The polymerase chain reaction pcr, first envisaged in 1984 by kary mullis, has revolutionized life sciences and has become an essential technique in many aspects of science, including clinical diagnostics, forensics and genetic engineering. Intersimple sequence repeatpolymerase chain reaction issrpcr zietkiewicz et al. Brain, nathaniel goodyer and pierre laneuville cancer res august 15 2003 63 16 48954898. Simple sequence repeat definition of simple sequence. Optimization of polymerase chain reaction for inter simple. Thirteen diverse strains of the silkworm bombyx mori were analysed using the simple sequence repeat anchored polymerase chain reaction ssranchored pcr or interssrpcr issrpcr. Inter simple sequence repeat issr polymorphism and its. Characterisation of dark septate endophytic fungi dse using. Holdenrieder 25 assumption that, thus far, could not be confirmed t. Rapid identification of trichoderma koningiopsis and. Genomic instability measured by intersimple sequence repeat.
To improve the reproducibility and quantitative aspects of this method, we used fluorescent dyelabeled primers for the intersimple sequence repeat fissrpcr reac. Sequencerelated amplified polymorphism and intersimple. Genomic instability of human aberrant crypt foci measured by. Twentyfive primer combinations preselected by their ability to polymerase chain reaction amplify ssrs from a set of test cowpea germplasm were evaluated. Repeat units of four and five nucleotides are referred to as tetra and pentanucleotide motifs, respectively.
For example, the nucleotide sequence of a particular gene can be determined. Samples were run on a 5% polyacrylamide gel and visualized using silver staining. To improve the reproducibility and quantitative aspects of this method, we used fluorescent dyelabeled primers for the inter simple sequence repeat fissrpcr reac. Simple sequence repeat synonyms, simple sequence repeat pronunciation, simple sequence repeat translation, english dictionary definition of simple sequence repeat. Inter simple sequence repeat analysis of genetic diversity.
Intersimple sequence repeat polymerase chain reaction issrpcr we have adapted the protocol of zietkiewicz et al. Leprosy is a contagious disease and was eliminated globally in 2002. Citeseerx document details isaac councill, lee giles, pradeep teregowda. Inter simple sequence repeat polymerase chain reaction issrpcr analysis is quick, simple and generates complex bind profiles. Issrs are amplified by pcr using microsatellite core sequences as primers with a few selective nucleotides as anchors into the nonrepeat adjacent regions 1618 bp. Inter simple sequence repeats issrs are regions in the genome flanked by microsatellite sequences. The intersimplesequence repeat issr primers that anneal to a simple repeat of various length and at nonrepetitive motifs at 3. Measurement of genomic instability in preleukemic p190bcrabl. Results showed that each primer gave rich and clear patterns with bands ranged from 179 to 1256 bp. Thirteen diverse strains of the silkworm bombyx mori were analysed using the simple sequence repeat anchored polymerase chain reaction ssranchored pcr or inter ssrpcr issrpcr. Molecular characterization of trichinella genotypes by. Molecular markers such as randomly amplified polymorphic dna rapd, simple sequence repeat ssr, amplified fragment length polymorphism aflp and intersimple sequence repeat issr have been applied in the estimation of genetic diversity, genetic relationship and germplasm management and conservation in v. Using dna fingerprinting to detect the genetic relationships. Characterisation of dark septate endophytic fungi dse.
Sekuen oligonukleotida ini akan menjamin bahwa primer hanya akan terikat pada satu ujung lokus ssr. A total of 188 polymorphic loci were detected, with. Measurement of genomic instability in preleukemic p190bcr. Nov 17, 2017 molecular markers such as randomly amplified polymorphic dna rapd, simple sequence repeat ssr, amplified fragment length polymorphism aflp and inter simple sequence repeat issr have been applied in the estimation of genetic diversity, genetic relationship and germplasm management and conservation in v. Citeseerx the utility of intersimple sequence repeat. Once the copies are made, the dna may more easily be studied. Kuntze cultivars were analyzed using the simple sequence repeat anchored polymorearse chain. Thus, intratumoral variability in genomic instability was observed. Simple sequence repeat anchored polymerase chain reaction amplification ssrpcr is a genetic typing technique based on primers anchored at the 5 or 3 ends of microsatellites, at high primer annealing temperatures. Intersimple sequence repeat issr markers are generated from singleprimer polymerase chain reaction pcr amplifications in which the primers are based on dinucleotide or trinucleotide repeat motifs. We assessed genetic diversity and structure in eight geographic populations of gypsy moths from china using five polymorphic inter simple sequence repeat markers, which produced reproducible banding patterns. Kuntze cultivars were analyzed using the simple sequence repeat anchored polymorearse chain reaction ssranchored pcr or inter ssrpcr issr. Thus, in the present study, we compared to molecular methods, namely rlep polymerase chain reaction rleppcr and inter simple sequence repeat pcr issrpcr taking afb microscopy as a gold standard for the detection of leprosy.
Inter simple sequence repeat issr is the genome region between microsatellite loci. The technique has been used widely for dna fingerprinting and. Furthermore, because researchers can specify a primers sequence to target a speci. Pdf assessment of genetic diversity of tea camellia. The polymerase chain reaction is used to amplify a segment of dna that lies between the two regions of known sequence, where two oligonucleotides as primers can bind the opposite strands of dna due to complementary nature of base sequence. Dominant marker intersimple sequence repeatpolymerase.
The two specific markers for amplifying a single unique band consistent witht. The technique has been used widely for dna fingerprinting a. Applicability of start codon targeted scot and inter. Characterisation of dark septate endophytic fungi dse using intersimple sequence repeat anchored polymerase chain reaction issrpcr amplification author links open overlay panel christoph r. Several pcr polymerase chain reaction based dna marker systems including rapd random amplified polymorphic dna, aflp amplified fragment length polymorphism and ssrs simple sequence repeats or microsatellites are available for genetic assessment,7 but each of the methods have some limitations. Genetic characterization of the silkworm bombyx mori by. Issrpcr very clearly distinguished the aforementioned species, demonstrating that it is an excellent molecular diagnostic tool. The reactions were carried out in a 25jxl volume containing 1. A comparative evaluation partha sarathi mohanty 1, avi kumar bansal 1, farah naaz 1, shripad a patil 1, mamta arora 1, manjula singh 2.
The number of dinucleotide or trinucleotide repeats varies but is generally sufficiently long to make a primer sequence of at least 14 nucleotides. For example, the sequence tatatatata is a dinucleotide microsatellite, and gtcgtcgtcgtcgtc is a trinucleotide microsatellite with a being adenine, g guanine, c cytosine, and t thymine. Cicadellidae with the following dna fingerprinting methods. Pdf molecular characterization of cultivated bromeliad. Simple sequence repeat or microsatellites, also known as simple sequence repeats ssrs or short tandem repeats strs, are repeating sequences of 2. Polymerase chain reaction pcr is a method used widely in molecular biology to make millions to billions of copies of a specific dna sample rapidly, allowing scientists to take a very small sample of dna and amplify it to a large enough amount to study in detail. Optimization of dna isolation and pcr protocol for issr. These mice ubiquitously express bcrabl for an average of 3 months before developing bcell type lymphomaleukemia.
Inter simple sequence repeat issrpcr is a technique, which involvesthe use of microsatellite sequences as primers in a polymerase chainreaction to generate multilocus markers. Pcr is based on using the ability of dna polymerase to synthesize new strand of dna complementary to the offered template strand. Characterisation of dark septate endophytic fungi dse using intersimplesequencerepeatanchored polymerase chain reaction issrpcr amplification c. Genetic diversity of 30 common vetch vicia sativa l. Improvement and application of fluorescence intersimple. Products of dna synthesis separated by electrophoresis. International journal of mycobacteriology free full text. The results reported here are from work conducted fiscal year 2004 to fiscal year 2005. Comparison of intersimple sequence repeat polymerase chain reaction patterns of lcm dna from adjacent foci of papillary and anaplastic tumors showed conserved genome alterations. Our study included 65 sporadic colorectal cancer patients diagnosed from 1987 to 1991 with last followup ascertained in 2006. Genetic diversity and phylogenetic relationships among 141 cowpea vigna unguiculata l. Sanger sequencing, also known as the chain termination method, is a technique for dna sequencing based upon the selective incorporation of multicolored fluorescentlabeled chain terminating dideoxynucleotides ddntps by dna polymerase during in vitro dna replication.
Dna was extracted from 100 blood samples of five populations using optimized and modified salting out method. We estimated an overall tally of alterations using the genomewide sampling technique of inter simple sequence repeat ssr polymerase chain reaction pcr, and evaluated its relationship with allcause survival. Genome scanning for insertions and deletions in samples of dna extracted from kidney and spleen tissues taken from preleukemic animals was performed using the inter simple sequence repeat pcr. In addition, issrs are considered useful in gene mapping studies.
After a single amplification carried out on a single larva with the primer 816canry. Dominant marker inter simple sequence repeat polymerase chain reaction versus codominant marker rlep polymerase chain reaction for laboratory diagnosis of leprosy. Issr marker is detected by means of repeaters anchored primers that are ampli. We observed 102 polymorphic loci across the 176 individuals sampled. Inter simple sequence repeat issr markers are generated from singleprimer polymerase chain reaction pcr amplifications in which the primers are based on dinucleotide or trinucleotide repeat motifs. It is a simple and quick methodthat combines most of the advantages of microsatellites ssrs andamplified fragment length polymorphism aflp to the universality ofrandom amplified polymorphic dna rapd. Changing temperature conditions, the amount of amplification cycles and concentration of reaction mixture components is allowed to vary the number of. Measurement of genomic instability in preleukemic p190 bcrabl transgenic mice using intersimple sequence repeat polymerase chain reaction julia m. A bulk analysis of intersimple sequence repeatpolymerase chain reaction issrpcr provides a quick, reliable, and highly informative system for dna banding patterns that permit species identification. The efficiency and reliability of the method was tested by assessing quantity and quality of the extracted dna by restriction analysis and polymerase chain reaction pcr using intersimple sequence repeat issr. And this allows, in principle, someone like me to go and to grab a single cell from you, take it to dna, and get. The aim of this study was to evaluate the diversity of kermani sheep using issr markers.
For the development of this technique, known today as the polymerase chain reaction or pcr, mullis was awarded the nobel. Conclusions at the genome level, thyroid tumors may be highly heterogeneous. In this study, inter simple sequence repeat issr polymerase chain reaction pcr tool was used to analyze the genetic alteration of treated seeds of v. Inter simple sequence repeat an overview sciencedirect topics. Characterization of beauveria bassiana isolates from japan.
A number of dna samples from specimens of littorina plena amplified using polymerase chain reaction with primers targeting a variable simple sequence repeat ssr, a. Inter simple sequence repeat an overview sciencedirect. Pcr analysis is quick, simple and generates complex bind profiles. In the current study, we tested the utility of the intersimple sequence repeat polymerase chain reaction issrpcr dna fingerprinting method in distinguishing geographic populations of the smoketree sharpshooter stss homalodisca liturata ball and in distinguishing several. And its something known as the polymerase chain reaction. Of the six issr primers tested, namely, ag8t, ag8c, ag8g, ca8a, tg8c and tg8g, only primer ag8t produced amplification of bp in the two nonpathogenic leptospira species. Nov 04, 20 simple sequence repeat or microsatellites, also known as simple sequence repeats ssrs or short tandem repeats strs, are repeating sequences of 26 base pairs of dna. Out of the 45 primers 12were chosen for final study.
Oct 10, 2006 read genomic instability of human aberrant crypt foci measured by inter simple sequence repeat pcr and arraycgh, mutation researchfundamental and molecular mechanisms of mutagenesis on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Assessment of genetic diversity and fingerprinting of. Summary inter simple sequence repeat issrpcr is a technique, which involves the use of microsatellite sequences as primers in a polymerase chain reaction to generate multilocus markers. Sequencing of simple sequence repeat anchored polymerase. The present study evaluates the applicability of this system to trichinella species identification. Teknik intersimple sequence repeat polymerase chain reaction issrpcr menggunakan primer tunggal yang memiliki sekuen mikrosatelit dan terletak pada salah satu ujung rantai 5 atau 3 dengan basa oligonukleotida zietkiewicz et al. This technique has already been used in studies of genetic variability of several organisms, using different primer designs. In the current study, we tested the utility of the intersimple sequence repeatpolymerase chain reaction issrpcr dna fingerprinting method in distinguishing geographic populations of the smoketree sharpshooter stss homalodisca liturata ball and in distinguishing several egg parasitoid species in the genus gonatocerus. Pcr amplification of these regions using a single primer yields multiple amplification products that can be used as a dominant multilocus marker system for the study of genetic variation in various organisms. In the current study, we tested the utility of the intersimple sequence repeat polymerase chain reaction issrpcr dna fingerprinting method in distinguishing geographic populations of the smoketree. A total of 168 clinically diagnosed leprosy patients were recruited in this study including 58. Abstract inter simple sequence repeat polymerase chain reaction issr. Anaplastic thyroid cancer evolved from papillary carcinoma. The objective of this study was to complete the molecular evaluation of five acacia species including first by determining the genetic diversity of the plants using the polymerase chain reaction pcrbased inter simple sequence repeat issr method.
Simple sequence repeat definition of simple sequence repeat. Measurement of genomic instability in preleukemic p190bcrabl transgenic mice using intersimple sequence repeat polymerase chain reaction. Intersimple sequence repeat issr markers academy of. Pcr is a method for performing dna replication in a test tube. Many, many copies of a small dna sequence may be amplified from a few starting copies.
Analisis keragaman plasmanutfah apel malus pumila mill. Assessment of the genetic diversity in cowpea vigna. Because of the multilocus fingerprinting profiles obtained, issr analysis can be applied in studies involving genetic identity, parentage, clone and strain identification, and taxonomic studies of closely related species. The present study evaluates the applicability of this system to trichinella. Inter simple sequence repeat polymerase chain reaction for. Using multiplexed intersimple sequence repeat issr primers, thousands of genomewide regions were amplified effectively from a wide variety of genomes, without prior genetic information. Twelve issr primers produced 342 bands, of which 287 84% were polymorphic, with polymorphic bands per primer ranging from 17 to 34. Because dna polymerase can add a nucleotide only onto a preexisting 3oh group, it needs a primer to which it can add the. Polymerase chain reaction optimization for inter simple sequence repeat primers is a key factor to obtain accurate and reproducible results for gene mapping, studying the genetic structure of populations, plant passporting, phylogenetic analysis.
Simple sequence repeat ssr or microsattelite youtube. Issrs are dna fragments of about 1003000 bp located between adjacent, oppositely oriented microsatellite regions. Genetic characterization of the gypsy moth from china. Issrpcr was found to be highly reproducible since dna amplification from several singlesporeisolate subcultures of the b. The technique has been used widely for dna fingerprinting and genetic distance analysis. Dna polymorphisms were detected in homalodisca coagulata say homoptera. Assessment of genetic diversity in vigna unguiculata l. A bulk analysis of intersimple sequence repeat polymerase chain reaction issrpcr provides a quick, reliable, and highly informative system for dna banding patterns that permit species identification.
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