Southern analysis

To minimize problems with the detection and analysis of a gene that exists as a single copy on an autosomal chromosome, technology of extreme sensitivity needs to be employed. Although the standard Southern analysis combines reasonable sensitivity with greater specificity, it is labor-intensive, requiring the use of radioisotopes such as 32P, and a few days are required to complete an analysis. Several pitfalls of the Southern procedure can be eliminated by substituting the PCR technique (M4). 

The standard technique for CF testing is DNA analysis by Southern blotting. Although the Southern analysis provides reasonable sensitivity with enhanced specificity, it is time- and labor-consuming. It requires handling of radioisotopes and approximately one week to complete. Some of these problems can be minimized by the use of PCR. The most common approach for the detection of... 

Much of the FLS biochemical and structure/fimction analysis has focused on a protein from C. unshiu (mandarin). A cDNA was isolated based on sequence homology to an Arabidopsis FLS EST (153O10T7) and used as a probe to determine regulation of gene expression [92]. Higher expression was observed in young leaf, flower, peel, and juice sac/segment epidermis tissues. Expression decreased with tissue age, as has been observed for citrus CHS, CHI, and F3H [57]. Southern analysis... 

Northern analysis is usually used to identify and quantitate specific mRNAs in a sample. Southern analysis is used to determine whether or not a gene of interest is present as well as its copy number. Other uses for Southern analysis include identifying restriction fragment length polymorphisms and changes in heterozygosity. 

Fig. 6. Physical detection of reciprocal translocations. The chromosomes shown are identical to those in Fig. S. Vertical arrows represent recognition sites for a single restriction enzyme horizontal arrows correspond to synthetic oligonucleotide primers and lines below the chromosomes indicate the sizes of restriction or PCR fragments. In (A), the alteration in restriction fragment size as a result of exchange is illustrated. Such alterations can be detected by Southern analysis using the duplicated sequence as a probe. In (B), the production of a PCR product from one of the exchange chromosomes is illustrated. Neither parental chromosome directs synthesis of a PCR product.

Given the latest advances in CE methodology and detection, on-line Southern analysis procedures with precolumn DNA hybridization in solution, followed by CE analysis of the resulting hybrid, have been demonstrated (Chen et al., 1991 Bianchi et al., 1994). A fluorescently tagged oligonucleotide of complementary sequence is used as a probe and mixed with a PCR product. The mixture is then heated to 100°C and ramped slowly down to room tempera-... 

AJ, Adomat SA. Comparison of cytogenetic analysis, southern analysis, and polymerase chain reaction for the detection of t(14 18) in folficular lymphoma. Am J Clin Pathol 1995 103 472-8. 

A bidirectional capillary transfer (Smith and Summers, 1980) (Table 9.8C) is feasible for Southern analysis of DNA present in high... 

Potentially transgenic mice are usually screened for the presence of the injected gene by performing PGR or Southern analysis with a DNA sample extracted from the tail or ear. Several protocols are available to extract DNA for these analyses and Protocols 11 and 12 show two excellent examples. 

The most widely used approach for the detection and analysis of DNA is Southern analysis (80,81,125), which is useful because it not only allows detection of the specific plasmid, but also assessment of degradation and conformation of the transfected plasmid. The method can be applied to DNA samples from in vitro or in vivo transfection. Southern analysis is sensitive enough to detect small amounts of plasmid DNA against a large background of genomic DNA. 

Figure 2. Southern analysis of mildew DNA restriction digests probed with plasmid pUC8 containing the sterol 14 a demethylase sequence from yeast, (pDC8-2860H), the whole coding sequence (A), 3 end (B) and 5 end (C).

Selected PER.C6 cell lines possess low copy numbers, typically below 10 copies per cell as measured by Southern analysis (Fig. 3.6). 

CIO. Cate, R. L., Ehrenfels, C. W., Wysk, M., Tizard, R., Voyta, J. C., Murphy, O. J., and Bronstein, I., Genomic southern analysis with alkaline phosphatase-conjugated oligonucleotide probes and the chemiluminescent substrate AMPPD. Genet. Anal. Tech. Appl. 8, 102-106 (1991). 

The first step in the conversion of tabersonine to vindoline is hydroxylation of the C-16, which is catalyzed by the enzyme tabersonine 16-hydroxylase (T16H), Fig. (6). Characterization of T16H indicated the enzyme is a cytochrome P-450 monooxygenase [91], what was confirmed by the molecular analysis of the isolated cDNA [92]. Southern analysis suggests the presence of at least two T16H genes in C. roseus. 

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