Gene Mapping by In Situ Hybridization

Gene Mapping by In Situ Hybridization

The previous mapping methods are indirect in that they provide information on the physical location of a gene on a particular chromosome but without actually visualizing the gene's map position. A more direct approach is in situ hybridization, which involves hybridizing DNA (or RNA) probes directly to metaphase chromosomes spread on a slide and visualizing the hybridization signal (and thus the location of the gene to which the probe hybridizes) under a microscope.

The DNA in metaphase chromosomes is denatured in place (hence, in situ) on the slide, and hybridization of a labeled probe is allowed to proceed. Methods for mapping single-copy gene sequences by in situ hybridization originally were laborious and slow, requiring long exposures of the slides under photographic emulsion to detect the location of hybridized probe that had been labeled with low-level isotopes, such as tritium. Mapping with confidence required analysis of many metaphase spreads to distinguish the real hybridization signal from background radioactivity. However, more sensitive techniques have now been developed that enable rapid detection of hybridized probes labeled non radioactively with compounds that can be visualized by fluorescence microscopy (Fig). Even in a single metaphase spread, one can easily see the position of the gene being mapped.

In combination with banding methods for chromosome identification, fluorescence in situ hybridization can be used to map genes to within 1 to 2 million base pairs (1000 to 2000 kb) along a metaphase chromosome. Although this degree of resolution is a considerable improvement over other methods, it is still substantially larger than the size of most individual genes.


Figure: Gene mapping by in situ hybridization of a biotin-labeled DNA probe for the human muscle glycogen phosphorylase gene (MGP) to a spread of human metaphase chromosomes. Location of the MGP gene is indicated by the bright spots seen over each chromatid at the site of the gene in band q13 of chromosome 11. The mapping of MGP to 11q13 also assigns the locus for McArdle disease, an autosomal recessive myoglobinuria caused by deficiency of MGP. (Photograph courtesy of Peter Lichter, Yale University)

The deafness gene

The deafness gene


The mutation of MicroRNA mir-96 leads to progressive loss of hearing if present in single copy, and deafness if present in two copies

The journal Nature Genetics have posted the results of a search conducted under the projects "Sirocco" and "Eurohear", funded by the European Union. The discovery concerns the association between a new type of gene and the progressive loss of hearing, because the mir-96 gene is a small piece of RNA that affect the process of generation of other molecules in sensory hair cells of the inner ear .

The results come from the collaboration of two research groups, one Spanish and one English.

Karen Steel, one of the coordinators of the team of British Sanger Institute, said "We were able to demonstrate relatively quickly if the mice that were carriers of one copy of the variant of this gene suffer from progressive loss of hearing, and if they were carrying both genes, they were suffering from severe hearing loss. The principal questions to be answered concerning the possibility to determine which variant was involved and how influences on hearing "

Having identified the chromosome 7, the possible location of the gene altered the two groups of researchers have sequenced the gene in each "homologous genomic regions in man and mouse that are associated with hearing loss" and showed the presence of a mutation in the gene mir - 96.

Miguel Angel Moreno-Pelayo, author of the study and researcher at the Hospital Ramon y Cajal in Spain, said "We know a number of genes associated with deafness in humans and mice, but we discovered with surprise that this belongs to a new class of MicroRNA genes defined. No one had observed a mutation that can cause disease in a couple of MicroRNA sequence. This is the first MicroRNA gene associated with hearing loss and e 'is significant that the first to be associated with a hereditary condition. "

Experts recognize that the MicroRNA may bind to the active messengers in the generation of cell protein, effectively stopping the process and now have discovered that it is possible to analyze the role of mutation in mice. It seems the sensory hair cells in the mutant mice are affected by mir-96 gene, while mice carrying two copies of the gene mutant hair cells are deformed from birth and cells subjected to a degeneration in the early stages of life.

Morag Lewis of the Sanger Institute, who discovered the mutation, commented "The mutation, or variation of a single letter of genetic code from A to T in this tiny extension, is sufficient to cause a serious loss in mice" .

This mechanism also occurs in humans but, according to analysis of the two families used as a sample, the mutation does not happen ever in the same regions where the mouse, although affect neighboring regions, and always very important for the proper functioning of mir-96 .

Race for the Double Helix

Race for the Double Helix

The Watson-Crick model of DNA was a Nobel Prize winning structure. But what differed from this one to all of the other DNA structures that already existed? Well, Watson and Crick discovered the DNA was double-helical. Previously it was believed that DNA had a triple helix. Watson and Crick knew that the phosphates could not be located on the outside while the bases were on the inside.

The novel feature of their structure was that the two chains were held together by a purine and pyrimidine base. They are joined in pairs, a single base from one chain being hydrogen-bonded to a single base from the other chain, so that the two lie side by side with identical z-co-ordinates. Adenine (purine) is paired with thymine  (pyrimidine), and guanine (purine) is paired with cytosine (pyrimidine). Many times it will be written like: A-T, G-C.

As we already know, DNA consists of two strands, and each of those strands consist of a phosphate-sugar-phosphate backbone with bases (A, T, C or G) on the sugar molecules. The two strands run opposite of each other, with the bases pointing inwards and (as mentioned above) adenine is always paired with thymine, and guanine is always paired with cytosine. Therefore, if you know the sequence of one strand, then you know the sequence of the other strand. They are mirror images in reverse.

RNA recreated in the laboratory from inorganic compounds

RNA recreated in the laboratory from inorganic compounds


Researchers at the University of Manchester, lead by John Sutherland, illustrated in an article published in Nature, the process which created ribonucleotides (units of RNA, the molecule basis of all life processes) starting from simple elements, such as those that were presumably the primordial soup, the aqueous solution from which have formed the first organic molecules four billion years ago.

So far, in experiments of this type are started immediately by adding a phosphate sugars and nitrogen bases. In this study, scientists started by the most simple and have played the environmental conditions by heating the solution. BY the solution is obtained a residue of hybrid molecules, which were re-hydrated and heated, allowed to evaporate and irradiated with UV light in order to reproduce the cycle of the ecosystem primordial environment.

At each step the resulting molecules seemed increasingly complex and the addition of phosphate end, which has functioned as a catalyst and regulator of acidity "Surprisingly it was a ribonucleotide format!" as reported by Sutherland and continues "We suspect that there is something out there good, but it took 12 years to discover.

In twenty years of attempts had shown no concrete evidence of the formation of RNA from the reaction of simple molecules and chemicals while known for some time.

Now because of this research is another step to demonstrate the validity of the theory that places the RNA as the starting point of the life on Earth, although still not shown that this method can create molecules of RNA complete.

Sutherland, however, hopes to further develop his research to solve the doubts remained.