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Teaching molecular genetics: chapter 4-positional cloning of genetic disorders.

Puliti A, Caridi G, Ravazzolo R, Ghiggeri GM - Pediatr. Nephrol. (2007)

Bottom Line: The genetic markers define an interval that is a function of their recombination frequencies with the disease, in which the disease gene is localised.The step following the definition of a critical genomic region is the identification of candidate genes that is based on the analysis of available databases from genome browsers.More often, positional cloning ends with the generation of mice with homologous mutations reproducing the human clinical phenotype.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, Istituto G. Gaslini, Genoa, Italy.

ABSTRACT
Positional cloning is the approach of choice for the identification of genetic mutations underlying the pathological development of diseases with simple Mendelian inheritance. It consists of different consecutive steps, starting with recruitment of patients and DNA collection, that are critical to the overall process. A genetic analysis of the enrolled patients and their families is performed, based on genetic recombination frequencies generated by meiotic cross-overs and on genome-wide molecular studies, to define a critical DNA region of interest. This analysis culminates in a statistical estimate of the probability that disease features may segregate in the families independently or in association with specific molecular markers located in known regions. In this latter case, a marker can be defined as being linked to the disease manifestations. The genetic markers define an interval that is a function of their recombination frequencies with the disease, in which the disease gene is localised. The identification and characterisation of chromosome abnormalities as translocations, deletions and duplications by classical cytogenetic methods or by the newly developed microarray-based comparative genomic hybridisation (array CGH) technique may define extensions and borders of the genomic regions involved. The step following the definition of a critical genomic region is the identification of candidate genes that is based on the analysis of available databases from genome browsers. Positional cloning culminates in the identification of the causative gene mutation, and the definition of its functional role in the pathogenesis of the disorder, by the use of cell-based or animal-based experiments. More often, positional cloning ends with the generation of mice with homologous mutations reproducing the human clinical phenotype. Altogether, positional cloning has represented a fundamental step in the research on genetic renal disorders, leading to the definition of several disease mechanisms and allowing a proper diagnostic approach to many conditions.

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Genetic markers: DNA single nucleotide polymorphisms and microsatellites. a Example of SNP. Left Fragments of DNA with the same sequence containing a difference only in a single nucleotide. The two alleles (G and T) give rise to three possible genotypes: TT homozygous, T/G heterozygous and GG homozygous. On the right-hand side: example of SNP hypothetical segregation in a pedigree. b Example of DNA microsatellite. Left Fragments of DNA with the same sequence containing a (CA) microsatellite. The polymorphism consists of different numbers of the dinucleotide (CA) present in each of the four possible alleles represented (N = 10 to N = 13). Right Example of DNA microsatellite polymorphism hypothetical segregation in a pedigree
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Fig2: Genetic markers: DNA single nucleotide polymorphisms and microsatellites. a Example of SNP. Left Fragments of DNA with the same sequence containing a difference only in a single nucleotide. The two alleles (G and T) give rise to three possible genotypes: TT homozygous, T/G heterozygous and GG homozygous. On the right-hand side: example of SNP hypothetical segregation in a pedigree. b Example of DNA microsatellite. Left Fragments of DNA with the same sequence containing a (CA) microsatellite. The polymorphism consists of different numbers of the dinucleotide (CA) present in each of the four possible alleles represented (N = 10 to N = 13). Right Example of DNA microsatellite polymorphism hypothetical segregation in a pedigree

Mentions: The logical basis for genetic mapping lies in the occurrence of genetic recombination of maternal and paternal homologues, which results from crossing over during meiosis. Linkage analysis tests whether specific disease characteristics segregate at meiosis independently or in association with genetic markers widespread in the different segments of the human genome. DNA single nucleotide polymorphisms (SNPs) and short tandem repeats, also called DNA microsatellites, are utilised as markers of the segment of DNA of interest. SNPs are the most common form of DNA sequence variation, occurring when a single nucleotide—A, T, C, or G—in the genome differs among individuals or between paired chromosomes in an individual. Changes can occur in both coding (gene) and non-coding regions of the genome, most of them showing no effect on cell function. DNA microsatellite markers consist of short sequences, typically from one to four nucleotides, repeated in tandem several times and therefore forming highly informative multiple alleles. On the other hand, SNPs are less informative and less useful for human genetic mapping (Fig. 2). In spite of this, SNPs have the advantage of being more diffusely present than microsatellites in the human genome (one every 100 to 300 bases compared with one every 1,000–3,000). In addition, while microsatellite analysis requires DNA sequencing, SNPs may be rapidly characterised by using DNA arrays. It is clear, however, that the choice of using SNP or microsatellites in linkage analysis depends on their availability in the genomic region of interest and on the variability among the members of families under study [1].Fig. 2


Teaching molecular genetics: chapter 4-positional cloning of genetic disorders.

Puliti A, Caridi G, Ravazzolo R, Ghiggeri GM - Pediatr. Nephrol. (2007)

Genetic markers: DNA single nucleotide polymorphisms and microsatellites. a Example of SNP. Left Fragments of DNA with the same sequence containing a difference only in a single nucleotide. The two alleles (G and T) give rise to three possible genotypes: TT homozygous, T/G heterozygous and GG homozygous. On the right-hand side: example of SNP hypothetical segregation in a pedigree. b Example of DNA microsatellite. Left Fragments of DNA with the same sequence containing a (CA) microsatellite. The polymorphism consists of different numbers of the dinucleotide (CA) present in each of the four possible alleles represented (N = 10 to N = 13). Right Example of DNA microsatellite polymorphism hypothetical segregation in a pedigree
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908434&req=5

Fig2: Genetic markers: DNA single nucleotide polymorphisms and microsatellites. a Example of SNP. Left Fragments of DNA with the same sequence containing a difference only in a single nucleotide. The two alleles (G and T) give rise to three possible genotypes: TT homozygous, T/G heterozygous and GG homozygous. On the right-hand side: example of SNP hypothetical segregation in a pedigree. b Example of DNA microsatellite. Left Fragments of DNA with the same sequence containing a (CA) microsatellite. The polymorphism consists of different numbers of the dinucleotide (CA) present in each of the four possible alleles represented (N = 10 to N = 13). Right Example of DNA microsatellite polymorphism hypothetical segregation in a pedigree
Mentions: The logical basis for genetic mapping lies in the occurrence of genetic recombination of maternal and paternal homologues, which results from crossing over during meiosis. Linkage analysis tests whether specific disease characteristics segregate at meiosis independently or in association with genetic markers widespread in the different segments of the human genome. DNA single nucleotide polymorphisms (SNPs) and short tandem repeats, also called DNA microsatellites, are utilised as markers of the segment of DNA of interest. SNPs are the most common form of DNA sequence variation, occurring when a single nucleotide—A, T, C, or G—in the genome differs among individuals or between paired chromosomes in an individual. Changes can occur in both coding (gene) and non-coding regions of the genome, most of them showing no effect on cell function. DNA microsatellite markers consist of short sequences, typically from one to four nucleotides, repeated in tandem several times and therefore forming highly informative multiple alleles. On the other hand, SNPs are less informative and less useful for human genetic mapping (Fig. 2). In spite of this, SNPs have the advantage of being more diffusely present than microsatellites in the human genome (one every 100 to 300 bases compared with one every 1,000–3,000). In addition, while microsatellite analysis requires DNA sequencing, SNPs may be rapidly characterised by using DNA arrays. It is clear, however, that the choice of using SNP or microsatellites in linkage analysis depends on their availability in the genomic region of interest and on the variability among the members of families under study [1].Fig. 2

Bottom Line: The genetic markers define an interval that is a function of their recombination frequencies with the disease, in which the disease gene is localised.The step following the definition of a critical genomic region is the identification of candidate genes that is based on the analysis of available databases from genome browsers.More often, positional cloning ends with the generation of mice with homologous mutations reproducing the human clinical phenotype.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, Istituto G. Gaslini, Genoa, Italy.

ABSTRACT
Positional cloning is the approach of choice for the identification of genetic mutations underlying the pathological development of diseases with simple Mendelian inheritance. It consists of different consecutive steps, starting with recruitment of patients and DNA collection, that are critical to the overall process. A genetic analysis of the enrolled patients and their families is performed, based on genetic recombination frequencies generated by meiotic cross-overs and on genome-wide molecular studies, to define a critical DNA region of interest. This analysis culminates in a statistical estimate of the probability that disease features may segregate in the families independently or in association with specific molecular markers located in known regions. In this latter case, a marker can be defined as being linked to the disease manifestations. The genetic markers define an interval that is a function of their recombination frequencies with the disease, in which the disease gene is localised. The identification and characterisation of chromosome abnormalities as translocations, deletions and duplications by classical cytogenetic methods or by the newly developed microarray-based comparative genomic hybridisation (array CGH) technique may define extensions and borders of the genomic regions involved. The step following the definition of a critical genomic region is the identification of candidate genes that is based on the analysis of available databases from genome browsers. Positional cloning culminates in the identification of the causative gene mutation, and the definition of its functional role in the pathogenesis of the disorder, by the use of cell-based or animal-based experiments. More often, positional cloning ends with the generation of mice with homologous mutations reproducing the human clinical phenotype. Altogether, positional cloning has represented a fundamental step in the research on genetic renal disorders, leading to the definition of several disease mechanisms and allowing a proper diagnostic approach to many conditions.

Show MeSH
Related in: MedlinePlus