Microsatellite Length Research Articles

Genetic evidence that both dNTP-stabilized and strand slippage mechanisms may dictate DNA polymerase errors within mononucleotide microsatellites.

Mononucleotide microsatellites are tandem repeats of a single base pair, abundant within coding exons and frequent sites of mutation in the human genome. Because the repeated unit is one base pair, multiple mechanisms of insertion/deletion (indel) mutagenesis are possible, including strand-slippage, dNTP-stabilized, and misincorportion-misalignment. Here, we examine the effects of polymerase identity (mammalian Pols α, β, κ, and η), template sequence, dNTP pool size, and reaction temperature on indel errors during in vitro synthesis of mononucleotide microsatellites. We utilized the ratio of insertion to deletion errors as a genetic indicator of mechanism. Strikingly, we observed a statistically significant bias toward deletion errors within mononucleotide repeats for the majority of the 28 DNA template and polymerase combinations examined, with notable exceptions based on sequence and polymerase identity. Using mutator forms of Pol β did not substantially alter the error specificity, suggesting that mispairing-misalignment mechanism is not a primary mechanism. Based on our results for mammalian DNA polymerases representing three structurally distinct families, we suggest that dNTP-stabilized mutagenesis may be an alternative mechanism for mononucleotide microsatellite indel mutation. The change from a predominantly dNTP-stabilized mechanism to a strand-slippage mechanism with increasing microsatellite length may account for the differential rates of tandem repeat mutation that are observed genome-wide.

Genotyping microsatellites in next-generation sequencing data

Background Microsatellites are short (2-6bp) DNA sequences repeated in tandem, which make up approximately 3% of the human genome [1]. These loci are prone to frequent mutations and high polymorphism with the estimated mutation rates of 10 10 events per locus per generation, orders of magnitude higher than other parts of the genome [2]. Dozens of neurological and developmental disorders have been attributed to microsatellite expansions [3]. Microsatellites have also been implicated in a range of functions such as DNA replication and repair, chromatin organisation and regulation of gene expression [4]. Traditionally, microsatellite variation has been measured using capillary gel electrophoresis [5]. In addition to being time-consuming, and expensive, this method fails to reveal the full complexity at these loci because it does not directly sequence the fragment but only measure the number of bases in the repeat. Next-generation sequencing has the potential to address these problems. However, determining microsatellite lengths using next-generation sequencing data is difficult. In particular, polymerase slippage during PCR amplification introduces stutter noise. A small number of software tools have been written to genotype simple microsatellites in next-generation sequencing data [6-8], however they fail to address the issues of SNPs and compound repeats, and in some cases provide only approximate genotypes. We have begun to develop a microsatellite genotyping algorithm that addresses these issues, providing high accuracy as well as more detailed analysis of microsatellite loci. We have validated it using high depth amplicon sequencing data of microsatellites near the AVPR1A gene.

Polymorphism of the IPK1 gene among members of the genus Glycine

The structural polymorphism of the IPK1 gene involved in the biosynthesis of phytic acid was studied in 30 samples from three species of the genus Glycine (G. max, G. soja, and G. gracilis). Single nucleotide polymorphisms in the sequences of the first and the second introns of the IPK1 gene were found, differences in the length ofmicrosatellite loci located in intron 2 were demonstrated, and a total of six polymorphic variants were identified. Dataon the distribution of the identified alleles among the samples from the working set and the analysis and discussion of these data are given.

Microsatellite Instability Testing in Colorectal Carcinoma: A Practical Guide

In this month’s “Road Ahead” article, we continue last month’s theme that focused on the role of pathology in the care of our GI patients. Many gastroenterologists have successfully incorporated pathology into their core practices either within a business infrastructure or as part of a larger health care system. Dr Gibson and her colleagues at Yale University School of Medicine have helped inform us about a particularly difficult management problem: how to handle the genetically high-risk patients we see frequently in our hospitals and endoscopy units. New comprehensive practice guidelines concerning management of hereditary colon cancer syndromes are in development, but this short article provides a clear and concise guide for the practicing gastroenterologist.

Functional Significance of Microsatellite Markers

The review summarizes literature data on the positive results of association studies between the length of microsatellite repeats and predisposition to pathologies. Actually, the data can be classified according to the localization of the microsatellite: in the gene promoter, in the part of exon 1 coding the signal sequence, in gene introns, in the coding areas of genes, and in 3'-untranslated regions. The functional significance of microsatellite length changes can be evaluated in many cases. The authors came up to the conclusion that further studies on microsatellite associations with diseases remain prospective as they reflect changes in the gene functional activity.

The role of avpr1a microsatellite length on reproductive success of female Microtus ochrogaster

Vasopressin and its’ interactions with the vasopressin 1a receptor (V1aR) are important in the formation of social attachments and parental behavior in male mammals but there also is evidence that vasopressin is involved in maternal behavior in female mammals. Laboratory studies have shown that female rats with greater expression of V1aR in particular brain regions display more maternal behavior than those with less V1aR expression. Previous data showed that variation in neural V1aR expression in males of several species of rodents was influenced by the length of microsatellite DNA within the regulatory region of gene (avpr1a) encoding V1aR. In male prairie voles (Microtus ochrogaster), the neural expression of V1aR differs between individuals with longer versus shorter avpr1a microsatellites. Males with longer avpr1a microsatellites had greater V1aR expression in particular brain regions, spent more time with their female social partner, and licked and groomed pups more than males with shorter avpr1a microsatellite lengths. If avpr1a microsatellite length is correlated with V1aR expression in females in a similar fashion to that in male prairie voles, females with longer avpr1a microsatellites should have greater reproductive success since maternal care should be critical for offspring survival. In addition, female prairie voles may benefit by living with males that have longer avpr1a microsatellites due to increased male presence at the nest and paternal care. We tested these predictions with prairie voles from semi-natural populations. Females that had summed avpr1a microsatellite allele lengths greater than the median produced more litters and total offspring than females with summed avpr1a lengths less than the median. Females with summed avpr1a microsatellite allele lengths greater than the median also produced offspring sired by more males. The avpr1a length of male social and genetic partners did not influence female reproductive success. This is the first field study showing a relationship between avpr1a microsatellite allele length and female reproductive success in any species.

Microsatellite Instability within the CD30 Gene Promoter Distinguishes between Normal and Neoplastic Cells in Lymphoma

Aims:As differences in promoter activity and CD30 surface expression between CD30+ lymphoid cell-lines and peripheral blood leukocytes have been shown previously to be independent of the size of the CD30 promoter microsatellite, in this study we investigate the instability within the region in a range of neoplasms including malignant lymphoma, breast and colon carcinoma and lung adenocarcinoma. Study Design:A representative sample of CD30+ and CD30lymphomas and cell lines as well as non-haemopoietic malignancies and normal tissues were typed for CD30 microsatellite length and compared. Place and Duration of Study: Department of Anatomical Pathology, Pathwest Laboratory Original Research Article British Journal of Medicine & Medical Research, 4(2): 755-765, 2014 756 Medicine WA, Perth, Australia and School of Chemistry & Biochemistry, University of Western Australia, 2000-2012. Methodology: DNA was prepared from archived biopsy specimens and used to PCR ampify the CD30 microsatellite region prior to size determination using an Genescan instrument (Applied Biosystems). Results: This study has identified instability within the CD30 promoter microsatellite region in DNA from the tumour tissue of cases of malignant lymphoma, colon carcinoma and lung adenocarcinoma but not in DNA from benign lymphoid cells or normal tissues. Conclusion: These findings indicate that variability in the length of the CD30 microsatellite may be a general characteristic of the neoplastic phenotype and may reflect defects in the mismatch repair system in these malignancies rather than a specific feature of CD30positive neoplasms. Our results suggest that the CD30-MS may be a useful marker to distinguish between normal lymphoid tissue and lymphoid malignancy.

Novel MicroRNAs and Microsatellite-like Small RNAs in Sexual and Apomictic Boechera Species

Apomixis refers to plant asexual reproduction through seeds that give rise to progeny which are genotypically identical to the maternal parent. It has evolved from many different sexual taxa although the underlying genetic factors remain unknown. Previous analyses of the over-representation of transcription factors, in a comparison of microdissected ovules from apomictic and sexual Boechera, showed that many transcription factor mRNAs possessed microRNA (miRNAs) binding sites, thus pointing to miRNAs as potentially important factors that may be involved in the regulatory switch from sexual to apomictic reproduction. A microarray-based approach was used to identify (1) 673 microsatellitelike small RNAs (misRNAs) containing predominantly 2-7 repeats of (GAA)n/(CUU)n, (GCA)n/(CGU)n, (GGA)n/(CCU)n, (GGU)n/(CCA)n and (UGA)n/(ACU)n, and (2) 166 more typical non-repeat small RNAs. In total, 87 small RNAs were found to be located in cDNAs that could fold into stem-loop structures and thus represent miRNA molecules. In addition, 109 Boechera small RNAs including both misRNAs and non-repeat small RNAs, showed significant homology to 407 Arabidopsis thaliana small RNAs including the A. thaliana pollen-specific ath-miR5021. This indicates that only a fraction of the identified small RNAs are unique to Boechera. Ten small RNAs were validated using a Northern blot assay on flower and leaf tissues, eight of which showed flower-specific expression with varying abundance. The potential binding sites of many of the misRNAs and non-repeat small RNAs occur predominantly in exonic regions. This feature coupled with their flower-specific pattern of expression is suggestive of their probable role in post-transcriptional gene regulation. We propose that quantitative variation for misRNA target binding (and hence post-transcriptional gene regulation) could arise via microsatellite length polymorphisms occurring either in misRNA precursors or in their gene targets.

Effects of experience and avpr1a microsatellite length on parental care in male prairie voles (Microtus ochrogaster)

Pair-bonded males often make substantial contributions to the care of their offspring. Male parental behavior may be affected by a range of factors, including previous experience (parental or alloparental), genetic influences, and contributions by the female partner. Previous studies have shown that a microsatellite polymorphism in the regulatory region of the avpr1a gene influences aspects of paternal behavior in male prairie voles (Microtus ochrogaster). Specifically, males with longer avpr1a microsatellites groomed offspring more than did males with shorter avpr1a microsatellites. Previous experience with alloparental care also appears to influence subsequent paternal care in prairie voles. We investigated the influence of avpr1a microsatellite length and previous parental experience on paternal behavior in prairie voles two generations from the field and specially bred to exaggerate differences in avpr1a microsatellite length. We found that avpr1a microsatellite length alone did not affect any of the paternal behaviors that we measured. In contrast, males differed in parental behavior between first and second litters. Regardless of avpr1a microsatellite length, males licked/groomed the second litter less, and retrieved pups more quickly during the second compared to the first litter. Our results show that previous paternal experience may play a more important role than the length of the microsatellite in the regulatory region of the avpr1a gene in influencing paternal care.

Molecular methods for strain typing of Candida albicans : a review

Candida albicans is one of the most medically important fungi because of its high frequency as a commensal and pathogenic microorganism causing superficial as well as invasive infections. Strain typing and delineation of the species are essential for understanding its biology, epidemiology and population structure. A wide range of molecular techniques have been used for this purpose including non-DNA-based methods (multi-locus enzyme electrophoresis), conventional DNA-based methods (electrophoretic karyotyping, random amplified polymorphic DNA, amplified fragment length polymorphism, restriction enzyme analysis with and without hybridization, rep-PCR) and DNA-based methods called exact typing methods because they generate unambiguous and highly reproducible typing data (including microsatellite length polymorphism and multi-locus sequence typing). In this review, the main molecular methods used for C.albicans strain typing are summarized, and their advantages and limitations are discussed with regard to their discriminatory power, reproducibility, cost and ease of performance.

Phenotypic and Molecular Characterization of <i>Phaseolus vulgaris</i> Plants from Non-Cryopreserved and Cryopre-served Seeds

The objective of this work was to evaluate if cryostorage of Phaseolus vulgaris L. seeds induced variations in regenerated plants at the phenotypic and molecular levels. A series of agricultural traits was measured on plants grown from control, non-cryopreserved and cryopreserved seeds, and the genetic stability of plants of the second generation was analysed at selected microsatellite loci. The phenotype of the second generation plants was evaluated as well. No statistically significant phenotypic differences were observed for the parameters measured, neither in the first nor in the second generations. Averaging both treatments, about 76% of the seeds had germinated 10 days after sowing. At harvest we recorded plants with about 73 cm in height, 13 stem internodes, 25 fruits, 103 grains and 4 grains per fruit. One hundred seeds weighted about 26 g. The genetic analyses performed on the second generation plants using six nuclear Simple Sequences Repeats (SSR) markers revealed no changes in microsatellite length between control and cryopreserved samples, implying that there was no effect of seed liquid nitrogen exposure on genome integrity. The phenotypic and molecular results reported here confirm that cryostorage is an efficient and reliable technique to conserve P. vulgaris seeds and regenerate true-to-type plants.

Identification of subpopulations of prairie voles differentially susceptible to peer influence to decrease high alcohol intake

Peer influences are critical in the decrease of alcohol (ethanol) abuse and maintenance of abstinence. We previously developed an animal model of inhibitory peer influences on ethanol drinking using prairie voles and here sought to understand whether this influential behavior was due to specific changes in drinking patterns and to variation in a microsatellite sequence in the regulatory region of the vasopressin receptor 1a gene (avpr1a). Adult prairie voles’ drinking patterns were monitored in a lickometer apparatus that recorded each lick a subject exhibited during continuous access to water and 10% ethanol during periods of isolation, pair housing of high and low drinkers, and subsequent isolation. Analysis of fluid consumption confirmed previous results that high drinkers typically decrease ethanol intake when paired with low drinkers, but that a subset of voles do not decrease. Analysis of bout structure revealed differences in the number of ethanol drinking bouts in the subpopulations of high drinkers when paired with low drinkers. Lickometer drinking patterns analyzed by visual and by cross-correlation analyses demonstrated that pair housing did not increase the rate of subjects drinking in bouts occurring at the same time. The length of the avpr1a microsatellite did not predict susceptibility to peer influence or any other drinking behaviors. In summary, subpopulations of high drinkers were identified, by fluid intake and number of drinking bouts, which did or did not lower their ethanol intake when paired with a low drinking peer, and these subpopulations should be explored for testing the efficacy of treatments to decrease ethanol use in groups that are likely to be responsive to different types of therapy.

Molecular Fingerprints to IdentifyCandidaSpecies

A wide range of molecular techniques have been developed for genotyping Candida species. Among them, multilocus sequence typing (MLST) and microsatellite length polymorphisms (MLP) analysis have recently emerged. MLST relies on DNA sequences of internal regions of various independent housekeeping genes, while MLP identifies microsatellite instability. Both methods generate unambiguous and highly reproducible data. Here, we review the results achieved by using these two techniques and also provide a brief overview of a new method based on high-resolution DNA melting (HRM). This method identifies sequence differences by subtle deviations in sample melting profiles in the presence of saturating fluorescent DNA binding dyes.

Using of Inter Microsatellite Polymorphism to evaluate gamma-irradiated<br>Amaranth mutants

Abstract Molecular markers ISSR were used to analyse γ-irradiated mutants of amaranth Ficha cultivar and K-433hybrid; tested six microsatellite - primed PCR markers. First analysis of gamma-irradiated amaranth mutant lines was done using the ISSR. In the study, primers that distinguish the mutant lines from control plant were reported as well as primer with the differentiation ability for all analysed mutant lines. The very specific changes in the mutant lines´ non-coding regions based on inter microsatellite length polymorphism were analysed. Mutant lines of the Ficha cultivar (C15, C26, C27, C82, C236) shared a genetic dissimilarity of 0.16and their ISSR profiles were more similar to the Ficha than those of K-433 hybrid mutant lines. The K-433mutant lines (D54, D279, D282) shared genetic dissimilarity of 0.22 but are more distinct to their control plant as a whole, as those of the Ficha mutant lines. Different ISSR fingerprints patters of the mutant lines when compared to the Ficha cultivar and K-433 hybrid ISSR specific profiles may be part of a consequence of the complex response of the intergenic space of mutant lines to the gamma-radiance.

Within‐ and trans‐generational plasticity affects the opportunity for selection in barbed goatgrass (Aegilops triuncialis)

Environments are composed of selective agents, and environments may also modify the efficacy of these agents. Environments affect the rate of maximum evolutionary change by influencing variation in relative fitness (i.e., the opportunity for selection, or I). Within- and transgenerational plastic environmental responses may affect I, speeding or slowing processes of local adaptation. • We determined whether environmental factors affected the opportunity for selection (I) in Aegilops triuncialis (barbed goatgrass) by measuring I as a within- and transgenerational plastic response to two maternal glasshouse environments (serpentine/dry and loam/moist). We also determined whether this species' two most common genetic lineages (determined by DNA microsatellite length polymorphism) varied in response to glasshouse treatments. • Opportunity for selection was less for plants grown in the dry serpentine environment than for plants grown in the moist loam environment. This response varied between genetic lineages. The east lineage exhibited a within-generation response to the dry serpentine environment. For both seed mass and average seed weight in this lineage, the opportunity for selection was lower in dry serpentine than in moist loam. The west lineage had a transgenerational response to the dry serpentine such that the opportunity for selection for seed number and seed mass was lower for plants produced by mothers grown in dry serpentine than for plants produced by mothers in moist loam. • Phenotypic variation in relative fitness is constrained by the dry serpentine environment, which leads to lower evolvability in this environment. Within- and transgenerational effects of the environment may slow local adaptation to serpentine soils.

Soluble CD30 Level and CD30 Promoter Microsatellite Length Polymorphism in Renal Transplantation

Soluble CD30 Level and CD30 Promoter Microsatellite Length Polymorphism in Renal Transplantation

DNA polymerase kappa microsatellite synthesis: Two distinct mechanisms of slippage‐mediated errors

Microsatellite tandem repeats are frequent sites of strand slippage mutagenesis in the human genome. Microsatellite mutations often occur as insertion/deletion of a repeat motif (unit-based indels), and increase in frequency with increasing repeat length after a threshold is reached. We recently demonstrated that DNA polymerase κ (Pol κ) produces fewer unit-based indel errors within dinucleotide microsatellites than does polymerase δ. Here, we examined human Pol κ's error profile within microsatellite alleles of varying sequence composition and length, using an in vitro HSV-tk gap-filling assay. We observed that Pol κ displays relatively accurate synthesis for unit-based indels, using di- and tetranucleotide repeat templates longer than the threshold length. We observed an abrupt increase in the unit-based indel frequency when the total microsatellite length exceeds 28 nucleotides, suggesting that extended Pol κ protein-DNA interactions enhance fidelity of the enzyme when synthesizing these microsatellite alleles. In contrast, Pol κ is error-prone within the HSV-tk coding sequence, producing frequent single-base errors in a manner that is highly biased with regard to sequence context. Single-nucleotide errors are also created by Pol κ within di- and tetranucleotide repeats, independently of the microsatellite allele length and at a frequency per nucleotide similar to the frequency of single base errors within the coding sequence. These single-base errors represent the mutational signature of Pol κ, and we propose them a mechanism independent of homology-stabilized slippage. Pol κ's dual fidelity nature provides a unique research tool to explore the distinct mechanisms of slippage-mediated mutagenesis.

Piecing together an integrative taxonomic puzzle: microsatellite, wing shape and aedeagus length analyses of Bactrocera dorsalis s.l. (Diptera: Tephritidae) find no evidence of multiple lineages in a proposed contact zone along the Thai/Malay Peninsula

Bactrocera dorsalis (Hendel) and B. papayae Drew & Hancock represent a closely related sibling species pair for which the biological species limits are unclear; i.e. it is uncertain if they are truely two biological species, or one biological species which has been incorrectly split taxonomically. The geographical ranges of the two taxa are thought to abut or overlap on or around the Isthmus of Kra, a recognised biogeographic barrier located on the narrowest portion of the Thai Peninsula. We collected fresh material of B. dorsalis s.l. (i.e. B. dorsalis s.s.+ B. papayae) in a north–south transect down the Thai Peninsula, from areas regarded as being exclusively B. dorsalis s.s., across the Kra Isthmus, and into regions regarded as exclusively B. papayae. We carried out microsatellite analyses and took measurements of male genitalia and wing shape, both used previously to separate the taxa. No significant population structuring was found in the microsatellite analysis, consistent with one, predominantly panmictic population. Both morphological datasets showed consistent, clinal variation along the transect, without disjunction. No evidence supported historical vicariance driven by the Isthmus of Kra, and no dataset supported the current taxonomy of two species. Rather, within and across the area of range overlap or abutment between the two species, only continuous morphological and genetic variation was recorded. Recognition that morphological traits previously used to separate these taxa are continuous, and that there is no genetic evidence for population segregation in the region of suspected species overlap, is consistent with a growing body of literature that reports no evidence of biological differentiation between these taxa.

Microsatellite polymorphism in the EGFR, NOTCH4 and E2F4 genes and their association with breast cancer risk

The sequences of many human genes that encode proteins involved in cancer contain polymorphic microsatellites. Variations in microsatellite length may constitute risk factors in several human diseases, a possibility that has been little explored in breast cancer. Among the genes that contain polymorphic microsatellites are EGFR, NOTCH4 and E2F4. The length of some of these microsatellites has been associated with breast cancer risk. To determine whether the length of the microsatellites (CA)n in EGFR, (CTG)n in NOTCH4 and (AGC)n in E2F4 was associated with breast cancer risk, we genotyped these 3 microsatellites in 212 women with breast cancer and a control group of 308 women from the general population who did not have this disease. The allelic distribution observed for the 3 microsatellites matched that found in other white populations, with the exception of some (AGC)n alleles in E2F4, which have not been described previously. The length of (CA)n in EGFR and (CTG)n in NOTCH4 was not associated with breast cancer (OR=0.99; 95% CI 0.59-1.37; p=0.619 and OR=1.08; 95% CI 0.71-1.65; p=0.725, respectively). Short alleles (<13 repeats) of (AGC)n in E2F4 were less frequent in women with cancer than in the control sample.

New chloroplast microsatellite markers suitable for assessing genetic diversity of Lolium perenne and other related grass species

Lolium perenne (perennial ryegrass) is the most important forage grass species of temperate regions. We have previously released the chloroplast genome sequence of L. perenne 'Cashel'. Here nine chloroplast microsatellite markers are published, which were designed based on knowledge about genetically variable regions within the L. perenne chloroplast genome. These markers were successfully used for characterizing the genetic diversity in Lolium and different grass species. Chloroplast genomes of 14 Poaceae taxa were screened for mononucleotide microsatellite repeat regions and primers designed for their amplification from nine loci. The potential of these markers to assess genetic diversity was evaluated on a set of 16 Irish and 15 European L. perenne ecotypes, nine L. perenne cultivars, other Lolium taxa and other grass species. All analysed Poaceae chloroplast genomes contained more than 200 mononucleotide repeats (chloroplast simple sequence repeats, cpSSRs) of at least 7 bp in length, concentrated mainly in the large single copy region of the genome. Nucleotide composition varied considerably among subfamilies (with Pooideae biased towards poly A repeats). The nine new markers distinguish L. perenne from all non-Lolium taxa. TeaCpSSR28 was able to distinguish between all Lolium species and Lolium multiflorum due to an elongation of an A(8) mononucleotide repeat in L. multiflorum. TeaCpSSR31 detected a considerable degree of microsatellite length variation and single nucleotide polymorphism. TeaCpSSR27 revealed variation within some L. perenne accessions due to a 44-bp indel and was hence readily detected by simple agarose gel electrophoresis. Smaller insertion/deletion events or single nucleotide polymorphisms detected by these new markers could be visualized by polyacrylamide gel electrophoresis or DNA sequencing, respectively. The new markers are a valuable tool for plant breeding companies, seed testing agencies and the wider scientific community due to their ability to monitor genetic diversity within breeding pools, to trace maternal inheritance and to distinguish closely related species.