Southern blots probe labeling

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

In 1987, CL started to be applied in DNA hybridization assays as an alternative to the use of radioactive tags. These assays are based on the specificity of a binding process that of DNA strands for each other. An unknown DNA can be identified with the Southern blot method in which the strands of the analyte are separated and allowed to interact with labeled probe DNA strands on nitrocellulose filter paper. If the label on the probe is detected, the DNA can be identified and, in some cases, quantitated. Conventionally, radioactive tags were used be-... 

Another similar technique to Southern blotting is Northern blotting. Here, instead of DNA fragments, mRNA fragments are probed with a labelled cDNA probe after separation by electrophoresis and transfer to nitrocellulose membranes. Northern blotting is used to detect and quantify mRNA from tissue extracts. 

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 firagments in the material to be analyzed (DNA, RNA, or protein) are separated by gel electrophoresis. The smaller molecules travel faster and appear nearer the bottom of the gd. The bands of material in the gel are transferred or blotted to the surface of a membrane. The membrane is incubated with a (usually radioactive) labeled probe that will specifically bind to the molecules of interest. Visualization of the labded probe (usually by autoradiography) will reveal which band interacted with the probe. The most common types of blots are compared in Table 1-7-1. Most typically, DNA restriction fragments are analyzed on a Southern blot. 

Western blots Western blots (also called immunoblots) are similar to Southern blots, except that protein molecules in the sample are separated by electrophoresis and blotted to a membrane. The probe is a labeled antibody, which produces a band at the location of its antigen. 

One way to select a desired segment of DNA from a digest of chromosomal DNA is to sort out the "restriction fragments" by gel electrophoresis.613 The DNA from the gel can be transferred to a nitrocellulose sheet while retaining the separation pattern using a method devised by Southern.560 614 615 In this Southern blot technique, solvent flows from a pool beneath the gel up through the gel and the nitrocellulose sheet into paper towels. The DNA is trapped on the nitrocellulose in the same pattern observed in the electrophero-gram. A suitably labeled probe such as cDNA with... 

Southern blotting involves electrophoresis of DNA molecules in an agarose gel and then blotting the separated DNA bands on to a nitrocellulose filter. The filter is then incubated with a labeled DNA probe to detect those separated DNA bands that contain sequences complementary to the probe. 

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