Electrophoresis Southern blotting

Eollowing agarose gel electrophoresis. Southern blotting can be used for more advanced analysis of separated DNA bands, allowing marking and identification of specific DNA sequences. 

M sodium citrate. A strip of nitrocellulose, soaked in the same solution, and is placed on top of the gel a piece of dry paper toweling is put on top of that. The compounds diffuse upward carried by the capillary action of the liquid. The DNA selectively binds to the surface of the nitrocellulose, and diffusion stops. The membrane is quite flexible and easy to handle. Figure 27-16 shows the sequence of steps. Southern blotting usually is done with agarose gels and horizontal electrophoresis. Southern blotting has become very popular since the use of DNA patterns for the identification of people. 

Additional DNA strands, or RNA strands, can be added to a denatured DNA sample and allowed to participate in the renaturation reaction. When the added DNA or RNA associates with one of the original DNA single strands this is called hybridization. Hybridization is a very powerful technique for detecting particular DNA or RNA sequences, especially in conjunction with electrophoresis (Southern blotting and northern blotting) or in primer annealing in the polymerase chain reaction (PCR, Chapter 22). 

The use of agarose as an electrophoretic method is widespread (32—35). An example of its use is in the evaluation and typing of DNA both in forensics (see Forensic chemistry) and to study heritable diseases (36). Agarose electrophoresis is combined with other analytical tools such as Southern blotting, polymerase chain reaction, and fluorescence. The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

Presented below are four increasingly stringent confirmatory techniques for PCR and a brief discussion of considerations, limitations and advantages of each. These four techniques are agarose gel electrophoresis, restriction analysis. Southern blotting and sequencing. 

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. 

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. 

Northern blots Northern blots are very similar to Southern blots (see Figure 32.12, p. 453), except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized to a... 

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. 

Southern blotting is a technique that can be used to detect specific genes present in DNA. The DNA is cleaved using a restriction endonuclease, the pieces are separated by gel electrophoresis and then transferred to a nitrocellulose membrane for analysis. The fragment of interest is detected using a probe. 

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. A method for detecting a specific DNA restriction fragment, developed by Edward Southern. DNA from a gel electrophoresis pattern is blotted onto nitrocellulose paper then the DNA is denatured and fixed on the paper. Subsequently, the pattern of specific sequences in the Southern blot can be determined by hybridization to a suitable probe and autoradiography. A northern blot is similar, except that RNA is blotted instead onto the nitrocellulose paper. 

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