Mutation detection by analysis of DNA heteroduplexes in TILLING populations of diploid species

In the beginning of mutation research, mutations could only be detected indirectly through the analysis of the phenotypic alterations that they caused. The detection of mutations at the DNA level became possible with the development of sequencing methods. Nowadays, there are many different methods and strategies that have been created for mutation detection, both in natural and mutagenised populations. The strategies differ in accuracy and sensitivity, as well as in the laboratory facilities, time, costs and efforts that are required. The majority of them involve the pooling of DNA samples and the amplification of a gene (fragment) of interest followed by heteroduplex formation. One of the popular strategies for mutation identification takes advantage of the specific endonuclease (e.g. CEL I) that recognises and cuts heteroduplexes precisely at the 3′ position of the mismatch site. The cleaved fragments are usually visualised through electrophoresis in a polyacrylamide gel using LI-COR sequencers, but agarose electrophoresis may also be used for this purpose, although with less sensitivity. A different mutation identification strategy, which is based on the high-resolution melting (HRM) technique, may be the method of choice when working with a short gene or a gene fragment whose length optimally does not exceed 400 bp

High-Throughput Detection of Induced Mutations and Natural Variation Using KeyPoint™ Technology

Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variants among mutant or natural populations. Current (pre-) screening methods such as TILLING and EcoTILLING are based on the detection of single base mismatches in heteroduplexes using endonucleases such as CEL 1. However, there are drawbacks in the use of endonucleases due to their relatively poor cleavage efficiency and exonuclease activity. Moreover, pre-screening methods do not reveal information about the nature of sequence changes and their possible impact on gene function. We present KeyPoint™ technology, a high-throughput mutation/polymorphism discovery technique based on massive parallel sequencing of target genes amplified from mutant or natural populations. KeyPoint combines multi-dimensional pooling of large numbers of individual DNA samples and the use of sample identification tags (“sample barcoding”) with next-generation sequencing technology. We show the power of KeyPoint by identifying two mutants in the tomato eIF4E gene based on screening more than 3000 M2 families in a single GS FLX sequencing run, and discovery of six haplotypes of tomato eIF4E gene by re-sequencing three amplicons in a subset of 92 tomato lines from the EU-SOL core collection. We propose KeyPoint technology as a broadly applicable amplicon sequencing approach to screen mutant populations or germplasm collections for identification of (novel) allelic variation in a high-throughput fashion

Лапаротомия в системе лечения перитонитов

ПЕРИТОНИТ /ХИРБРЮШИНЫ БОЛЕЗНИ /ХИРЛАПАРОТОМИЯХИРУРГИЧЕСКИЕ ОПЕРАЦИИ /МЕТОДЫРЕЛАПАРОТОМИ

Prevalence, risk factors and disability associated with fall-related injury in older adults in low- and middle-income countries: results from the WHO Study on Global AGEing and Adult Health (SAGE)

In 2010 falls were responsible for approximately 80 % of disability stemming from unintentional injuries excluding traffic accidents in adults 50 years and over. Falls are becoming a major public health problem in low- and middle-income countries (LMICs) where populations are ageing rapidly. Nationally representative standardized data collected from adults aged 50 years and over participating in the World Health Organization (WHO) Study on global AGEing and adult health (SAGE) Wave 1 in China, Ghana, India, Mexico, the Russian Federation and South Africa are analysed. The aims are to identify the prevalence of, and risk factors for, past-year fall-related injury and to assess associations between fall-related injury and disability. Regression methods are used to identify risk factors and association between fall-related injury and disability. Disability was measured using the WHO Disability Assessment Schedule Version 2.0 (WHODAS 2.0). The prevalence of past-year fall-related injuries ranged from 6.6 % in India to 1.0 % in South Africa and was 4.0 % across the pooled countries. The proportion of all past-year injuries that were fall-related ranged from 73.3 % in the Russian Federation to 44.4 % in Ghana. Across the six countries this was 65.7 %. In the multivariable logistic regression, the odds of past-year fall-related injury were significantly higher for: women (OR: 1.27; 95 % CI: 0.99,1.62); respondents who lived in rural areas (OR: 1.36; 95 % CI: 1.06,1.75); those with depression (OR: 1.43; 95 % CI: 1.01,2.02); respondents who reported severe or extreme problems sleeping (OR: 1.54; 95 % CI: 1.15,2.08); and those who reported two or more (compared with no) chronic conditions (OR: 2.15; 95 % CI: 1.45,3.19). Poor cognition was also a significant risk factor for fall-related injury. The association between fall-related injury and the WHODAS measure of disability was highly significant (P<0.0001) with some attenuation after adjusting for confounders. Reporting two or more chronic conditions (compared with none) was significantly associated with disability (P<0.0001). The findings provide a platform for improving understanding of risk factors for falls in older adults in this group of LMICs. Clinicians and public health professionals in these countries must be made aware of the extent of this problem and the need to implement policies to reduce the risk of falls in older adults.