Southern blots hybrid selection

Fig. 1. Time-course study of the accumulation of DHBV CCC DNA in PDH congenitally infected with DHBV. PDH cultures were harvested from d 1 to 15 postplating and selectively extracted to enrich for viral CCC DNA. The DNA was analyzed by Southern blot hybridization using a full length, double-stranded DHBV probe labeled with 32P.

Following transformation of mutant S. 6803 T-1,2,3 [4] with plasmid pSF8.1 colonies were selected for photoautotrophic growth. DNA from potoautotrophic transformants were then analyzed by Southern blot hybridization to confirm the insertion of the A. hybridus psbA gene. Fig. 2 shows the results from one selected transformant, S. 6803 SF. 

It s also possible to select your DNA before you put it in the vector. If you know the sequence (or even part of it), DNA pieces (from genomic DNA or cDNA) with this sequence can be purified on a gel and identified by hybridization to an oligonucleotide using a Southern blot. Alternatively, if you know the sequence of the ends of your DNA, you can amplify it specifically by the polymerase chain reaction. There are lots of clever ways to find your DNA. 

Acridinium ester—labeled chemiluminescent probes have been utilized to detect the specific protein-coding transcripts and to distinguish between transcripts that code for the 190-kDa protein and the two closely related 210-kDa proteins. The assay is called the hybridization protection assay (D3). In this assay, RNA isolated from the patient s white blood cells is first amplified by PCR. The amplified product is incubated with the chemiluminescent probe. The unhybridized probe is removed by selective hydrolysis in sodium tetraborate buffer, containing surfactant Triton X-100 at pH 8.5, in an incubation step at 60°C for 6 min. After the sample is cooled to room temperature, the chemiluminescence of the hybridized probe is measured in a luminometer. The procedure is reported to detect one leukemic cell in a population of a million or more normal cells. It is also rapid, requiring less than 30 min. Its reliability has been attested to by correlation with results obtained on karyotypic and Southern blot analysis (D3). 

The localization of bNOS to the human genome was accomplished by Kishimoto and coworkers70. These investigators used a rat cerebellar cDNA to obtain a human cDNA from Clontech. This cDNA was hybridized to Southern blots containing DNA from a battery of human-rodent somatic cell DNA. Since the blots had been shown to be selective, the authors showed that the cDNA hybridized to chromosome 12. By using restriction nucleases EcoRI and Hind in the assignment was made to 12 ql4-qter. One or two copies were indicated in vivo but reducing the hybrid conditions showed more bands. It is necessary to conduct further studies to see whether the other cDNA are derived from this or another clone. 

A number of methods are available for analyzing tumor HER-2 status. The selection of the method depends on the target molecule to be detected. The target molecules are DNA mRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot (Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al 1993), fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH) on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction (Gramlich et al., 1994). Assays to determine mRNA oveiexpiession include Northern blot (Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) and ISH (Naber et al., 1990). Methods to assess HER-2/mcm protein product overexpression... 

Fig. 2. Mutagenesis strategy. A neo gene replaced one-third of the ORF and was used as a positive selectable marker. The HSV thymidine gene (HSV-ffc) was used for negative selection. The vector was linearized with Xbal digestion. The targeted events were screened by Southern blot. Two Xbal sites are in the outside of the targeting vector (the 3 -end one is just at the end of the vector, which cannot be digested by Xbal if the vector is randomly inserted). An outside probe is located at the 5 -end. The genotype of the mice can be easily identified based on the size of the hybridized bands, 6.7 kb (mutant) vs 6 kb (wild-type).

The most representative method for the analysis of DNA hybridization is southern blotting in which separated DNA fragments are blotted onto a nitrocellulose physical support and radioisotopically labeled DNA probes are annealed to the complementary targets. It has good sensitivity and selectivity, however, this method requires a large amount of radioisotopic labeling. 

Nylon, particularly the positively charged membranes, has largely replaced nitrocellulose or diazotized paper for hybrid selection. DNA, e.g., from linearized plasmid, can be applied in 0.4 N NaOH to denature the DNA and to promote a strong fixation. The nylon membrane (2-cm squares) is soaked for 5 min in water and then 30 min in 0.4 N NaOH. Concurrently, DNA is denatured for 10 min in 0.4 N NaOH. DNA is then applied repeatedly with drying between applications. The membrane is then washed twice with 1 M NH4OAC and twice with 1 X SSG. The membrane is blotted and dried (may contain > 100 p,g/cm ). An alternative method is presented in Section 8.3.1. Disks of 0.5 cm are then punched out with a sterile one-hole paper punch. It is also possible to use Southern blots, or fragments thereof, for hybrid selection. 

Fig. 4.2 Detection of homologous recombinants by Southern blot. Restriction enzyme A cuts once in the homologous side arms of the targeting construct and once within the neomycin selection cassette. For Southern blot, the genomic DNA is digested with restriction enzyme A and hybridized with an external probe which stains bands of different size for homologous recombinants or the wild-type gene. In case of heterologous recombi...

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