MATERIALS 10x Amplification buffer Include 0.01% (w/v) gelatin in the buffer. 10x TBE electrophoresis buffer Use TBE at a working strength of 1x (89 mM Tris-borate, 2 mM EDTA) for polyacrylamide gel electrophoresis. 6x Gel-loading buffer I Formamide loading buffer Human genomic DNA to be screened for point mutations Dissolve the DNA at 10 μg/ml in TE (pH 7.6). Oligonucleotide primers, forward a...

We reported a new approach of ABO genotyping by a polymerase chain reaction and restriction fragment length polymorphism method. Instead of amplifying the loci containing the positions of nucleotides 258 and 700 of cDNA of the A transferase separately, we successfully amplified these 2 loci together in one reaction mixture using 2 sets of primers. The amplified DNA products were digested at the...

DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris-HCl...

AIMS To investigate the associations between the Rsa I, Dra I, and Taq I genetic polymorphisms of cytochrome p4502E1 and susceptibility to alcoholic liver disease or to hepatocellular carcinoma. METHODS DNA samples isolated from 61 patients with alcoholic liver disease, 46 patients with hepatocellular carcinoma, and 375 healthy controls were subjected to polymerase chain reaction amplificatio...

Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold DNA Polymerase, in multiplex and time-release...

We describe a method for direct cycle sequencing of PCR fragments amplified from genomic DNA or cDNA. DNA sequencing template is amplified using PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent sequencing primers, Sanger dideoxy sequencing chemistry and an enzyme mixture of a mutant Taq DNA polymerase and therm...

The Stoffel fragment of Taq DNA polymerase lacks the 5' to 3' exonuclease activity that hydrolyzes potentially blocking DNA strands during primer extension. We there-fore asked whether by using this fragment in the PCR, non-extendable, base-paired oligonucleotides could inhibit amplification in a sequence-dependent manner. Model targets were chosen from the partially conserved ribosomal 16S rDN...

To detect occult lymphoma, the polymerase chain reaction (PCR) technique was used to amplify joined bcl-2/JH DNA sequences at the juncture of the t(14:18) translocation in follicular lymphoma. Using the heat-stable DNA polymerase Taq and automated cycling of the reaction, we were able to detect as few as one to two copies of bcl-2/JH. Under these conditions, PCR proved to be at least 10,000-fol...

The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S rRNA genes. The method was used prior to a PCR that amplified 10 f...