Cloning methods

The most efficient strategy may be to use the topoisomerase cloning method in conjunction with the Crs-lox or X recombination systems. In this way, PCR products can be efficiently inserted into the Univector or Entry Clone with minimal additional sequences added to PCR primers. Once the genes are inserted into the starting vectors they can rapidly be converted to functional vectors using either Cre-lox or X recombination. 

In the following, a short overview of the expression cloning method with Xenopus laevis oocytes is given. Protocols for standard procedures are provided in the Appendix, while those methods that are subject to frequent modification are described in general and the reader is referred to the corresponding handbooks for further details. 

Cell Line Source, species, history, characteristics, cloning methods, vectors used, and genotype and phenotype of host cell system... 

For other production hosts (yeast, insect, and mammalian cells), standard promoter formats have been used in combination with FITP cloning methods to produce vectors for expression screening (see Section 2.3.2). A particularly interesting development is the use of multipromoter plasmids for expression in two or more hosts from a single vector. The construction of a dual E.coli (T7 promoter) and baculovirus transfer vector (polH promoter) for expression in insect cells has been described (Chambers et al., 2004). A three-promoter vector (T7, plO, and hCMV or CAG promoter) is available from Novagen (pTrlEX ) and its use reported for comparing protein expression in E. coli and insect cells (Xu and Jones, 2004). 

Berrow, N. S., Alderton, D., Sainsbury, S., Nettleship, J., Assenberg, R., Rahman, N., Stuart, D. 1. and Owens, R. J. (2007). A versatile Ugation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res. 35, E45,1-12. 

Marsischky, G. and LaBaer, J. (2004). Many paths to many clones a comparative look at high-throughput cloning methods. Genome Research 14,2020-2028. 

Nakajima, D., et al. (2005) Preparation of a set of expression-ready clones of mammalian long cDNAs encoding large proteins by the ORF trap cloning method. DNA Res. 12, 257-267. Nagase, T., et al. (2008) Exploration of Fluman ORFeome Fligh-throughput preparation of ORF clones and efficient characterization oftheir protein products. DNA Res 15, 137-149. 

Preparation of a Set of Expression-Ready Clones of Mammalian Long cDNAs Encoding Large Proteins by the ORF Trap Cloning Method DNA Res 12, 257-267. 

Procedures for construction of the expression clone follow classical molecular cloning methods. The overall cloning strategy is shown in Fig. 2. 

The polymerase chain reaction (PCR) is a test tube method for amplifying a selected DNA sequence that does not rely on the biologic cloning method described on p. 446. PCR permits the synthesis of millions of... 

The major advantages of PCR over cloning as a mechanism for amplifying a specific DNA sequence are sensitivity and speed. DNA sequences present in only trace amounts can be amplified to become the predominant sequence. PCR is so sensitive that DNA sequences present in an individual cell can be amplified and studied. Isolating and amplifying a specific DNA sequence by PCR is faster and less technically difficult than traditional cloning methods using recombinant DNA techniques. 

What are the major advantages of the polymerase chain reaction (PCR) method for amplifying defined segments of DNA as opposed to the use of conventional cloning methods How might the PCR method be used to test for infection with the AIDS virus and... 

Kamada, S., H. Kusano, H. Fujita, M. Ohtsu, R.C. Koya, N. Kuzumaki, and Y. Tsujimoto. 1998. A cloning method for caspase substrates that uses the yeast two-hybrid system cloning of the antiapoptotic gene gelsolin. Proc Natl Acad Sci USA. 95 8532—7. 

Kadono-Okuda, K. and Andres, D. A. (1997) An expression cloning method to identify monomeric GTP-binding proteins by GTP overlay. Anal. Biochem. 254, 187-191. 

Dilute so that 5 ml contains between 50 and 250 cells and quickly inoculate into a 5 or 6 cm dish (see cloning, method 1 below). 

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