Southern blot analysis specific

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). 

Two types of screening procedures are used to identify ES cell clones carrying a targeted integration of the construct DNA polymerase chain reaction (PCR) and Southern blot analysis (Southern, 1975). Both methods rely upon the specific juxtaposition of vector components and target locus sequences after homologous recombination. 

D. Restriction length fragment polymorphism analysis detects mutations in the DNA that either introduce or eliminate a recognition site for a restriction enzyme. This is detected by performing a Southern blot analysis on patient DNA after incubation with a specific restriction enzyme and comparing it to one performed on DNA obtained from a normal gene. 

In the laboratory. DNA can be cut into small pieces by enzymes called restriction endonucleases. The.se enzymes are restricted to cutting the DNA at specific base pair sequences. Tbe presence of polymorphic sites, where there are differences in the base pair sequence of the DNA, may create or abolish a cutting site for a particular restriction endonuclease. In these instances, the polymorphisms are known as restriction fragment length polymorphisms (Rf-LPs) as they lead to the production of DNA fragments of different lengths after enzyme action. DNA fragments may be separated by electrophoresis, transfered to a nylon membrane and hybridized with a DNA probe labelled with a radioactive or optically active marker. This is called. Southern blot analysis (Fig. 1). 

Strategies to experimentally detect translocations are important because of the numerous cases of genes in leukemia-associated translocations. Such methods include Southern blot analysis, which is not as sensitive as PCR, karyotype analysis and fluorescence in situ hybridization (FISH) with specific probes. Reverse transcriptase (RT)-PCR with gene-specific primers detects only a fraction of translocations because there are no primers available for many of the genes involved. 

For the isolation of psliG2, Hindlll digested total DNA was separated on a 10 -30 % sucrose gradient. Fractions enriched in fragment sizes of 5 - 6 kb were subcloned in Bluescript KS. About 700 colonies were screened by Southern blot analysis with a psbG specific probe under low stingency conditions and six positive clones were identified. 

Figure 7.4 Compensatory relationship between horA- and horC-specific determination methods for beer-spoilage ability of lactic acid bacteria (LAB) strains. A total of 88 strains belonging to various beer-spoilage species were examined by polymerase chain reaction and Southern blot analysis. It was shown that beer-spoilage LAB strains possess at least one of the genetic markers, indicating that horA and horC are excellent genetic markers for comprehensibly determining beer-spoilage ability of LAB.

If the genetic lesion is understood and a specific probe is available, prenatal diagnosis is possible. DNA from cells collected from as little as 10 mL of amniotic fluid (or by chorionic villus biopsy) can be analyzed by Southern blot transfer. A fetus with the restriction pattern AA in Figure 40-10 does not have sickle cell disease, nor is it a carrier. A fetus with the SS pattern will develop the disease. Probes are now available for this type of analysis of many genetic diseases. 

RFLP analysis by the Southern blot procedure and detection with probe specific for each VNTR are the basis of DNA fingerprinting. Using just four DNA probes, paternity can be excluded with a cumulative probability of 99.9% (J2). 

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... 

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