University of Pennsylvania School of Medicine, Philadelphia, PA, USA
DNA Cloning without Restriction
Enzyme and Ligase BioTechniques 27:1240-1244 (December 1999)
One common problem in using the tradi-tional DNA cloning procedure is that suit-able natural restriction sites are often un-available for a given task. Creating new restriction sites is often time consuming. Here, I describe a simple technique of pro-ducing "customized cohesive ends" by a combination of PCR primer design and l exonuclease digestion. These complemen-tary cohesive ends can form hybrids to link two sequences. Because the overhangs cre-ated by lexonuclease are slightly longerthan the complementary sequence, after hy-brid formation, a stretch of single-strand gap remains, which then is repaired by Klenow (3' - 5' exo - ) enzyme. The repair process also stabilizes the linkage. Because of the independence from natural or artifi-cial restriction sites, this method allows rapid and precise insertion of one DNA fragment into another at virtually any posi-tion. It also simplifies the planning of a cloning strategy, increases recombinant fre-quency and is suitable for automation.
Protein-binding matrices for phenol-free extraction of DNA and RNA modifying enzymes: applications for genetic engineering and high throughput.
Clonogen Ltd., St. Petersburg, Russia
Isolation of Intact High Molecular Weight Chromosomal DNA from Desulfovibrio spp.
Vitaly Zinkevich* and Iwona B Beech
School of Pharmacy and Biomedical Sciences, Microbiology Research Laboratory,
University of Portsmouth, St. Michael's Building, White Swan Road, Portsmouth PO1
2DT, United Kingdom.
Molecular Biology Today (2000) 1(1): 29-33.
A simple, reproducible method to extract high quality DNA from sulphate-reducing bacteria (SRB) is described. SRB isolated from patients with ulcerative colitis, and from environmental samples, were used for purification of chromosomal DNA. DNA yield purified by this method was more than one order of magnitude higher compared with the Marmur method, phenol-chloroform or QIAGEN procedures. A 10ml culture was enough for isolation of sufficient DNA to perform hundreds of PCR-based reactions, and can be used in other DNA manipulation techniques such as restriction digestion and DNA cloning despite a low yield of cells (1x10 7 - 1x10 8 cells/ml).
Specificity-Enhanced Hot-Start PCR: Addition of Double-Stranded DNA
Fragments Adapted to the Annealing Temperature
BioTechniques 28:278-282 (February 2000)
A new method to produce hot-start con-ditions in PCR is described. Short double-stranded DNA fragments were found to in-hibit the activity of DNA polymerases from Thermus aquaticusand Thermus flavus. This inhibition is not sequence specific, but exclusively dependent on the melting tem-perature of the fragments as shown by its correlation to their melting curves as mea-sured. This property is exploited by adding fragments of the appropriate length to the PCR mixture during the reaction setup and thereby preventing the DNA polymerases from extending primers annealed nonspecif-ically at lower than the optimal temperature. By amplifying ten copies of phage lDNA in the presence of 2 mg of nonspecific DNA, it is shown for three different primer pairs how the melting temperatures of the double-stranded DNA fragments have to be adapted to the cycle profiles to obtain predominantly specific products in the 0.5 mg range.
Preliminary Characterization of zntA, a Gene Which Encodes
a Zn(II)/Cd(II)-Export Protein in Escherichia coli
Dayle K. Blencowe, Samantha J. Marshall and Andrew P. Morby*
School of Molecular and Medical Biosciences
University of Wales Cardiff, Museum Avenue, Cardiff CF1 3US UK
© Biotechnology et alia, 1997 2:1-6
An increasing number of proteins from both prokaryotic and eukaryotic organisms which transport and/or bind transition metal ions contain sequences corresponding to the conserved heavy metal associated motif (GMTCXXC). Those proteins in this group which translocate metal ions across membranes also show primary sequence similarity to the P-type ATPase family of membrane transport proteins. Database analysis identified a gene encoding a candidate metal ion transport ATPase in the E. coli genome (yhho). The gene has been isolated and expressed in E. coli from a heterologous promoter. Data supports the function of this polypeptide as a Zn(II)/Cd(II) export protein and we designate the gene, zntA.