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About this sample
About this sample
Words: 1915 |
Pages: 4|
10 min read
Published: Apr 11, 2019
Words: 1915|Pages: 4|10 min read
Published: Apr 11, 2019
Understanding 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 from that gene is being transcribed and translated. The reason to know the location of the gene is very important that in a population this product can various phenotypes, each of which might have an advantage over the other. In order to locate the gene, scientists have introduced a molecular process, which involves genetic mapping, forward and reverse mutations that uses techniques such as suitable marker genes and other procedures and other series of techniques like cross-species hybridization, potential exons fragment trapping, methylated/unmethylated CpG island characterization which altogether are applied in ‘Positional Cloning’.
Not only we can 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 protein or disease-causing protein, or even no protein production. Hence, the main purpose of ‘Positional Cloning’ is to identify any genetic disorders and its location that has passed on to the next generation using the idea of Mendelian inheritance, through which we can trace the origin. In short, the procedures involve selection of 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 substitute method that includes inverse genetics, where genotypes are recognized followed by phenotype characterization.
The very first application of this technique was the identification of a disease-causing gene in human genome. This later in 1986 became a common procedure to identify any sorts of genetic mutation, which causes Chronic Granulomatous disease. Using Positional Cloning, many human genetic diseases that can be passed onto next generation have been identified such as cystic fibrosis, Duchenne muscular dystrophy, fragile X syndrome and also breast cancer.
It is very important to have correct data because any inappropriate collection of data can result in the wrong analysis. This collection of data includes all sorts of known genetic disorders in a patient as well as in that individual’s family members. All these together can help patient get ideas about his or her gene mutations and clinical treatments corresponding to a specific genetic disease. This procedure also includes extensive clinical study of family members baring the same clinical symptoms. This type of data collection is commonly known as ‘Pedigree analysis.’ Pedigree analysis must make sure that complex traits such as environmental factors should not be associated with the Mendelian concept, which is also a part of the clinical study. To carry on the research, DNA samples must be collected from all the family members of the patient especially who were accounted in the Pedigree Analysis.
Identifying the disease-causing gene from the patients who have chromosomal anomalies is the first task. Translocations or inversion 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 due to balanced translocation, there are some incidents where the abnormal phenotype caused by balanced translocation suggested that this characteristic is not balanced and ultimately some DNA quantity might be lost during chromosome rearrangement.
On the other hand, there is evidence, which suggested that the balanced translocation either can enhance a diseased gene to become active or might 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 encodes a 14-kb transcript was found to be damaged by chromosome translocation. Furthermore, the mother and daughter who were suffering from this disease and showing clinical symptoms have a balanced translocation but the parents of the mother showed no signs of renal cysts and were cytogenetically normal. It was later found that there was a breakpoint in the isolated gene, which caused mutations and finally caused the PKD1 disease.
Translocation or inversion cannot only promote aberrations; certain conditions like deletions also promote chromosomal aberrations, which can simultaneously support positional cloning. ‘Contiguous Gene Syndrome’ is a condition where few or several genes are deleted in a chromosome, which affects important organs in the body. One such example is Mental Retardation that is often caused due to deletions of large genes from the chromosome. In certain diseases which are caused by deletions, it was found that the site of deletions was in close distance with the region of the disease causing gene, which becomes easy to identify the candidate gene as they will be near 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 oligonucleotide-array fluorescence probes are used to in situ hybridizes DNA sample from a patient cell and a reference sample (control). Images from a software tells us about the ratio of the two fluorescence signals which differ in both reference and patient sample DNA. From this, we can detect the amount of loss or gain of DNA material or any kinds of imbalance translocation, inversion, duplication, and even presence aneuploidy in the genome.
Recently, is used to analyze and detect genes, which are involved in tumor formation. A newly identified gene, WTX found on chromosome X cause Wilms tumor in children. 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 causes the WTX to increase the copy number in Wilms tumor male patients causing nephroblastomas.
Using genetic linkage analysis, we can also identify the mutations whether it was simple Mendelian trait or complex trait. This procedure involves single markers or multiple markers to the linkage in siblings. This was possible in only limited applications. Through this technique involves genetic markers along where we can understand the characteristics of independent chromosomal segregation at meiosis that can further result in maternal and parental homologues.
DNA microsatellites such as Single Nucleotide Polymorphisms (SNP) and Short Tandem Repeats are used to locate the gene of interest according to the designed microsatellites. SNP have the most common variation in their DNA sequence where one single nucleotide (A, T, G, C) can be changed which also brings a changed in the genome as well as the individuals phenotype. Such SNP can be found in both coding and non-coding regions and most of the SNPs’ do not have a direct effect on the cell functions.
On the other hand, DNA markers are small sequences of two-to-four nucleotides and can be repeated numerous times forming tandem repeats. These tandem repeats are very useful for genetic mapping. Both of these are used for linkage analysis which depends on the variability of the family members (SNP) and availability of the gene of interest in the individual (tandem repeats) who are under the study. The probability from linkage analysis suggests how the diseased gene and the genetic markers are related.
Candidate gene is the target gene region which is associated with genetic variation along with phenotype variation that is associated with the disease. To be precise, looking for the disease-causing gene and the variation of the gene in the population which can be done using gene mapping from databases followed by series of steps.
For this procedure, Bulk Segregant Analysis is used to identify gene 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 healthy trait. Then both of the collected genomic samples are analyzed using Restriction Fragments Length Polymorphism and Random Amplification of Polymorphic DNA (RAPD) to make DNA fragments using restriction enzymatic sites and then amplifying it using 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 various loci of the genome.
One such limitation is that, sometimes the genes are completely unknown for which it cannot be cloned nor do geneticists know the restriction sites. For such cases, paralogous and orthologous genes derived from mutant mouse can be used. These genes 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 arrays the database of the candidate genes are identified. Once the candidate genes and their sequence are identified, they can be used as genetic markers to locate the regions of the mutant gene in the diseased individual, and can be tested for the presence of polymorphisms.
The work is still not complete after identifying the presence of disease-causing gene in the individual. The genome of a human contains introns which are spliced out while the exons remains. The process ‘Fragment Exon Trapping” is used to locate the exons from the genome of the patient. The candidate region fragment is inserted into the intron version of a ‘splicing expression vector’ that has a known exon segment in it. When the vector is expressed, it will splice out the introns from the inserted genome fragment forming a mature mRNA. mRNA is collected to detect if the size of mRNA increased which indicated that the disease-causing region is present and expressed.
Now all the information of the disease causing gene are gather together, such as the genetic markers collected from RT-PCR, blotting and expression arrays and DNA sequence from Sanger sequencing, probes can be designed according to the complementary region and can be used to localize the DNA segment with high specificity. For this process, Fluorescence in situ Hybridization is the common procedure to locate the segment. The probes are attached with a either a radioactive label (32P) or a fluorescence molecule (Digoxenin).
The samples are washed with DAPI (4`,6-diamidino-2-phenylindole) and digoxygenin-labelled probes. The probes bind to the specific segments at the metaphase situation while DAPI strongly binds to the A-T rich regions. These are later observed under fluorescence microscope where the probes will bind to the target genes which are basically the disease causing genes. Then by the process of gene mapping, position of the target gene can be determined whether it is in the long arm or short arm or near the centromere, and so on and even we can study on which chromosome the gene is located according to mutational analysis. This is how geneticists use all the procedures in a Positional Cloning technique to localize a gene.
Through the techniques of positional cloning, recently many genes are being identified that are causing various nephritic syndromes. Through mutational analysis, candidate genes are targeted in nephrology because mutational analysis shows use the frameshit mutations as well as any sort of non-sense recombination. Mutated Phospholipase c Epsilon (PLCE1) shows abnormal production of protein in nephrons.
In order to locate the gene, Zebrafish with a functional PLCE1 protein activity in glomeruli was selected that was later knocked down artificially to study the effect on the organism itself. In a knock-out mouse model, the mouse showed no signs of abnormal renal phenotype because of the presence of modifier genes for which the mouse made use of it to enhance the protein activity. This is one challenge when using animal models for human diseases. As a result, Zebrafish was used to locate and sequence PLCE1 gene in diseased human. In humans, the gene location on the chromosome is 10q23.32-q24.1 which contains more 43 genes.
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