Showing papers by "Stratagene published in 1993"

Transgenic systems for in vivo mutation analysis.

Abstract: Transgenic mice carrying shuttle vectors containing the lacI gene as the target permit the in vivo measurement of mutations in multiple tissues and have been used to test the mutagenic effects of several compounds. Tissue-specific and time-dependent responses have been observed, and the spectrum of mutations determined by sequencing allows analysis of the role of expression time in mutagenesis. The results obtained from sequencing analysis have demonstrated spectra paralleling those observed in alternative in vivo assays. In addition to color screening, modifications to this system have permitted direct selection for mutations in the lacI target by a variety of methods. Transgenic rats containing the same lambda/lacI shuttle vector have been developed for inter-species comparison of mutagenesis testing results, which may offer a better understanding of the specific mechanisms involved in mutagenesis at the molecular level in vivo.

Induction of hepatic mutations in lacI transgenic mice

Abstract: Transgenic B6C3F1 and C57BL/6 mice containing a lambda shuttle vector that carries a lacI target and an alpha lacZ reporter gene have been constructed for use in in vivo mutagenesis assays. After chemical treatment of mice carrying the lacI target gene, genomic DNA is isolated and the shuttle vector is recovered by exposing the DNA to lambda phage packaging extracts in vitro. Mutations in the lacI target gene that inactivate the repressor gene allow expression of the alpha lacZ reporter gene, resulting in blue mutant plaques. We have examined the ability of two genotoxic agents, dimethylnitrosamine (DMN) and methylmethane sulfonate (MMS), to induce mutations in these transgenic mice. Both compounds induce a variety of DNA adducts in mouse liver; DMN is a hepatocarcinogen that induces significant hepatic cell proliferation, but MMS is not hepatocarcinogenic and does not induce hepatic cell proliferation. The effects of animal age, differences in strain and dosing regimen, and length of expression time were evaluated. Mice were treated for 5, 14 or 21 days and were sacrificed 1, 8 or 22 days after the final dose to evaluate the effects of increased expression time on mutant frequency in liver. In 3 week old mice, DMN (2 mg/kg/day) produced 10- to 20-fold elevations in mutant frequency that increased with expression time and the number of treatments. In contrast, MMS (20 mg/kg/day) failed to increase the mutant frequency. DMN failed to induce mutations in 6 week old mice at 2 mg/kg/day, but 4 mg/kg/day yielded significant elevations in hepatic mutations. Sequencing results indicate that treatment of mice with DMN produced predominantly C:G-->T:A transitions.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenesis testing using transgenic non-human animals carrying test DNA sequences

Abstract: An assay for monitoring and assessing the mutagenic potential of agents which involves creating transgenic non-human animals carrying a test DNA sequence or sequences that can be quickly recovered and examined for mutations following exposure to one or more suspected mutagenic agents.

Purified thermostable Pyrococcus furiosus DNA ligase

Abstract: Purified thermostable DNA ligase is described that catalyzes template-dependent ligation at temperatures of about 30° C. to about 80° C., and which substantially retains its catalytic ability when subjected to temperatures of from about 85° C. to about 100° C. The thermostable DNA ligase has an estimated molecular weight of 50,000 to 70,000 daltons. A preferred thermostable DNA ligase is described that was isolated from the archaebacteria Pyrococcus furiosus. Also described are plasmid vectors for producing recombinant thermostable DNA ligase.

Oligonucleotide libraries useful for producing primers

Abstract: A library of oligonucleotides is described comprising a plurality of different oligonucleotides each in separate containers. The oligonucleotides are typically of the same length of from about 5 to 10 nucleotides in length, and each oligonucleotide in the library has the same nucleotide sequence of from 1 to 3 nucleotides in length at the 5' terminus of all the oligonucleotides in the library. In addition methods are described for using the oligonucleotide library for producing oligonucleotides of preselected nucleotide sequence for use in DNA sequencing and primer extension reactions.

Method for the directional cloning of DNA

Abstract: A method for directionally cloning an insert DNA fragment into a target sequence using differential phosphorylation is disclosed, Monophosphorylated PCR fragments are directionally cloned into a monophosphorylated plasmid, Methods for directionally cloning non-PCR fragments into target DNA sequences are also discussed.

Polynucleotide amplification mycoplasma assay, primers, and kits therefore

Abstract: The invention described herein consists of methods and materials for the detection of mycoplasma infections. Mycoplasma infections may be detected in cell cultures and in animals. The subject methods use polynucleotide primer pairs that are capable of hybridizing to mycoplasma tRNA genes so as to provide for the generation of a distinctive set of amplification products when the primers are used in a cyclic amplification synthesis reaction, such as PCR (polymerase chain reaction). In addition to detecting mycoplasma infections, the subject methods may be used to identify the particular species of mycoplasma causing the infection. The subject invention also provides for primers and kits for performing the subject methods.

Method of double stranded DNA synthesis

Abstract: The present invention provides an improved method for the synthesis of double stranded DNA, particularly complementary DNA for the construction of directional complementary DNA libraries. The method comprises synthesizing a first strand of DNA complementary to a selected RNA or DNA template by contacting with the template a linker/primer comprising a selected restriction site, a suitable RNA or DNA dependent DNA polymerase, and substrates comprising a deoxyribonucleotide triphosphate analog. The linker/primer and deoxyribonucleotide triphosphate analog are selected such that incorporation of the nucleotide analog in the first strand substantially protects the double stranded DNA from cleavage, under conditions sufficient to cleave or substantially cleave the linker/primer, at the selected restriction site.

Nucleic acid construct encoding a nuclear transport peptide operatively linked to an inducible promoter

Abstract: A system for regulating expression of eukaryotic genes in cells is described. The system contains two recombinant DNA molecules, a first molecule that encodes a nucleus-targeted inducible repressor polypeptide, and a second molecule that encodes an operator-regulated reporter polypeptide. Transgenic animals containing the system, and methods for using the system are also described.

Push column chromatography method

Abstract: A method for chromatography of DNA, RNA, proteins and other molecules includes the use of a column adapted to hold a chromatography material and a sample to be filtered. A pneumatic pressure differential is applied across the column and the sample is urged through the chromatography material. A selected portion of the sample may then be collected.

Directional cloning of blunt-ended PCR products.

Abstract: A method that allows the directional cloning of blunt-ended polymerase chain reaction (PCR) fragments is described One PCR primer must be 5' phosphorylated Extra bases are not required on either PCR primer A linearized vector is enzymatically processed to contain a single 5'-terminal phosphate The monophosphorylated vector is amenable to recombinant-insertion during ligation when the fragment is in the correct orientation Increased recombinant yield results from incubating the monophosphorylated vector with a restriction enzyme (SrfI) that relinearizes nonrecombinant plasmids during the ligation reaction

Push column and chromatography apparatus

Abstract: An apparatus for chromatography of DNA, RNA, proteins and other molecules includes the use of a column adapted to hold a chromatography material and a sample to be filtered. A pneumatic pressure differential is applied across the column and the sample is urged through the chromatography material. A selected portion of the sample may then be collected.

TRT1 polynucleotides, host cells and assays

Abstract: A rapid method for generating a set of discrete DNA amplification products characteristic of a genome as a “fingerprint” comprises the steps of: priming target nucleic acid of a genome or from a cellular RNA preparation with an single-stranded primer to form primed nucleic acid such that a substantial degree of internal-mismatching occurs between the primer and the target nucleic acid; amplifying the primed nucleic acid by performing at least one cycle of polymerase chain reaction amplification; and amplifying the product of step (2) by performing at least about 10 cycles of polymerase chain reaction amplification. The method is known as the arbitrarily primed polymerase chain reaction (AP-PCR) method and is suitable for the identification of bacterial species and strains, mammals and plants. The method of the present invention can identify species, cell types or tissues rapidly, and does not require knowledge of the nucleotide sequence or other molecular biology of the nucleic acids of the organisms to be identified. The polynucleotide sequence LF9.5m, associated with normal growth of ovary cells, and the polynucleotide TRT1, associated with arrested cell growth, are specifically provided.