Here we will cover in greater detail the topic of Southern transfers that were mentioned and briefly described in Chapter 2. At the same time, since the concepts are almost the same, we will also mention the so-called Northern and Western transfers. Southern transfers once were a necessary step in chromosome mapping, but their use has been superseded by techniques based on the polymerase chain reaction. To review, DNA fragments can be separated according to size by electrophoresis through gels, denatured, transferred to a nylon or paper mem brane and immobilized. Then the membrane can be immersed in a buffer containing a labeled oligonucleotide or DNA fragment and incubated under conditions permitting hybridization between complementary nucleic acid sequences (Fig. 1). The labeled fragment will therefore hybridize to its complementary sequence. The portion of the membrane carrying the fragment will then become radioactively labeled, and can be detected by autoradiography. This part of the process is analogous to plaque and colony screening described in the previous chapter. This simple technique, named a Southern transfer for Southern who first devised it, can be used in the analysis of chromosome structure.

Fig1. Southern transfer methodology. After electrophoretic separation according to size, the fragments are denatured and electrophoretically transferred to a membrane before hybridization with radioactive probe.

Consider the problem of learning whether the two nearest EcoRI cleavage sites on either side of a segment of DNA are at the same location in two nearly homologous chromosomes. If they are not, the situation is described as a restriction fragment length polymorphism, RFLP, and the nucleotide differences producing this polymorphism can be used as a genetic marker. If the restriction fragment containing the sequence is the same size from both chromosomes, then the nearest EcoRI cleavage sites are likely to be in the same locations. To search for RFLPs, a DNA sample containing the two chromosomes is cut with a restriction enzyme, separated by electrophoresis, and “probed” with a radioactively labeled segment of the region (Fig. 2). A difference in the sizes of corresponding fragments indicates the presence of an RFLP.

Fig2. Detection of an RFLP by Southern transfer in which the radioactive probe was fragment S. The chromosome A generates band A and the chromosome B which lacks the first EcoRI cleavage site on the right generates band B. A heterozygous individual shows both bands in a Southern transfer probed with the segment S.

Northern transfers are the converse of Southern transfers in that it is RNA rather than DNA that is separated by electrophoresis, transferred, and immobilized on membrane to preserve the original pattern.

Membrane with immobilized RNA can then be used in hybridization, just like paper with immobilized DNA.

What kinds of questions can be answered with immobilized RNA? One concerns the in vivo state of various RNAs. Transient precursors of a mature RNA molecule can easily be detected because they will be larger than the mature RNA and will be separated during electrophoresis. This permits tracking the maturation process of an RNA molecule. Not only can the changing sizes of the maturing species be monitored, but fates of specific regions that are removed can also be followed by probing with appropriate sequences.

Transfer-like technology can also be used to purify specific RNAs or DNAs. Either single-stranded RNA or single-stranded DNA can be bound to the paper. Then either RNA or DNA fragments complementary to the immobilized RNA or DNA can be isolated from a mixture by hybridization followed by elution. As an application, messenger RNA eluted from such immobilized DNA can be translated in vitro to provide a definitive identification of a candidate clone for a specific gene.

Western transfers involve proteins, not nucleic acids. The principle is the same as for Northern and Southern transfers. A pattern of proteins that have been separated by electrophoresis is transferred to paper or a membrane and then specific proteins are visualized. Some DNA- or RNA-binding proteins can easily be detected after transfer. These proteins partially renature despite being stuck to the paper. Then the paper with the immobilized proteins is incubated with the radioactive nucleic acid which binds to the immobilized protein. After washing the paper to remove unbound radioactive nucleic acid, autoradiography of the paper reveals the location of the immobilized protein. More often, the position of a specific protein is revealed by antibody probing as described  in the previous section.

مواضيع ذات صلة

Finding Clones Using Antibodies Against a Protein

Finding Clones from a Known Amino Acid Sequence

Enzymatic DNA Sequencing

Chemical DNA Sequencing

Targeting of Nuclear Proteins

Ribosome Disorders

The Process of Translation

RNA Granules and Membraneless Organelles

RNA Interference

Balancing Synthesis of Ribosomal Components

Regulation of Ribosome Synthesis

Proportionality of Ribosome Levels and Growth Rates