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H B Strack

Publications and source records attributed to H B Strack.

11 recordsLinked to original sources
PubMed1992

In vitro amplification of DNA fragments greater than 10 kb.

The synthesis of a 10.9-kb DNA fragment from a bacteriophage lambda template was used in the search for conditions to extend the range for the polymerase chain reaction (PCR). Using the same primer sequences and conditions (denaturation at 94 degrees C, 1 min; annealing at 57 degrees C, 1 min; polymerization at 70 degrees C, 20 to 30 min) as published by W. Rychlik, W. J. Spencer, and R. E. Rhoads [(1990) Nucleic Acids Res. 18, 6409-6412], unsatisfactory results were obtained with AmpliTaq and native Taq polymerase (poor reproducibility, low product yield, nonspecific products), whereas Tub polymerase completely failed to amplify this fragment. Only after changes in the following parameters were reliable results obtained but only with Tub polymerase: A two-step PCR procedure with primer annealing and extension at 65 degrees C followed by DNA denaturation at 94 degrees C for 1.5 min was performed. The DNA fragment desired was specifically amplified when the enzyme concentration was reduced to 0.4 U/50 microliters and extension times as low as 4 to 12 min with an optimum at 8 min were used. A prolongation to 20 min or more resulted in an accumulation of unspecific products with a concomitant reduction in the yield of the fragment. Under the conditions described above it was also possible to amplify a DNA fragment even significantly longer (15.6 kb).

Bacteriophage lambda↗
PubMed1989

A modified primer extension procedure for specific detection of DNA-RNA hybrids on nylon membranes.

We have developed a modified primer extension procedure for specific detection of mRNA. Alkali-fragmented total cellular RNA or some RNA fraction is hybridized to single-stranded or double-stranded M13 DNA containing the insert of interest which is immobilized on nylon membranes. Hybridized RNA is then detected by incubation of membranes with Escherichia coli RNase H and DNA polymerase I. RNase H is used for nicking the RNA in the hybrids. The resulting 3'-OH groups can subsequently be used by DNA polymerase I to synthesize a labeled complementary strand. The method described is both relatively fast and sensitive and particularly useful for screening large numbers of DNA clones for their representation in RNA populations. Using total cellular RNA as hybridization probe and single-stranded M13 DNA as template as low as 0.25 ng of a specific mRNA was detected (2.5-fold background) when adding 1 microCi [3H]dCTP or 2.5 microCi [32P]d-CTP alternatively as radioactive precursor for the labeling reaction. The detection limit increased to 1 ng (2-fold background) with denatured replicative form double-stranded M13 DNA as template.

Animals↗
PubMed1977

Analysis of the phase variation in lambda reduced immunity lysogens.

Two distinct phases characterized by different levels of immunity that appear in some E. coli strains lysogenic for reduced immunity mutants of bacteriophage lambda are identified as single and tandem double lysogens respectively on the basis of DNA-DNA hybridization experiments and the requirement of the phage xis function for the transition from a single to a double, and of the host recA function for the transition from a double to a single lysogen (in a xis- condition). Rim lysogens with a further increase in immunity, containing some 5 copies of the lambda genome per host genome, have also been observed. It is argued that the different levels of immunity are a direct reflection of the CI gene dosage effect. An unexplained finding is that rim single lysogens yield double lysogens with a frequency of near 1% per generation, whereas cured cells fail to appear even at a frequency 100 times lower.

Cell Division↗