Abstract: The subject invention provides novel compositions containing a mixture of (a) an enzyme that possesses substantial 3'-5' exonuclease activity (b) a DNA polymerase with less 3'-5' exonuclease activity than the enzyme with substantial 3'-5' exonuclease activity. Preferably, the DNA polymerase for inclusion in the compositions are DNA polymerases that substantially lack 3'-5' exonuclease activity. A preferred embodiment of the invention is a composition comprising the Taq DNA polymerase (isolated from Thermus aquaticus) and the Pfu DNA polymerase (isolated from Pyrococcus furiosus). Another aspect of the invention is to provide methods for synthesizing polynucleotides, typically DNA, using compositions comprising an enzyme that possesses substantial 3'-5' exonuclease activity and a DNA polymerase with less 3'-5' exonuclease activity than the enzymes possessing substantial 3'-5' exonuclease activity, preferably a DNA polymerase that substantially lacks 3'-5' exonuclease activity.

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.

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 end 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, including Staphylococcus and Streptococcus species, mammals and plants.

Abstract: A method of irradiating a biological specimen with ultraviolet, in particular a polynucleotide specimen selected from DNA or RNA, or optionally a protein. In the case where the specimen is DNA or RNA, or potentially proteins, the specimen is irradiated to cross-link the specimen to a substrate. In the case where the specimen is DNA, the specimen can also be irradiated to form thymine dimers. The method uses an apparatus which permits relatively precise control of the total ultraviolet dose received by the specimen, despite any changes of ultraviolet flux from the lamps which may occur from during any one experiment, or between a number of experiments. Thus, the method allows relatively highly reproducible results to be obtained.

Abstract: An improved method of determining the nucleotide base sequence of DNA is disclosed. The method comprises preparing a DNA substrate comprising a set of molecules, each having a template strand and a primer strand, wherein the 3' ends of the primer strands terminate at about the same nucleotide position on the template strands of the molecules within each set. DNA synthesis is induced to obtain labeled reaction products comprising newly synthesized DNA complementary to the template strands using the 3' ends of the primer strands to prime DNA synthesis, labeled nucleoside triphosphates, and at least one modified nucleoside triphosphate, wherein the modified nucleoside triphosphate is selected to substantially protect newly synthesized DNA from cleavage. The labeled reaction products are cleaved at one or more sites to obtain labeled DNA fragments wherein the newly synthesized DNA is substantially protected from cleavage at the selected site or sites.

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.

Abstract: A method of (and apparatus for) electrophoresis in which the gel temperature is controlled automatically. A sensor may measure a gel temperature and a processor may control a power supply for the electrophoresis process in response to that measurement. Measurements may be made at predetermined times (e.g., periodically) and the processor may determine control settings for the power supply based on a preferred temperature for the electrophoresis process. The preferred temperature for the electrophoresis process may be a predetermined temperature which is chosen so that the electrophoresis process operates at or near the highest speed which is unlikely to break the glass plates.

Abstract: A method of irradiating a biological specimen with ultraviolet, in particular a polynucleotide specimen selected from DNA or RNA, or optionally a protein. In the case where the specimen is DNA or RNA, or potentially proteins, the specimen is irradiated to cross-link the specimen to a substrate. In the case where the specimen is DNA, the specimen can also be irradiated to form thymine dimers. The method uses an apparatus which permits relatively precise control of the total ultraviolet dose received by the specimen, despite any changes of ultraviolet flux from the lamps which may occur from during any one experiment, or between a number of experiments. Thus, the method allows relatively highly reproducible results to be obtained.

Abstract: Vectors are described that circumvent traditional DNA cloning and subcloning procedures, and that contain a unique DNA cartridge that permits both cloning of DNA directly into DNA sequences present within the cartridge, and in vivo removal and circularization of the cartridge thereby yielding an autonomously replicating structure. Because the DNA cartridge can include a wide variety of functional DNA sequences, the cloned DNA can be subjected to a plethora of molecular biological procedures without having to remove the cloned DNA from the cartridge thereby obviating the need to perform additional subcloning techniques. A particularly useful example of this type of vector is bacteriophage lambda containing the DNA cartridge.

Abstract: A method for detecting a new Gaucher disease mutation in an allele in a human having an insertion mutation of a guanine nucleotide adjacent to nucleotide position 57 in the normal glucocerebrosidase gene exon 2 is provided. Identification of the mutation is accomplished by first amplifying, with a polymerase chain reaction (PCR) primer, a region of human genomic DNA containing nucleotide positions 57 and 58 of glucocerebrosidase gene exon 2 followed by detection of the mutation.

Abstract: Vectors are described that circumvent traditional DNA cloning and subcloning procedures, and that contain a unique DNA cartridge that permits both cloning of DNA directly into DNA sequences present within the cartridge, and in vivo removal and circularization of the cartridge thereby yielding an autonomously replicating structure. Because the DNA cartridge can include a wide variety of functional DNA sequences, the cloned DNA can be subjected to a plethora of molecular biological procedures without having to remove the cloned DNA from the cartridge thereby obviating the need to perform additional subcloning techniques. A particularly useful example of this type of vector is bacteriophage lambda containing the DNA cartridge.

Abstract: An apparatus and method for the transfer of macromolecules from a source medium to a transfer medium employ positive pressure to urge a sample from the source medium to the transfer medium. The source and transfer media are disposed in adjacent relation, a positive pressure such as air pressure, fluid pressure, air and fluid pressure or mechanical pressure is applied against the source medium, and the molecule is recovered from the transfer medium.