Total genomic DNA

Myers, R.M., Lumelsky, N., Lerman, L.S. and Maniatis, T. (1985c) Detection of single base substitutions in total genomic DNA. Nature 313, 495-498. 

Amplified fragment length polymorphism (AFLP), developed by Vos et al. [453], is also a new technique for DNA fingerprinting. This technique is based on the selective amplification of restriction fragments (SARE) by PCR from a digest of total genomic DNA. 

Lonvaud-Funel et al. (1989, 1991a) described the identification of LAB during vinification and wine storage by DNA-DNA hybridization. Genomic DNA of the strain to identify was hybridized with total genomic DNA probes extracted from reference strains. They found that this method was particularly efficient when used in colony hybridization to study mixed populations at least five different species can be detected in a mixture with this system (Lonvaud-Funel et al. 1991b). 

The most precise tests are the genotypic including characterisation of chromosomal, plasmid or total genomic DNA. In this way it is possible to study the polymorphism and separate the various clones. An accurate analysis of the methods available is made in this paper. 

Figure 51.2 The amount of modified DNA bases in total genomic DNA of peripheral blood of goitrous (SMOID-G) and control (NMID-C) children. Source-. Giray and Hincal, 2002, with kind permission from Taylor Francis. 1 nmol/mg of DNA corresponds to approximately...

Isolate total genomic DNA using DNeasy tissue kit (Qiagen) and quantify. 

A crucial aspect of virtually all DNA-DNA hybridization studies is that they involve only so-called single-copy DNA (scDNA) or "unique DNA. Total genomic DNA is made single-stranded by heating and then allowed to reassociate into duplex DNA after cooling. Repeated copies of DNA sequences re-anneal faster than single-copy sequences due to their higher concentration in the solution. Thus, under controlled conditions and time, it is possible to remove excess copies of repeated sequences. 

If density centrifugation of total genomic DNA separates one or more clearly distinguishable satellite bands, these can be isolated and used for cloning... 

Figure.5 Filter hybridization of Eco RI fragments of total genomic DNA of the Drosophila sp ies indicated above with a cloned satellite repeat of D. ambigua. A ladderlike hybridization pattern appears for the closely related species D. ambigua, D. tristis and D. obscura. No hybridizations can be seen in any of the other species. Lane 1 D. melanogaster, lane 2 D. azteca lane 3 D. pseudoobscura lane 4 D. ambigua lane 5 D. tristis lane 6 D. obscura lane 7 D. bifasciata lane 8 D. subsilvestris lane 9 D. subobscura lane 10 D. madeirensis lane 11 D. guanche.

This procedure has been used with success on a wide variety of plant groups and even some animals. The method is used to isolate total genomic DNA (nuclear, chloroplast, and mitochondrial). It is a rapid, inexpensive method that is suitabie for use in conjunction with other protocois, such as isolation of DNA enriched for cpDNA. it is also easy to scale down for use in population sampling, using 0.01 g or less of fresh tissue. Other applications include isolation of DNA from herbarium specimens (Doyle Dickson, 1987. Taxon 36 715-722), and isolation of RNA. A brief word on the history of the protocol is in order. This procedure was modified by us (Doyle and Doyle, 1987. Phytochemical Bulletin 19 11-15) for use with fresh piant tissue from a method of Saghai-Maroof et al. (1984, PNAS USA 81 8014-8019) who used lyophilized tissue. They in turn had developed their procedure from earlier protocols. We were recently asked to publish a slightly modified version of our procedure (Doyle and Doyle, 1990 Focus 12 13-15). We recently learned from Brian Taylor (Texas A M University, USA) that he had published a virtually identical procedure for fresh tissue, also in Focus, in 1982 (Taylor Powell, Focus 4 4-6) of which we (and apparently the editors of Focus ) were entirely unaware. It is indeed a useful procedure, thus independently confirmed. 

Fig. 1. Direct fluorescent double in situ hybridization to a partial metaphase of a hexaploid wheat variety canying a rye translocation (IB/IR). (A) Bright fluorescence of the rye chromosome arms probed with rhodamine-labeled total genomic DNA from rye after green excitation (anow heads), with the wheat chromosomes fluorescing weakly. (B) Strong fluorescence of the fluorescein-labeled rDNA under blue excitation. rDNA sites can be distinguished on several wheat chromosomes (arrows) and the two rye arms (open arrows). Bar = 10 pm.

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