Simple Mathematical Model Of Pathologic Microsatellite Expansions: When Self-Reparation Does Not Work

We propose a simple model of pathologic microsatellite expansion, and describe an inherent self-repairing mechanism working against expansion. We prove that if the probabilities of elementary expansions and contractions are equal, microsatellite expansions are always self-repairing. If these probabilities are different, self-reparation does not work. Mosaicism, anticipation and reverse mutation cases are discussed in the framework of the model. We explain these phenomena and provide some theoretical evidence for their properties, for example the rarity of reverse mutations

Polymerase Chain Reaction (PCR): Principle and Applications

The characterization of the diversity of species living within ecosystems is of major scientific interest to understand the functioning of these ecosystems. It is also becoming a societal issue since it is necessary to implement the conservation or even the restoration of biodiversity. Historically, species have been described and characterized on the basis of morphological criteria, which are closely linked by environmental conditions or which find their limits especially in groups where they are difficult to access, as is the case for many species of microorganisms. The need to understand the molecular mechanisms in species has made the PCR an indispensable tool for understanding the functioning of these biological systems. A number of markers are now available to detect nuclear DNA polymorphisms. In genetic diversity studies, the most frequently used markers are microsatellites. The study of biological complexity is a new frontier that requires high-throughput molecular technology, high speed computer memory, new approaches to data analysis, and the integration of interdisciplinary skills

Manual on application of molecular tools in aquaculture and inland fisheries management. Part 2. Laboratory protocols and data analysis

The aim of this manual is to provide a comprehensive practical tool for the generation and analysis of genetic data for subsequent application in aquatic resources management in relation to genetic stock identification in inland fisheries and aquaculture. The material only covers general background on genetics in relation to aquaculture and fisheries resource management, the techniques and relevant methods of data analysis that are commonly used to address questions relating to genetic resource characterisation and population genetic analyses. No attempt is made to include applications of genetic improvement techniques e.g. selective breeding or producing genetically modified organisms (GMOs). The manual includes two ‘stand-alone’ parts, of which this is the second volume: Part 1 – Conceptual basis of population genetic approaches: will provide a basic foundation on genetics in general, and concepts of population genetics. Issues on the choices of molecular markers and project design are also discussed. Part 2 – Laboratory protocols, data management and analysis: will provide step-by-step protocols of the most commonly used molecular genetic techniques utilised in population genetics and systematic studies. In addition, a brief discussion and explanation of how these data are managed and analysed is also included. This manual is expected to enable NACA member country personnel to be trained to undertake molecular genetic studies in their own institutions, and as such is aimed at middle and higher level technical grades. The manual can also provide useful teaching material for specialised advanced level university courses in the region and postgraduate students. The manual has gone through two development/improvement stages. The initial material was tested at a regional workshop and at the second stage feedback from participants was used to improve the contents

Quantifying Intestinal Stem Cell Dynamics Using Microsatellite Sequencing

The intestinal epithelium is rapidly renewing throughout life. A population of stem cells exist within the intestinal crypt that drive rapid cell renewal and replace each other by a pattern of neutral drift. Perturbation of these dynamics through oncogenic mutation can predispose the epithelium to neoplastic transformation. Understanding the factors that govern these dynamics will give insight into the early stages of oncogenesis. Continuous clonal labelling, whereby DNA strand slippage leading to the contraction or expansion of a microsatellite during mitotic replication, can be employed to enable the detection of a single clone. Previous studies have shown that quantification of clone size over time allows inference of the functional stem cell number and stem cell replacement rate within intestinal crypts. Current continuous labelling techniques require the introduction of a transgenic microsatellite into a model system genome, such as mouse, that leads to reporter expression following mutation. This obviously precludes human studies. Alternative somatic alterations techniques used for continuous labelling in humans requires spontaneous loss of a protein, or change in methylation status, within a single clone that can act as a clonal mark. Though these techniques have given insight into the spread of mutations within the intestinal epithelium and enabled inference of adenoma clonality, the true neutrality of these changes are currently unknown. We propose that the small changes in endogenous microsatellite length will act as a neutral clonal mark within the intestinal epithelium and allow an unbiased approach to quantifying intestinal stem cell dynamics in human intestinal tissues. To overcome the many technical challenges associated with accurate measurement of microsatellite length, a stepwise approach was taken to develop a technique for the multiplexed high throughput sequencing of up to 21 native dinucleotide repeats in hundreds of single crypts. Furthermore, a novel method was developed for the quantification of clone size from data generated from the targeted re-sequencing of microsatellites in single crypts. This protocol was validated in vitro and in vivo in mouse. Furthermore, proof of principle sequencing in human crypts was performed to show that this method is suitable for larger scale quantification of intra-cryptal clone size in human tissue. This, and similar approaches, may be the only way to quantify intestinal stem cell dynamics within the healthy human colon or, dysplastic or adenomatous patient tissue. These measurements should give a unique insight into the dynamics of healthy, pre-neoplastic and neoplastic human intestinal stem cells.Cancer Research UK University of Cambridg

Molecular Mapping In Tropical Maize (zea Mays L.) Using Microsatellite Markers. 1. Map Construction And Localization Of Loci Showing Distorted Segregation.

Microsatellites have become the most important class of markers for mapping procedures. Primarily based on restriction fragment length polymorphism (RFLP) markers, several molecular genetic maps of maize have been developed, mainly using temperate inbred maize lines. To characterize the level of polymorphism of microsatellite loci and construct a genetic map in tropical maize, two elite inbred lines, L-08-05F and L-14-4B, were crossed to produce 400 F(2) individuals that were used as a mapping population. A survey of 859 primer pair sequences of microsatellites was used. The polymorphism screens of each microsatellite and genotype assignment were performed using high-resolution agarose gels. About 54 % of the primer sets gave clearly scorable amplification products, 13 % did not amplify and 33 % could not be scored on agarose gels. A total of 213 polymorphic markers were identified and used to genotype the mapping population. Among the polymorphic markers, 40 showed loci deviating from expected Mendelian ratios and clusters of deviating markers were located in three chromosome regions. Non-Mendelian scoring was present in 19 markers. The final genetic map with 117 markers spanned 1634 cM in length with an average interval of 14 cM between adjacent markers.13996-10

Fine-scale genetic structure and parentage in Urocitellus beldingi

Urocitellus beldingi (Belding\u27s ground squirrel), previously known as Spermophilus beldingi, is a social, montane rodent that occupies alpine and subalpine meadows in the Sierra Nevadas (Helgen et al. 2009). The Tioga Pass meadow (Mono Co. CA) population has been studied behaviorally and demographically for several decades. Microsatellites were developed for this species and conditions for polymerase chain reaction (PCR) were defined. Nine microsatellite loci amplified adequately and were in Hardy-Weinberg equilibrium, displaying an average heterozygosity of 0.67±0.19. These nine microsatellite loci were analyzed via PCR to elucidate the fine scale genetic structure, offering insight into population health, stochastic events, and dispersal. Two populations were identified, corresponding to the elevation differences between the sites (FST = 0.054605 pm) of 8.66. These populations appear to maintain a stable size and are not adversely affected by inbreeding. Urocitellus beldingi maternity can be deduced by observing female burrows usage during gestation and which offspring first emerge from the burrow associated with that particular female. Paternity, however, can only be posited by observation of mating because the species exhibits multiple mating in both sexes, though not all matings necessarily result in offspring. Multiple paternity has been previously verified through allozyme analysis. Microsatellites were used to verify maternity and determine paternity. Analysis of ten litters demonstrated an average of 1 father per 1.35 pups in a litter. Sibship deduced from maternity and paternity analysis was then compared with previously collected behavioral data. While previous observations indicate preference of fullsib play partners in juveniles, our findings do not corroborate this, though the results were not significant