Polymorphism Of DNA Fragments Research Articles

Applicability of Inter – Simple Sequence Repeats ( Issr) for Testing Water Quality Through Cluster Analysis in Onion Genome Treated with Wastewaters تطبیق تقنیة التتابعات القصیرة المتکررة التى تتخلل الجینوم فى إختبار جودة المیاه من خلال التحلیل العنقودی لجینوم البصل المعامل بالمخلفات المائیة

In this investigation, three drainage water samples were collected from selected agricultural wells in Dakhalia and Kafr El–Sheikh Governorates. The drainage water samples were analyzed for physico – chemical properties. Six ISSR primers were used to assesst genetic diversity induced on the molecular level due to the treatments of onion roots with wastewaters from the different resources. This technique exhibits a great diversity in onion genotype particularly on the molecular level. The purpose of the present investigation was to evaluate the level of water quality on the molecular level using ISSR biomarkers, in addition to compare chemical composition of drainage water with the standards of WHO guidelines for drinking water quality. Onion roots , as biological indicator can be used to measure the genotoxic effects of water pollutants on DNA molecule via measuring the polymorphism of DNA fragments in treated roots. Results suggested that a qualitative measurement revealed changes in ISSR profiles. A distinct distance appeared between the band profile of treated roots and control samples. The comparison between treated and untreated genomes revealed that ISSR analysis could be used as a new tool to evaluate the level of water quality through how the drainage water modifies the structure of DNA molecule in living organisms. This study indicated that extreme application of chemical fertilizers had a severe impact on water quality. The physic-chemical analysis of drainage water quality used in this study revealed that the drainage water quality does not meet the WHO standards about direct reusing it in irrigation.

Genetic structure of three-way cross of rabbits in comparison with parental breeds

The comparative analysis of polymorphism of DNA fragments, flanking by the inverted repeats of microsatellites (ISSR-PCR markers) in three-way cross of rabbits and three parental breeds of rabbits was carried out. 26 loci are revealed by using three trinucleotide ISSR-markers ((GAG)6C, (ACC)6G и (CTC)6C), 20 of them being polymorphous. The most changeable there were the spectra of amplification products of primer (CTC)6C. Relative increased consolidation of three-way cross of rabbits in comparison with parental breeds such as White Giant, Soviet Chinchilla and Californian rabbits as also unique characteristics of their genetic structure in comparisons with parental breeds have been found.

ФРАГМЕНТЫ ГОМОЛОГИИ ЭНДОГЕННЫХ РЕТРОВИРУСОВ В ГЕНОМАХ РАСТЕНИЙ И ЖИВОТНЫХ

Using sequences of mobile genetic elements in order to mark polymorphic sites in their insertion might be the most effective approach to identify specific features of gene pools of different groups of organisms and control their dynamics, which is especially important in dealing with agricultural species. Sireviruses ( Pseudoviridae ) one of the oldest LTR-retrotransposons are widespread throughout the plant kingdom. SIRE-1 is one of the largest and most detailed studied retrotransposon. Its analysis showed that insertions of the retroelement (particularly in the genome of maize) have occurred recently. We studied the polymorphism of DNA fragments flanked by LTR-retrotransposon SIRE-1 (IRAP-PCR markers) in the genomes of different taxonomic groups. The objects of our study were Triticum aestivum (Moscovskaya 39, Mironovskaya 808, Omskaya 36 varieties), Glycine soja (five wild populations of Primorskii region of Russia) and G. max (China), as well as representatives of the factory and indigenous breeds of cattle Black-and-White cattle improved by Holstein cattle, Ayrshire, Yakut and Red Estonian cattle (97 animals in total). A terminal site of SIRE-1 was chosen as primer IRAP-PCR (GCA-GTT-ATG-CAA-GTG-GGA-TCA-GCA). The data indicate that the multiloci genotyping by IRAP-PCR using retrotransposon LTR SIRE-1 as a marker reliably differentiates not only representatives of the monocotyledonous and dicotyledonous plants, but also their varieties. Spectrum of DNA fragments (13 to 16 depending on the breed) obtained in studies different breeds of cattle using IRAP-PCR markers was in the length range of 330-1470 bp. The highest polymorphism of DNA fragments was observed in the middle part of the spectrum (760-980 bp) in Ayrshire and Black-and-White cows. According to the obtained dendrogram one of the groups of Black-and-White cows and Ayrshire cattle were closer to each other, while the other with Red Estonian and Yakut cattle became isolated in a separate subcluster. Identified differences in heterogeneity in the two studied groups of Black-and-White from different farms possibly could be explained by the peculiarities of breeding work carried out or with other factors of artificial and/or natural selection. The possibility of using LTR-retrotransposons as molecular genetic markers for polyloci genotyping plants and animals is discussed.

THE FRAGMENTS OF HOMOLOGIES OF ENDOGENOUS RETROVIRUSES IN THE GENOMES OF PLANTS AND ANIMALS

Using sequences of mobile genetic elements in order to mark polymorphic sites in their insertion might be the most effective approach to identify specific features of gene pools of different groups of organisms and control their dynamics, which is especially important in dealing with agricultural species. Sireviruses (Pseudoviridae) one of the oldest LTR-retrotransposons are widespread throughout the plant kingdom. SIRE-1 is one of the largest and most detailed studied retrotransposon. Its analysis showed that insertions of the retroelement (particularly in the genome of maize) have occurred recently. We studied the polymorphism of DNA fragments flanked by LTRretrotransposon SIRE-1 (IRAP-PCR markers) in the genomes of different taxonomic groups. The objects of our study were Triticum aestivum (Moscovskaya 39, Mironovskaya 808, Omskaya 36 varieties), Glycine soja (five wild populations of Primorskii region of Russia) and G. max (China), as well as representatives of the factory and indigenous breeds of cattle — Black-and-White cattle improved by Holstein cattle, Ayrshire, Yakut and Red Estonian cattle (97 animals in total). A terminal site of SIRE-1 was chosen as primer in IRAP-PCR (GCA-GTT-ATG-CAA-GTG-GGA-TCA-GCA). The data indicate that the multiloci genotyping by IRAP-PCR using retrotransposon LTR SIRE-1 as a marker reliably differentiates not only representatives of the monocotyledonous and dicotyledonous plants, but also their varieties. Spectrum of DNA fragments (13 to 16 depending on the breed) obtained in studies different breeds of cattle using IRAP-PCR markers was in the length range of 330-1470 bp. The highest polymorphism of DNA fragments was observed in the middle part of the spectrum (760-980 bp) in Ayrshire and Black-and-White cows. According to the obtained dendrogram one of the groups of Black-and-White cows and Ayrshire cattle were closer to each other, while the other with Red Estonian and Yakut cattle became isolated in a separate subcluster. Identified differences in heterogeneity in the two studied groups of Black-and-White from different farms possibly could be explained by the peculiarities of breeding work carried out or with other factors of artificial and/or natural selection. The possibility of using LTR-retrotransposons as molecular genetic markers for polyloci genotyping plants and animals is discussed.

ISSR-PCR маркеры и мобильные генетические элементы в геноме домашней лошади Equus caballus

The patterns and causes of the differences in the polymorphism of individual microsatellites, and species specificity of this variability are still not enough studied. Test systems developed by the International Association of Animal Genetics (ISAG) for genetic certification of agricultural animals, assessment of interbreed relations, the level of animal group consolidation, include a small number of genetic markers. The use of microsatellite loci fragments as PCR primers for polylocus genotyping and genome scan of agricultural animal species is more simple and effective methodical approach. We proposed a method for identifying positioning of microsatellite inverted repeat areas in genome sequences and for comparisons of this calculated areas to experimental DNA spectra amplified in PCR with the use of microsatellite loci as primers (ISSR-PCR markers). Here we report the results of the comparisons carried out for domestic horses ( Equus caballus ) of Altai breed ( n = 96), Trotter breed ( n = 48), Karachai breed ( n = 34). The DNA fragments (99 sites) in the sequenced genome of the domestic horse, flanked by inverted repeat (GA) 9C, were revealed and the presence of homologous sequences to mobile genetic elements was analyzed. The nucleotide sequences were examined in the DNA fragments (expected size/obtained size) of different location: 1250-1350/1326 bp (chromosome 17), 1250-1350/1302 bp (chromosome 2), 1250-350/1296 bp (Х-chromosome), 1250-1350/1287 bp (chromosome 20), 980/986 bp (chromosome 23), 800-900/900 bp (chromosome 24), 800-900/876 bp (Х-chromosome), 800-900/871 bp (chromosome 18), 800-900/857 bp (chromosome 3), 800-900/832 bp (chromosome 6), 800-900/823 bp (chromosome 19), 740/739 bp (chromosome 16), 580/585 bp (chromosome 1), 550/637 bp (Х-chromosome), 550/572 bp (chromosome 10), 500/494 bp (chromosome 1), 490/489 bp (chromosome 28), 380/378 bp (chromosome 11), 380/377 bp (chromosome 21), 360 (370)/364 bp (chromosome 5), 310/309 bp (chromosome 5). The PIC (polymorphic information content) values for loci were calculated. In the representatives of several breeds, the conservative and polymorphic DNA fragments were observed in the amplification product spectra obtained in the polymerase chain reaction with a primer sequence (GA) 9C. It was turned out that the polymorphous DNA fragments in different animals, in most cases, contained the homologous sequences to fragments of retrotransposons (to ERV III, particularly), while the conservative ones contained homologous sequences to DNA transposons. An increased density of nucleotide sequences, potentially prone to the G 4 quadruplex formation, was observed in the studied horse genomic DNA fragments, flanked by the inverted repeat (GA) 9C, that could be associated with an increased recombination in these areas. A secondary structure in the DNA regions flanked by a microsatellite repeat can influence the results of genotyping in case the ISSR-PCR markers are used. The data obtained suggest the relationship between the polymorphism of DNA fragments, flanked by inverted repeats of microsatellites, and a mobility of retrotransposons.

Increased genomic instability in somatic cells of the progeny of female mice exposed to acute X-radiation in the preconceptional period

The level of genome instability (GI) was studied in the progeny of female mice exposed in the preconceptional period to radiation doses of 0.5, 1, and 2 Gy in comparison to that in the progeny of the same parent pairs born before irradiation of the females. To assess the level of genome instability, we analyzed polymorphism of DNA fragments from postmitotic (blood and brain) and proliferating (spleen and tail tip) tissues amplified by AP-PCR (PCR amplification with an arbitrary primer). It was found that polymorphism of the spectrum of AP-PCR products, which is a multilocus genetic marker (MGM), in the genome of somatic cells in the progeny of female mice exposed to 2 Gy was higher than in the progeny of male mice exposed to the same doses. In the progenies of female mice born before and after irradiation, tissue-specific variations in the level of DNA polymorphism were detected. The maximum value of this polymorphism (with respect to the frequency of "nonparental bands") was determined for peripheral blood DNA in comparison with the other tissues. Estimations of the MGM polymorphism with the AP-PCR method demonstrate an increased level of genome instability in somatic cells of offsprings from female mice exposed to a single acute dose of X-rays (0.5, 1, and 2 Gy) in the pre-conceptional period. Radiation-induced transgenerational genome instability with an increase in the dose of preconceptional irradiation of female mice was more pronounced in DNA of the postmitotic tissues (blood and brain DNA) than in DNA of the proliferating tissues (spleen and tail tip epithelium).

Structural and functional particularities of microsatellites in cattle and sheep genomes

A comparative analysis of polymorphism of DNA fragments flanked by microsatellite loci of cattle and sheep is performed. In all, 102 loci revealed in the polymerase chain reaction with the use of fragments of di- and trinucleotide microsatellite fragments as primers are analyzed. It is found that polymorphism of the spectra of amplification products depends substantially on the nucleotide sequence of the primer. The dependence of polymorphism of DNA fragments on the structural and functional organization of their flanks is discussed.

Molecular genetic markers of DNA polymorphism and their genomic positioning

A comparative analysis of polymorphism of DNA fragments flanked by terminal regions of retrotransposon-like elements of the R175 family in rice and wheat varieties is performed. Differences are revealed in the distribution and polymorphism of such regions, attesting to the characteristics of their mutual positioning. The need in gene pool investigations to take into account the belonging of molecular genetic markers to various families of genomic elements differing in localization, structural and functional organization, conservatism/polymorphism, and evolution is shown.

Identification of genetic characteristics of maize (Zea mays L) using genetic markers

Different genetic markers are used for estimation of breeding material, its characteristics and potential for ultimate aim - heterosis of hybrids. They also point out to the qualitative seed traits at the level of linkage with genes responsible for desirable agronomic traits. This program encompasses testing methodologies for the new seed technology. Genetic analysis of breeding material during certain phases is comprised of isozymic gene expression and degrees of their variability, but it is continued (in order to be evaluated) until determination of presence or absence of some genes existing or introduced for certain traits. Using combination of different molecular methods such as PCR, RAPD and AFLP based on polymorphism of DNA fragments, the definite aim - identification of newly created products of improvement is achieved. Testing of traits of breeding material, its genetic variability and diversity is the first stage in analysis of the maize genome. It is also the condition for determination of presence of certain genes, used for obtaining the ultimate aim - attest of identity of the genotype.

Specific genomic DNA fragment analysis of different geographical clinical samples of shrimp white spot syndrome virus

White spot syndrome (WSS) has been found in many species of shrimp and crabs, not just In Asia but globally. The causative agent is known as white spot syndrome virus (WSSV). In order to clarify the relatedness of WSSV from various geographic regions, we compared the viral DNA of a number of clinical samples of WSSV. (1) China96-116A from Penaeus chlnens~s, (2) India95-314 from Penaeus monodon, (3) grocery store95-204 and grocery store96-l15 from P. rnonodon possibly originating from Thailand, (4) crayfish97-25 from Orconectespunctimanuscollected from the U.S. National Zoo, (5) Thailand95-46 from experimentally infected Penaeus vannamei. (6) South Carolina97-64 from P. vannamei, and (7) Texas95-242 and Texas96-7 from P. vannarnei. These specimens were first examined by dot hybridization analysis with nucleic acid probes derived from a WSSV Taiwan isolate. Although the intensity of the hybridization signals varied, and although some specimens of India95314, crayfish97-25, Texas95-242 and Texas96-7 failed to give a detectable hybriduation signal with certain probes, the broad consistency of dot hybridization data suggests that these WSSV clinical samples from different geographical locations are closely related. Following this analysis, all the specimens were examined using 10 virus-specific polymerase chain reactions (PCR). The amplification products were subsequently digested with Cfo I, Hae 111, Hpa I1 and Rsa I restriction endonucleases to determine if there were any DNA fragment polymorphisms in the WSSV clinical samples. The results highlighted the genetic relatedness of all the WSSV clinical samples with the possible exception of a series of Texas viral samples which could be distinguished from the other geographic samples in some of the PCR-based tests.

Identification of Hop Cultivars by DNA Marker Analysis

We applied random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism of hazy associations (ALPHA) to the identification of hop cultivars. The polymerase chain reaction (PCR) using random primer or sequence-tagged-sites (STS) was found to give a variety of specific fingerprints from hop DNA, requiring no hop DNA sequence information. Varietal identification of hops was performed using the fingerprints that included DNA fragment polymorphisms. Subsequently, these RAPD (or ALPHA) fragments were sequenced, and specific primers for the individual RAPD (or ALPHA) fragments were synthesized. The fragments amplified by the specific primers allow for a much clearer identification of hop cultivars. Eleven typical hop cultivars were successfully distinguished from one another using these specific primers. This method was also useful for the detection of mixed samples of hop cultivars.

Use of denaturing gradient gel blots to screen for point mutations in the factor VIII gene.

Denaturing gradient gel electrophoresis (DGGE) is commonly used to search for point mutations in DNA fragments amplified in vitro by the polymerase chain reaction (PCR). For the complete detection of mutations in large genes with many exons, the DGGE-PCR approach, or any other PCR-based method, requires many primer sets and amplification reactions to scan the entire protein-coding sequence. We previously demonstrated that DGGE analysis using DNA blots detects mutations in Drosophila genes and sequence polymorphisms in human genes without prior PCR amplification. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemophilia A families were analyzed for mutations in the factor VIII (FVIII) gene. Restriction enzyme digested DNA samples were subjected to DGGE and transferred to nylon blots. Hybridization of the DGG blots with FVIII cDNA probes revealed mutant and polymorphic DNA sequence differences. Among 26 affected families that were not carriers of intron 22 inversion mutations, 18 family-specific DNA fragment polymorphisms and one multiexon deletion were mapped. DNA sequencing of eight patient-specific polymorphic DNA fragments revealed six single base change mutations, one 4 bp deletion, and one 13 bp duplication.

Simple sequence repeat‐based single primer amplification of genomic DNA in random bred populations of turkeys and chickens

Abstract The ubiquity and polymorphism of simple sequence repeats (SSR) or microsatellites have made them the DNA markers of choice for linkage, diversity and phylogenetic studies in eukaryotes. In this study, we investigated the utility of a SSR‐based single primer amplification procedure (SPARs) for developing DNA markers in turkeys and chickens. The fifteen primers used to evaluate the average number, size, and polymorphism of DNA fragments within SPARs patterns consisted of one di‐, six tri‐ and eight tetra‐nucleotide repeats. Genomic DNA samples from a total of 60 and 40 random bred turkeys and chickens, respectively, were used. In addition, turkey reference families developed from a wild x Commercial Orlopp‐line reciprocal cross was included in the analyses. Cycling conditions for the polymerase chain reaction were optimized for each primer by testing for amplification using annealing temperatures of 35, 45 and 55°C. Molecular weight, number and level of polymorphism of the amplified DNA fragments w...

Characterization of Vitis germplasm using random amplified polymorphic DNA markers

An analysis of the amplification fragments polymorphism of DNA coming from different accessions of germplasm belonging to species and cultivars of the genus Vitis, was carried out using 40 primer decamers of arbitrary sequence. The RAPD profiles showed a great intraspecific diversity. In many cases a single primer produced a unique pattern for each species. A phylogram tree based upon presence/absence data of the principal DNA bands divided the species according to their geographical origins. The intraspecific polymorphism of DNA fragments was not sufficient for an unambiguous identification of Vitis vinifera cultivars but the RAPD profiles turned out to be highly reproducible. The high capacity of this technique to generate DNA markers offers a new possibility for the study of the genetic relationships in the genus Vitis.

Parentage determination in maize hybrids using the arbitrarily primed polymerase chain reaction (AP-PCR)

Using a novel procedure based on the polymerase chain reaction, we have developed a rapid, efficient, and economical method for identifying plant genotypes. The arbitrarily primed polymerase chain reaction (AP-PCR) generates reproducible fingerprints from any organism, without the need for DNA sequence information. These fingerprints include DNA fragment polymorphisms that can be (1) used for varietal identification and parentage determination, (2) followed in segregating populations produced by crosses, (3) used as markers for the construction of genetic maps, and (4) used to generate dendograms of phylogenetic relationships, especially at the intraspecific level. AP-PCR requires only minute quantities of DNA (10-25 ng per reaction) and therefore can be used in situations in which DNA is limiting. We demonstrate the use of AP-PCR to identify inbred parents of hybrid maize plants in double-blind experiments.

Multiresistant serotype O 12Pseudomonas aeruginosa: evidence for a common strain in Europe

A survey was made of serotype association and multiple antibiotic resistance in strains of Pseudomonas aeruginosa in Europe. Of 208 epidemiologically distinct strains from 16 laboratories in 10 countries, 48 were resistant to carbenicillin (MIC greater than 128 micrograms/ml) and gentamicin (MIC greater than 4 micrograms/ml), and 12 of these strains were of serotype O 12. Representative O 12 strains from different countries were compared with two multiresistant O 12 strains isolated 5 years apart, from a British burns unit and the antibiotic sensitive serotype reference strain. All O 12 strains were similar by phage and pyocin typing but lysogenic phage profiles indicated that two strains (the later burns isolate and the serotype strain) were distinct from the others. Electrophoretic characterization of outer membrane proteins, lipopolysaccharides and esterase enzymes corroborated the relationship of the strains and restriction fragment length polymorphism of DNA fragments hybridized with a cDNA probe copy of rRNA from P. aeruginosa provided further proof of their relatedness. We propose that the uniformity of characters of multiresistant O 12 P. aeruginosa in Europe is suggestive of a common origin for the strains.

Multiple DNA fragment polymorphisms associated with immunoglobulin mu chain switch-like regions in man.

DNA probes containing the switch region (S) associated with the human immunoglobulin heavy chain mu gene were used to investigate polymorphisms in the germ-line human DNA. Six polymorphisms, detected by a single restriction enzyme (Sst I) are described. Linkage studies in 29 families show that five of the six polymorphisms, although relatively unassociated in random individuals, segregate in complete linkage one to the other and to Gm allotypes (markers on the heavy chain of IgG), while the sixth segregates independently. Altogether, when one considers the DNA markers at the five closely linked loci and the IgG1 and IgG3 heavy chain allotypes, 33 different haplotypes have been described; of these, 28 are detected by the DNA polymorphism alone. Study of 158-187 random haplotypes showed strong linkage disequilibrium only between one DNA polymorphism (Sst A) and Gm. Of the polymorphic Sst I loci, one, Sst E [associated with 2.2- to 2.7-kilobase (kb) fragments], is included in the mu chain S region (S mu); another, Sst A (6.8-7.4 kb), must be very close to the gamma 1-gamma 3 chain gene cluster. Based on studies of an IgE human myeloma, a third polymorphism, Sst C (4.8-5.5 kb), should map 3' of the active epsilon chain gene. An Sst I restriction enzyme map of phage clones carrying the two alpha chain genes indicates that Sst A and Sst C loci probably overlap with the alpha 1 and alpha 2 S regions, respectively. Both deletion/duplications and point mutations were detected.