Table of ContentsIntroductionData and sample collectionCytogenetic studyGenetic linkage analysisCandidate region/gene identificationFinal mappingCurrent applications in nephrologyIntroductionUnderstanding any functionally biological product is very important. For this we need to understand and know the location of that particular gene, from where the product of that gene is transcribed and translated. The reason why it is necessary to know the location of the gene is very important because in a population this product can present different phenotypes, each of which can have an advantage over the other. In order to locate the gene, scientists introduced a molecular process, which involves genetic mapping, forward and reverse mutations that uses techniques such as appropriate marker genes and other procedures and other series of techniques like cross-species hybridization, potential trapping of exon fragments, Characterization of methylated/unmethylated CpG islands which are all applied in “positional cloning”. Say no to plagiarism. Get a tailor-made essay on “Why violent video games should not be banned”?Get an original essayNot only can we understand the functions and location of the gene and its corresponding product, but we can also study any gene associated with disease, resulting in a defective protein or a disease-causing protein, or even no protein production. Therefore, the main objective of “positional cloning” is to identify any genetic disorder and its location passed on to the next generation using the idea of ​​Mendelian inheritance, through which we can trace the origin. Briefly, the procedures involve selecting a disease-causing gene and locating its position, followed by the types of mutations in that gene and its corresponding phenotypes. Apart from this, there is a surrogate method that includes reverse genetics, where genotypes are recognized, followed by phenotypic characterization. The very first application of this technique was the identification of a pathogenic gene in the human genome. Later, in 1986, this procedure became common to identify any type of genetic mutation causing chronic granulomatous disease. Through positional cloning, many human genetic diseases that can be passed on to the next generation have been identified, such as cystic fibrosis, Duchenne muscular dystrophy, fragile X syndrome and also breast cancer. Data and sample collection It is very important to have correct data because any inappropriate collection of data can result in poor analysis. This data collection includes all kinds of known genetic disorders in a patient as well as their family members. All of these elements combined can help the patient get an idea of ​​their genetic mutations and the clinical treatments corresponding to a specific genetic disease. This procedure also includes a thorough clinical study of family members with the same clinical symptoms. This type of data collection is commonly referred to as “genealogical analysis.” Pedigree analysis should ensure that complex traits such as environmental factors should not be associated with the Mendelian concept, which is also part of the clinical study. To continue the research, DNA samples should be collected from all members of the patient's family, especially those who were considered in the genealogical analysis. Cytogenetic study The identification of the gene responsible for the disease in patients with chromosomal abnormalities is thefirst task. Translocations or inversions of DNA segments are targeted in this procedure because they do not result in any loss or gain of DNA material in a patient. Even if no DNA fragments are lost due to balanced translocation, there are some incidents where the abnormal phenotype caused by balanced translocation suggests that this characteristic is not balanced and ultimately a some amount of DNA could be lost during chromosomal rearrangement. evidence, which suggests that balanced translocation can either enhance a diseased gene to become active or could have dissociated it from its regulatory expression region/gene. For example, autosomal dominant polycystic kidney disease (PKD1) was first identified in 1994 in a Portuguese family. The isolated gene encoding a 14 kb transcript was found to be damaged by chromosomal translocation. Additionally, the mother and daughter who suffered from this disease and showed clinical symptoms had balanced translocation but the mother's parents showed no signs of renal cysts and were cytogenetically normal. It was later discovered that there was a breakpoint in the isolated gene, which caused mutations and ultimately caused PKD1 disease. Translocation or inversion may not only promote aberrations; certain conditions like deletions also favor chromosomal aberrations, which can simultaneously favor positional cloning. “Contiguous gene syndrome” is a disease in which few or many genes are deleted in a chromosome, affecting important organs in the body. One such example is mental retardation which is often due to deletion of large genes from the chromosome. In some diseases caused by deletions, the site of deletions has been found to be close to the region of the disease-causing gene, which becomes easy to identify the candidate gene as it is close to the regions flanking the disease. deletion break. point.To detect the level of chromosomal abnormality, comparative genomic hybridization (CGH) is commonly used. In this process, comparative oligonucleotide array fluorescence probes are used to in situ hybridize a DNA sample from a patient cell and a reference (control) sample. The software images tell us about the ratio of the two fluorescence signals that differ in both the reference DNA and the patient sample. From there we can detect the amount of loss or gain of DNA material or any type of translocation imbalance, inversion, duplication and even presence of aneuploidy in the genome. Recently, it is used to analyze and detect genes involved in tumor formation. . A newly identified gene, WTX, present on the X chromosome, causes Wilms tumor in children. The researchers looked for changes in DNA copy number in 51 tumor samples by performing CGH. It was found that there were deletions in chromosome Genetic linkage analysis Using genetic linkage analysis, we can also identify mutations, whether it is a simple Mendelian trait or a complex trait. . This procedure involves single markers or multiple markers for sibling bonding. This was only possible in limited applications. Thanks to this technique, we use genetic markers that allow us to understand the characteristics of independent chromosome segregation during meiosis,which may further result in maternal and parental counterparts. DNA microsatellites such as single nucleotide polymorphisms (SNPs) and short tandem repeats are used to locate the gene of interest according to the designed microsatellites. SNPs have the most common variation in their DNA sequence, where a single nucleotide (A, T, G, C) can be changed, which also results in a change in the genome as well as the phenotype of individuals. Such SNPs can be found in both coding and non-coding regions and most SNPs do not have a direct effect on cellular functions. On the other hand, DNA markers are small sequences of two to four nucleotides and can be repeated several times forming tandem repeats. These tandem repeats are very useful for genetic mapping. These two elements are used for linkage analysis which depends on the variability of family members (SNPs) and the availability of the gene of interest in the individual (tandem repeats) under study . The probability from linkage analysis suggests how the diseased gene and genetic markers are linked. Candidate Region/Gene Identification The candidate gene is the region of the target gene that is associated with genetic variation as well as phenotypic variation associated with the disease. To be precise, searching for the gene causing the disease and the variation in the gene in the population can be done using genetic mapping from databases followed by a series of steps . For this procedure, mass segregant analysis is used to identify genetic markers to detect mutant phenotypes. These are two groups of phenotypes, one of them being the diseased trait and the other being the normal trait or healthy trait. Then, the two collected genomic samples are analyzed using restriction fragment length polymorphism and random amplification of polymorphic DNA (RAPD) to create DNA fragments using enzyme sites of restriction, then amplifying them by PCR followed by gel electrophoresis and sequenced by Sanger sequencing. This will help the geneticist to initially locate and detect the mutant gene compared to the wild-type phenotype and find the differences and similarities at different loci in the genome. One of these limitations is that sometimes the genes are completely unknown and cannot be cloned and geneticists also do not know the restriction sites. For such cases, paralogous and orthologous genes derived from mutant mice can be used. These genes are derived from mutant mutants and are expressed in tissues to achieve the same diseased phenotype. Using RT-PCR, Northern Blot, Southern Blot, in situ hybridization and expression arrays, the candidate gene database is identified. Once candidate genes and their sequence are identified, they can be used as genetic markers to locate mutant gene regions in the diseased individual, and can be tested for the presence of polymorphisms. The work is still not finished after identifying the presence. of pathogenic genes in the individual. The human genome contains introns that are spliced ​​while exons remain. The “Fragment Exon Trapping” process makes it possible to locate the exons of the patient’s genome. The candidate region fragment is inserted into the intron version of a "splicing expression vector" which contains a known exon segment. When the vector is expressed, it separates the introns from the inserted genome fragment, forming a mature mRNA. The mRNA is collected to detect whether the size of the mRNA has increased, indicating that the disease-causing region is present and.