Hla typing with reference strandmediated conformation analysis. Conformational methods, such as the reference strand mediated conformational analysis rsca, have shown to achieve highresolution results without the ambiguities seen in the previously mentioned methods. Hlab typing by reference strand mediated conformation. Pcrrsca reference strandmediated conformational analysis although pcrsbt is able to detect unknown hla polymorphisms, the problem of not being entirely able to resolve novel arrangements of known polymorphisms, also known as ambiguity, can be overcome by the pcrrsca technique. Reference strandmediated conformational analysis rsca, which is increasingly used in studies of natural populations with multiple mhc genes, is. The ipd project stores all the data in a set of related databases.
Referencestrandmediated conformation analysis of mhc alleles. Pcr fingerprinting and reference strand mediated conformational analysis 20, 21. Characterization of the major susceptibility region for. Reference strand mediated conformational analysis rsca is yet another method used for hla typing. Recipients and donors are considered to be compatible or well matched if they share a high degree of genetic similarity at their hla loci. Genotypic characterization of an mhc class ii locus in lake trout salvelinus namaycush from lake superior by singlestranded conformational polymorphism analysis and reference strandmediated conformational analysis. Pdf rapid highresolution mhc class i genotyping of.
Hla typing by reference strand mediated conformation analysis. Genotyping of black grouse mhc class ii b using reference strandmediated conformational analysis rsca by tanja m strand and jacob hoglund topics. Several methods are available for genetic analysis of the polymorphic protein coding loci including direct sequencing, restriction fragment length polymorphism rflp analysis, denaturing gradient gel electrophoresis dgge, reference strandmediated conformational analysis rsca, and singlestranded. We have utilised a method, reference strand mediated conformation analysis rsca, which is technically simple and allows high resolution matching for all hla loci, for the typing of 48 patients. Kennedy lj, quarmby s, fretwell n, martin aj, jones pg, jones ca, ollier we. Development and optimization of a hybridization technique. Conformational methods, such as the referencestrandmediated conformational analysis rsca, have shown to achieve highresolution results without the ambiguities seen in the previously mentioned methods. Pcr fingerprinting and reference strandmediated conformational analysis 20, 21. Hla typing by reference strand mediated conformation analysis rsca rev immunogenet. This is a major technical breakthrough in hla typing. The use of reference strandmediated conformational analysis for the study of cheetah acinonyx jubatus.
Feline leucocyte antigen class ii polymorphism and. Genotyping of black grouse mhc class ii b using reference strand mediated conformational analysis rsca the major histocompatibility complex mhc is a cluster of genes involved in the vertebrate immune system and includes loci with an extraordinary number of alleles. A high resolution hla class i and class ii matching method. The pigtail macaque mhc class i allele mane presents an. Major histocompatibility complex diversity in the endangered ethiopian wolf canis simensis tissue antigens, 2011.
Highresolution characterization of the canine dladrb1 locus. Rapid high resolution mhc class i genotyping of chinese rhesus macaques by capillary reference strand mediated conformational analysis alex j. This report describes the application of reference strand mediated conformational analysis rsca, a novel dna typing technique, for the identification of clinically significant fungal pathogens. Reference strandmediated conformational analysis rsca is yet another method used for hla typing. We chose to use a hybridization technique, reference strandmediated conformational analysis rsca, to examine the chirab1 family, with a moderate number of genes dispersed across the microchromosome. Pdf genotyping mhc classiib in nonmodel species by. Rsca is limited by the number of hla reference alleles available since the hla region is so diverse. Hla typing with reference strandmediated conformation. Hla typing by reference strand mediated conformation. To date mhcgenotyping by reference strandmediated conformational analysis rsca has been used in only a few model species where extensive prior information on mhcgene sequences was available.
Reference strand conformational analysis rsca is a valuable tool in identifying mhcdrb sequences in three species of aotus monkeys. Genomic dna sequencingbased typing sbt methodology utilizes locus or groupspeci. Based on this technique, reference strand mediated conformation analysis rsca has been devel. The method of reference strand mediated conformation analysis rsca, formerly known as doublestrand conformation analysis relies upon the separation of homo and heteroduplexes in a gel matrix.
Fipthe cat had histopathologically confirmed andor clinically diagnosed fip. Highresolution characterization of the canine dladrb1. The use of reference strand mediated conformational analysis for the study of cheetah acinonyx jubatus feline leukocyte antigen class ii drb polymorphisms. Here we adapt this method to two nonmodel species, eurasian perch perca. Highresolution characterization of the canine dladrb1 locus using reference strandmediated conformational analysis. Platelet associated immunoglobulin igg and igm was assessed by an inhouse platelet immuno. We optimize the various steps, validate the analysis using wellcharacterized chicken mhc haplotypes, apply the system to type some experimental lines and. Identification of medically important pathogenic fungi by reference. Reference strandmediated conformational analysis rsca is a conformational method for highresolution typing class i and ii genes using native polyacrylamide gels. Genotyping mhc classiib in nonmodel species by reference. For the latter, the cat had to fulfil the following criteria. Reference strandmediated conformational analysis is. The use of reference strandamediated conformational analysis for.
Recently, reference strandmediated conformation analysis rsca has been shown to be effective for characterizing major histocompatibility complex genes in humans, sheep, horse, and cats. Allogeneic hematopoietic cell transplantation hct provides a curative treatment option for individuals with hematologic diseases. Pdf identification of medically important pathogenic fungi by. Mhc class i allele frequencies in pigtail macaques of. Since amplified alleles have the same length, accurate genotyping is difficult. Identification of medically important pathogenic fungi by reference strand mediated conformational analysis rsca brian p. Future perspectives in hla typing technologies intechopen. For statistical analyses, the hla data from the two groups of samples were tested using a modi. Rhesus macaques macaca mulatta provide wellestablished models for studying human disease pathogenesis and vaccine development. Pdf major histocompatibility complex diversity in the. Reference strand mediated conformation analysis rsca is a novel conformational method that offers high resolution and high sample throughput. We chose to use a hybridization technique, reference strand mediated conformational analysis rsca, to examine the chirab1 family, with a moderate number of genes dispersed across the microchromosome. Until recently, mhc genotyping was mainly done by cloningsanger sequencing or in species with low copy numbers by dnabased methods using a gel matrix for allele separation, such as single strand conformation polymorphism sscp, denaturing gradient gel electrophoresis dgge or reference strand mediated conformational analysis rsca combined.
Rapid high resolution mhc class i genotyping of chinese. Development and optimization of a hybridization technique to. Briefly, fragments containing exon 2, intron 2, and exon 3 of hlab were amplified with locus. Reference strandmediated conformational analysis rsca, which is increasingly used in studies of natural populations with multiple mhc genes, is a genotyping method capable to provide high. Manea 10 in outbred pigtail macaques were developed. The method of reference strand mediated conformation analysis rsca, formerly known as double strand conformation analysis relies upon the separation of homo and heteroduplexes in a gel matrix. In those cases, rsca was found to be a highly accurate and reliable method. We describe a typing protocol for classical chicken class i bf and class ii b blb genes, based on a hybridization method called reference strand mediated conformational analysis rsca. The authors of this article work for the university of liverpool, university of manchester, and the national museums of scotland. This technique differs from conventional sequencebased typing methodologies in that the hla type is assigned on the basis of accurate measurement of. Rsca is a heteroduplexbased conformational method which relies on detecting differences in the dna conformation of heteroduplexes generated in this study by the. However, whereas pcr fingerprinting uses mini and microsatellite sequences as targets for the primers and reference strand mediated conformational analysis is. Pdf rapid highresolution mhc class i genotyping of chinese. Historically, hct was limited to using hlaidentical related donors.
Reference strand conformational analysis 8 and commercial pcrssop sequence speci. Pcr and pcrdgge techniques have been evaluated to monitor biodiversity indexes within an atad autothermal thermophilic aerobic digestion system treating domestic sludge for land spread, by examining microbial dynamics in response to elevated temperatures during treatment. Identification of medically important pathogenic fungi by. Bifunctional chicken immunoglobulinlike receptors with a. This problem can be overcome by separating the alleles by groups or allelespecific pcr, cloning, or by the use of conformational techniques. When challenged with infectious agents, macaques exhibit individual differences in susceptibility. Pdf this report describes the application of reference strandmediated conformational analysis rsca, a novel dna typing technique, for the. Major histocompatibility complex susceptibility genes and. Baquero je, miranda s, murillo o, mateus h, trujillo e, suarez c, patarroyo me, parralopez c. Human leucocyte antigen typing wiley online library. Sequencing studies have suggested rapid divergence and little overlap between chir haplotypes, so we wished to begin to understand their genetics.
Krebs kc, jin z, rudersdorf r, hughes al, oconnor dh. Genotyping of black grouse mhc class ii b using reference strandmediated conformational analysis rsca tm strand, j hoglund bmc research notes 4 1, 183, 2011. Pdf genotyping of black grouse mhc class ii b using. Rsca is a cheap and rapid method, ideal for large epidemiological studies. Recipients receiving better hlamatched hematopoietic cell. Hla typing by reference strand mediated conformation analysis rsca. Genotyping of black grouse mhc class ii b using reference. Allelespecific high resolution hlab genotyping was determined by reference strand mediated conformational analysis previously described as double strand conformational analysis. Genespecific facs sorting method for target selection in. Two other universally applicable methods for the identification of candida species have been described. In this method, an unknown hla sample is mixed with a reference allele and run in a gel by electrophoresis. This report describes the application of reference strandmediated conformational analysis rsca, a novel dna typing technique, for the identification of. Pdf identification of medically important pathogenic.
Highresolution characterization of the canine dladrb1 locus using reference strand mediated conformational analysis. Reference strand mediated conformational analysis rsca, which is increasingly used in studies of natural populations with multiple mhc genes, is a genotyping method capable to provide high resolution and accuracy in such cases. The ipd project works with specialist groups andor nomenclature committees, which each curate a different section of the project. Mhc genotyping of nonmodel organisms using nextgeneration. Findings we adapted the rsca method to genotype multiple mhc class ii b blb genes in black grouse. We describe a typing protocol for classical chicken class i bf and class ii b blb genes, based on a hybridization method called reference strandmediated conformational analysis rsca. The macaques were derived from three separate breeding colonies in the usa, indonesia and australia, and allele frequencies were analysed within and between these groups.
Role of hla in hematopoietic stem cell transplantation. Use of amplified fragment length polymorphism analysis to. However, whereas pcr fingerprinting uses mini and microsatellite sequences as targets for the primers and reference strandmediated conformational analysis is. Mhc class i allele frequencies in pigtail macaques of diverse. Manea10, an allele that restricts the immunodominant siv gag epitope kp9, was the most common allele. Reference strandmediated conformational analysis rsca, which is increasingly used in studies of natural populations with multiple mhc genes, is a genotyping method capable to provide high resolution and accuracy in such cases. Reference strand mediated conformation analysis rsca is a novel conformational method that offers high resolution and high sample throughput for typing the hla class i and class ii genes. The atad process utilises a thermophilic population to generate heat and operates at elevated. High resolution hla class i typing by reference strand. The immuno polymorphism database ipd is a set of specialist databases related to the study of polymorphic genes of the immune system.
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