Cloning chain reaction

The discovery and exploitation of enzymes in aldoxime-nitrile pathway nitrile hydratase, amidase, nitrilase, aldoxime dehydratase, etc., are shown along with the use of methodologies, such as organic chemistry, microbial screening by enrichment and acclimation culture techniques, enzyme purification, gene cloning, molecular screening by polymerase chain reaction (PCR). 

Orlandi, R., Gussow, D.H., Jones, P.T., and Winteg G. (1989) Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86, 3833-3837. 

Phosphoinositase C (i.e. phosphoinositide-specific phospholipase C [PLC]) enzymes are found in the vast majority of mammalian cells. Molecular cloning of these enzymes, analysis of their predicted amino acid sequences and immunological cross-reactivity indicate that at least three major forms of the enzyme exist PLC-/I, -8 and -y. Each of these enzyme types is encoded by a distinct gene. More recent experiments using the polymerase chain reaction and molecular cloning have revealed even greater enzyme di-... 

Schaefer, B.C. Revolutions in rapid amplification of cDNA ends New strategies for polymerase chain reaction cloning of full-length cDNA ends. Anal Biochem 227 255-273, 1995. 

One of the in vitro (in the test tube) processes used to clone DNA is called the polymerase chain reaction (PCR). A vial in which PCR is to be carried out contains all the necessary components for DNA duplication the piece of DNA to be cloned large quantities of the four nucleotides, A, T, C, G large quantities of a primer sequence, a short sequence of about 20 nucleotides synthesized by the primase enzyme and DNA polymerase.To conduct the process, the vial is hrst heated to 90-95°C for 30 seconds to separate the two DNA chains in... 

Fig. 5. Selection scheme for the in vitro selection of RNA libraries. The RNA library is subjected to a selection criterion suitable for the enrichment of functionally active sequences. The few selected individual sequences are amplified by reverse transcription (RT) and polymerase chain reaction (PCR). The PCR-DNA is then subjected to in vitro transcription with T7 RNA polymerase. The resulting enriched and amplified RNA library can be used as the input for the next selection cycle. This process is repeated until active sequences dominate the library. At this point, individual sequences can be obtained by cloning and their sequence can be determined by sequencing...

Armed with this new tool, Schena et al. (1996) created a microarray of 1,046 human cDNAs of unknown sequence. They were derived from human peripheral blood lymphocyfes fransformed wifh Epsfein-Barr virus. Suitably sized inserts [>600 base pairs (bp)] were cloned into a lambda vector, subsequently infected into an Escherichia coli strain, and finally amplified by polymerase chain reaction (PCR) using 5 -amino-modified primers. The resulting 5 -amino-modified cDNA amplicons were then arrayed onto sily-lated microscope slides. Next, the expression levels in human Jurkat cells undergoing heat shock or phorbol ester induction were examined. 

ACC 71 synthase, i. e. (S)-adenosylmethionine methylthioadenosine lyase (EC 4.4.1.14), has been purified from several plant tissues [116]. Recently, ACC synthase cDNA clones have been isolated and sequenced from wounded fruit tissues of tomato, winter squash, zucchini, ripening apple and tomato fruit. Using the polymerase chain reaction (PCR), four different ACC synthase gene fragments were obtained by amplification of cDNA derived from mRNA of tomato... 

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