Important in understanding the regulation of gene transcription is the elucidation of specific protein-DNA interactions that occur in vivo. One strategy for such in vivo footprinting involves the treatment of whole cells with dimethyl sulphate (DMS), which leads to methylation of guanine residues in DNA at the N7 position (1, 2). This N7 atom lies in the major groove and its susceptibility to m...

Polymerase slippage during DNA synthesis by the Klenow fragment of DNA polymerase across A, C, G and T repeats (30 bases) has been studied. Within minutes, duplexes that contain only repeats (30 bp) expand dramatically to several hundred base pairs long. Rate comparisons in a repeat duplex when one strand was expanded as against that when both strands were expanded suggest a model of migrating ...

For the detection of RNA transcripts by RT-PCR, prior removal of genomic DNA must be performed. To remove genomic DNA, RNA is often prepared by DNase I digestion following phenol extraction. Recently, one-tube or onebuffer systems of RT-PCR were developed to prevent loss of RNA and to reduce the risk of contamination (2,6). In these methods, DNase I is added before RT. Taq DNA polymerase prepar...

To investigate the influence of the pyrimidine 2-keto group on selection of nucleotides for incorporation into DNA by polymerases, we have prepared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely 2-amino-5-(2'-deoxy-beta-d-ribofuranosyl)pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy- beta-d-ribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate (d*TTP) respectively. Bot...

The preparation of cDNA libraries usually involves multiple steps, such as isolation of poly A mRNA, synthesis of cDNA, cloning of cDNA into plasmid, and amplification of cloned plasmid. The last step, the amplification of insert DNA fragment after cloned in dephosphorylated plasmid vector, is done in either of two ways. The recombinant DNA can be extracted after amplification in host cells, or...

Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher amplicon yields and distinguished suppression of nonspecific amplification after pre-incubation of PCR mix with quantum dots between 30 ̊C and 56 ̊C. DNA targets were well amplified even ...

DNA binding of the Type 1 DNA polymerase from Thermus aquaticus (Taq polymerase) and its Klentaq large fragment domain have been studied as a function of temperature. Equilibrium binding assays were performed from 5 to 70 degrees C using a fluorescence anisotropy assay and from 10 to 60 degrees C using isothermal titration calorimetry. In contrast to the usual behavior of thermophilic proteins ...

Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is some 10-100-fold inhibi...