By Michael M. Burrell
Presents key details on quite a lot of enzymes conventional as instruments in molecular biology, supporting to reduce the time a scientist spends studying the literature to get reactions to paintings successfully and permitting the nonenzymologist to layout an scan. each one bankruptcy supplies heritage info at the enzyme chosen and people parameters vital in its use, describes either the resource and alertness of the enzyme, and offers info at the measurement and constitution of the protein. particular parameters crucial for attaining an optimized response are mentioned, besides exemplary sensible approaches an protocols.
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Additional resources for Enzymes of Molecular Biology (Methods in Molecular Biology Vol 16)
85 Rochhtz, C F , Scott, G. , Dodson, J M , and Benz, C C. (1988) Use of the polymerase chain reaction technique to create base-specific ras oncogene mutations. DNA 12,5 15-5 19 86. , Reiss, A , and Adesmk, M. (1989) Construction of mutant and chimeric genes using the polymerase chain reactton. Nucleic Acids Res 17,723-733. Landgraf and Wolfes 87. Mole, S. , Iggo, R. , and Lane, D. P. (1989) Using the polymerase cham reaction to modify expression plasmtds for epitope mapping. Nuclerc Acids Res 17,33 19 88.
Sometimes it is difficult to detect a single mutation in longer fragments using denaturation gradient gel electrophoresis (DGGE). This drawback can be overcome by attachment of a 40-bp GC-rich stretch to the primers (60). The analysis of the PCR product, melting at a higher temperature, will enhance the sensitivity of this method. Mixed oligodeoxynucleotide primers designedon the basis of the amino acid sequenceof urate oxidase were used successfully to clone the gene for this enzyme (34). If the primers crosshybridize to repetitive sequences, even an optimal adaptation of the annealing temperature will not eliminate unspecific bands.
1988) DNA sequencing with Thermusaquaticus DNA polymerase and direct sequencmg of polymerase cham reaction-amplified DNA Proc. Nat1 Acad Sci USA 85,9436-9440. 9 Keohavong, P. and Thtlly, W. G (1989) Fidehty of DNA polymerases m DNA amplification Proc. Nat1 Acad. Sci. USA 86, 9253-9257 10 Dunning, A. , Talmud, P , and Humphries, S E (1988) Errors m the polymerase cham reactton Nucleic Acids Res 16, 10,393. Taq Polymerase 11. Mullis, K. B. (1989) The polymerase cham reaction: why it works, in Current Communicattons in Molecular Btology* Polymerase Chain Reactton (Erhch, H.