end automation of the complete cycle of walking sequencing, where no human intervention will be required between the walks. Such closed-end automation has until now been precluded due to the need to synthesize a new primer for each walk. As a part of the closed cycle automation development, this report deals with modular primers used for sequencing with fluorescent dye terminators. The success rate and the quality of automated sequencing with modular primers are found similar to those with the conventional 17-20-base-long primers. One of the advantages of the described technique is simple purification without any proteins that need to be removed.

Base Sequence↗
PubMed1990

DNA trapping electrophoresis.

Attempts to improve the size separation of single-stranded DNA in polyacrylamide gels by field-inversion gel electrophoresis (FIGE) have met with limited success. Here we show that attaching a neutral globular protein, streptavidin, to one end of a single-stranded DNA molecule profoundly alters the DNA mobility pattern and increases the band separation manyfold within a size range controlled by voltage and pulse cycle. In constant field, short modified fragments are only slightly retarded but long molecules are retarded dramatically above a 'threshold size' of 0.6 kilobases at 60 V per cm. At this voltage, molecules above a 1.2-kilobase 'cut-off' do not enter the gel. Both the threshold and the cut-off sizes decrease as the voltage increases. In FIGE, the longer the reverse pulse, the larger the modified fragments that enter the gel. We interpret these results as the trapping by the gel matrix of the protein attached to the DNA. The probability of release then depends on the balance between the electric field and thermal motion: the larger the DNA and the higher the voltage, the harder it is to release.

DNA↗
PubMed1986

Curved DNA: design, synthesis, and circularization.

Curved DNA molecules and unusually small circles have been obtained by ligation of synthetic 21-base precursors: (sequence in text). The ligation resulted in the formation of double-stranded oligo-(precursor)s possessing a strong 10.5-base-pair (bp) periodicity of the runs of adenines. Two-dimensional polyacrylamide gel electrophoresis of the ligation products showed two distinct families of spots: (i) noncircular oligo(precursor)s of 21 to 231 bp (1- to 11-mers) and (ii) four circles from 105 to 168 bp (eluted and analyzed by denaturing gel electrophoresis). The noncircular oligomers exhibited anomalously slow migration, as if they were as much as three times longer than they actually are. The amount of circular products peaked sharply at approximately equal to 126 bp, near which size the circles have been estimated to be nonconstrained both torsionally and in terms of bending. The nonconstrained circularization provides a technique for the direct measurement of the inherent curvature of DNA in solution. From the size of the circles, an estimate of 8.7 degrees is obtained for the absolute value of the AA X TT wedge angle (roll and tilt combined).

DNA↗