The rDNA genes coding for ribosomal RNA (rRNA) in animals are repeat sequences with high GC content and complicated structure. Based on the sequences of human ribosomal DNA repeat unit and transcription unit and the long and accurate PCR method with LA Taq DNA polymerase and GC buffer, we were able to amplify the complicated repeat sequences of rDNA genes in Bos taurus. Three rDNA genes and 2 i...

Polymerase chain reaction (PCR) is an important molecular biological tool for the amplification of nucleic acids. PCR process can be divided into three phases according to the amplification rate: exponential, quasi-linear, and plateau. We investigated the cause of the plateau phenomenon through real-time monitoring of the amplification profile and computerized simulation. Possible limiting comp...

We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease HinfI, using a simple PCR procedure to synthesize DNA of known sequence in which every cytosine is methylated at the 5 position. We find that HinfI cannot digest cytosine methylated DNA at the concentrations normally used in restriction digests. Complete digestion is possible using a vast exce...

Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments. We show that Pfu DNA polymerase polishing of Taq DNA polymerase-generated fragments increases the yield and efficiency of cloning. Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-specific primer, both the presence and orientation...

Two restriction-modification systems have been previously discovered in Thermus aquaticus YT-1. TaqI is a 263-amino acid (aa) Type IIP restriction enzyme that recognizes and cleaves within the symmetric sequence 5'-TCGA-3'. TaqII, in contrast, is a 1105-aa Type IIC restriction-and-modification enzyme, one of a family of Thermus homologs. TaqII was originally reported to recognize two different ...

A highly efficient cloning vector was constructed for cloning PCR products by inserting an 80 bp DNA fragment into pGEM-5zf (+) vector. The Xcm I digestion of this vector gave rise to a 3' overhanging deoxythymidine offering the possibility of cloning PCR products with 3' adenosine overhang created by Taq DNA polymerase. Furthermore, two EcoR I sites were added to the construct for identificati...

Although the thermophilic bacterium Thermus aquaticus grows optimally at 70 degrees C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20-37 degrees C. This activity is a bane to some PCRs, since it catalyzes non-specific priming. We report mutations of Klentaq (an N-terminal deletion variant) DNA polymerase that have markedly reduced activity at 37 deg...

Polymerase chain reaction (PCR) methodology (1) has become a routine method for selectively amplifying segments of DNA from a wide variety of sources. Amplification of specific sequences is dependent upon an exact match between the template DNA and the oligonucleotide primers. Mismatches at the 3' terminus lead to greatly reduced amplification, with no detectable product when amplified under th...

Amplification of genomic sequences flanked by delta elements of retrotransposons TY1 and TY2 is a reliable method for characterization of Saccharomyces cerevisiae strains. The aim of this study is to evaluate the usefulness of microfluidic electrophoresis (Caliper LabChip) to assess the factors that affect interlaboratory reproducibility of interdelta sequence typing for S. cerevisiae strain de...