Recombinant DNA

  • Recombinant DNA

    Posted by Encyclios on May 16, 2023 at 1:34 PM

    In experimental organisms commonly used for genetic analysis (Bacteria, yeast, and Drosophila) there are phenotypic markers to map mutations, but in the case of genes associated with communicable genetic diseases this is not possible. However, with the use of recombinant DNA techniques, there are now a large number of molecular markers among which the most widely used are the so-called RFLPs (Restriction Fragment Length Polymorphism).

    This technique is based on the principle that variations on DNA sequences occur throughout the genome, and since much of the genome does not code for proteins, fairly large sequence variability is possible even in humans. It has been estimated that a sequence change occurs approximately every 200 nucleotides. These changes are referred to as polymorphisms and can be referred to different analysis systems. If substitutions occur in the specific sequences recognized by restriction enzymes, the site in question will no longer be cut and thus a map of restriction sites in a specific region of the genome will contain fragments of different lengths.

    Based on such variations, it is possible to draw specific maps of the chromosome and associate a particular genetic disorder with a specific restriction fragment. What is the origin of polymorphisms is a very interesting problem in modern genetics. Many mutations form spontaneously along the DNA and can affect regions and sequences that code for proteins. In this case, if their effect is very severe, the mutations will not be passed on to offspring because the one who carries them will die. Others occur in noncoding regions and will have no effect on cellular functions and thus will be passed on without problem to subsequent generations.

    In humans, polymorphisms appear to be associated with single base changes in DNA. The dinucleotide CpG (Cytosine phosphor Guanine), for example, is a hot spot for single sequence changes, so restriction sites containing that sequence are often polymorphic. These polymorphic sequences are often organized repetitively, from 14 up to 70 base pairs, and are encountered on average along the genome, once every 40 kilobases.

    Using a molecular biology technique called PCR (polymerizing chain reaction), which is capable of many-fold amplification of DNA, it is possible to determine the exact location of these repeated sequences, in defined regions of DNA, and thus map genes that are adjacent to them. Many families can be identified that are at risk for genetic disease because both parents are heterozygous for a recessive mutation associated with a particular genetic disease. By analyzing the DNA of these families and studying the frequency with which the polymorphic marker (e.g., the CA sequence, repeated 14 times) segregates along with the disease-causing mutant gene, one has a measure of their closeness.

    The greater the number of families studied, the more precise the mapping of the disease-associated gene. However, because in humans the number of generations that can be analyzed in a family is limited (grandparents, children, grandchildren), the location of a particular disease is very approximate. To overcome these limitations, the use of a new strategy called linkage disequilibrium was introduced. The method is based on the principle that a genetic disease is most likely caused by a mutation that has occurred over many generations.

    The chromosome of this ancestor will contain markers very close to the mutated gene that will be passed on generation after generation. In contrast markers that are quite distant from the mutated gene will tend to become more and more distant due to recombination. By studying the distribution of specific markers in all diseased individuals in a population, it is possible to identify the genetic locus of the disease, in small regions. In recent years, this methodology has been widely used and, for example, in the Finnish population, where dystrophic dysplasia is common, it has been possible to locate the gene in a 60-kilobase region.

    Encyclios replied 2 weeks, 2 days ago 1 Member · 0 Replies
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