Index IntroductionData and sample collectionCytogenetic studyGenetic linkage analysisIdentification of candidate genes/regionsFinal 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 which the product of that gene is transcribed and translated. The reason for knowing the location of the gene is very important because in a population this product can present different phenotypes, each of which could have an advantage over the other. To locate the gene, scientists introduced a molecular process, which involves genetic mapping, forward and reverse mutations using techniques such as suitable marker genes and other procedures, and other sets of techniques such as cross-species hybridization, trapping of potential exon fragments, Characterization of methylated/unmethylated CpG islands which are applied overall in “Positional Cloning”. Say no to plagiarism. Get a tailor-made essay on "Why Violent Video Games Shouldn't Be Banned"? Get an original essay Not only can we understand the functions and location of the gene and its corresponding product, we can also study any disease-associated gene, which results in a defective or pathogenic protein, or even no protein production at all. Therefore, the main purpose of "Positional Cloning" is to identify any genetic disorders and their location that have been passed on to the next generation using the idea of ​​Mendelian inheritance, through which we can trace the origin. In short, the procedures involve selecting a disease-causing gene and pinpointing its location, followed by the types of mutations in that gene and its corresponding phenotypes. Apart from this, there is a replacement method that includes reverse genetics, where genotypes are recognized followed by phenotype characterization. The very first application of this technique was the identification of a pathogenic gene in the human genome. Later, in 1986, this became a common procedure to identify any type of genetic mutation that causes chronic granulomatous disease. Using positional cloning, many human genetic diseases have been identified that can be passed on to the next generation, such as cystic fibrosis, Duchenne muscular dystrophy, fragile X syndrome, and even breast cancer. Data and Sample Collection It is very important to have correct data because any inappropriate data collection can lead to incorrect analysis. This data collection includes all types of genetic disorders known in a patient and their family members. All these together can help the patient get an idea about the genetic mutations and clinical treatments corresponding to a specific genetic disease. This procedure also includes in-depth clinical studies on family members presenting with the same clinical symptoms. This type of data collection is commonly known as “genealogical analysis.” Pedigree analysis must ensure that complex traits such as environmental factors are not associated with the Mendelian concept, which is also part of the clinical study. To carry out the research it is necessary to collect DNA samples from all the patient's family members, in particular from those included in the genealogical analysis. Cytogenetic study The first task is the identification of the pathogenic gene in patients presenting with chromosomal anomalies. Translocations or inversions of DNA segments are targeted in this procedure because they do not cause any loss or gain of DNA material in a patient. Even if no DNA fragments are lost acause of balanced translocation, there are some incidents where the abnormal phenotype caused by balanced translocation suggests that this feature is not balanced and eventually some amount of DNA may be lost during chromosomal rearrangement. On the other hand, there is evidence, which suggests that the balanced translocation may enhance the disease gene to become active or may 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. Furthermore, the mother and daughter who suffered from this disease and showed clinical symptoms had a 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 the PKD1 disease. Translocation or inversion cannot only promote aberrations; some conditions such as deletions also promote chromosomal aberrations, which can simultaneously support positional cloning. “Contiguous gene syndrome” is a condition in which a few or more genes are deleted in a chromosome, affecting major organs in the body. One such example is mental retardation which is often caused by the deletion of large genes from the chromosome. In some diseases caused by deletions, the site of the deletions was found to be a short distance from the region of the gene causing the disease, which makes it easy to identify the candidate gene since it will be close to the flanking regions of the deletion breaking point. To detect the level of chromosomal abnormality, comparative genomic hybridization (CGH) is commonly used. In this process, comparative fluorescent oligonucleotide probes are used to in situ hybridize the DNA sample from a patient cell and a reference (control) sample. The images coming from a software tell us the relationship between the two fluorescence signals that differ both in the reference DNA and in the patient sample. From this, we can detect the amount of loss or gain of DNA material or any type of translocation, inversion, duplication and even presence of aneuploidy in the genome. Recently, it is used to analyze and detect genes involved in tumor formation. A recently identified gene, WTX, found on the X chromosome, causes Wilms tumor in children. The researchers looked for changes in DNA copy number in 51 tumor samples by conducting CGH. It was found that there were deletions in chromosome Xq11.1 that cause WTX to increase copy number in male patients with Wilms tumor causing nephroblastomas. Genetic Linkage Analysis Using genetic linkage analysis, we can also identify mutations whether it was a simple Mendelian trait or a complex trait. This procedure involves single or multiple indicators for sibling linkage. This has only been possible in limited applications. Through this technique, genetic markers are implicated along which we can understand the characteristics of independent chromosome segregation at meiosis which can further result in maternal and parental homologs. DNA microsatellites such as single nucleotide polymorphisms (SNPs) and short tandem repeats are used to locate the gene of interest depending on the designed microsatellites. SNPs have the most common variation in the DNA sequence in which a single nucleotide (A, T, G, C) can be changed, whichit also involves a change in the individual's genome and phenotype. Such SNPs can be found in both coding and noncoding regions, and most SNPs do not have a direct effect on cellular functions. On the other hand, DNA markers are small sequences of two or four nucleotides and can be repeated numerous times forming tandem repeats. These tandem repeats are very useful for genetic mapping. Both 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 resulting from linkage analysis suggests how the diseased gene and the genetic markers are related. Candidate Region/Gene IdentificationThe candidate gene is the target gene region associated with genetic variation along with phenotypic variation associated with disease. To be precise, you can search for the gene that causes the disease and the variation of the gene in the population using genetic database mapping followed by a series of steps. For this procedure, bulk segregant analysis is used to identify genetic markers to detect mutant phenotypes. It involves two groups of phenotypes where one of them is the diseased trait and the other is the normal trait or the healthy trait. Then both collected genomic samples are analyzed using restriction fragment length polymorphism and random amplification of polymorphic DNA (RAPD) to create DNA fragments using restriction enzyme sites and then amplifying them using PCR followed by gel electrophoresis and sequenced by Sanger sequencing. This will help the geneticist to initially localize and detect the mutant gene compared to the wild type phenotype and find the differences and similarities in various loci of the genome. One of these limitations is that, sometimes, the genes are completely unknown so it is not possible to identify them. cloned, nor do geneticists know the restriction sites. For such cases, paralogous and orthologous genes derived from the mutant mouse can be used. These genes are derived from mutant mutants and expressed in tissues to achieve the same diseased phenotype. Using RT-PCR, Northern Blot, Southern Blot, in situ hybridization and Expression array the database of candidate genes 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 not yet complete after identifying the presence of the gene that causes the disease in the individual. The genome of a human contains introns that are separated while exons remain. The “Fragment Exon Trapping” process is used to locate exons from the patient's genome. The candidate region fragment is inserted into the intron version of a “splicing expression vector” that contains a known exon segment. When the vector is expressed, it will splice introns from the inserted genome fragment into a mature mRNA. The mRNA is collected to detect whether the size of the mRNA has increased, which indicates that the disease-causing region is present and expressed. Final Mapping Now all the information of the disease-causing gene is collected together, such as genetic markers collected by RT-PCR, blotting and expression arrays, and DNA sequence by Sanger sequencing, probes can be designed based on the complementary region and can be used to locate the DNA segment with high specificity. For this process, fluorescent in situ hybridization is the common procedure to locate the segment. The.