Typing forHLA-DPB1 * 03 andHLA-DPB1 * 06 using allele-specific DNA in vitro amplification and allele-specific oligonucleotide probes. Detection of “new” DPB1*06 variants

DP gene typing using in vitro DNA amplification combined with sequence-specific oligonucleotide probes has recently been reported. The resulting DNA amplification was specific for theHLA-DPB locus. Typing for the individualDPB alleles was exclusively dependent on the hybridizations of the probes but hampered by close sequence homology between differentDP alleles yielding complex patterns of reactivity with a panel of probes. We report the combined use of allele-specific DNA in vitro amplification and allele-specific oligonucleotides in typing forDPB1 ^* 03 andDPB1 ^* 06. Complete concordance with PLT typing was observed for theDPB1 ^* 03 alleles, while in the DPB1^*06 group, at least three variantDPB1 ^* 06 alleles were identified which have not been described previously.     

Positive correlation between oligonucleotide typing and T-cell recognition of HLA-DP molecules

The identification of 19 different HLA-DPB1 sequences implicates the existence of more DP specificities than can be typed for with cellular methods. How many of the DP sequences can be specifically recognized by T cells, and which of the polymorphic regions can contribute to the specificity of allorecognition, is not known. In order to investigate the distribution and the immunological relevance of recently described DPB1 alleles, we have typed a panel of 98 randomly selected Dutch Caucasoid donors for the HLA-DPB1 locus by oligonucleotide typing. Comparison of the typing results with primed lymphocyte typing (PLT) defined DP specificities shows an extremely good correlation. Moreover, additional alleles could be defined by oligonucleotide typing reducing the number of DP blanks in the panel. By selecting the appropriate responder stimulator combinations we were able to show that distinctive PLT reagents against oligonucleotide defined specificities DPB1*0401, DPB1*0402, DPB1*0901, and DPB1*1301 can be generated. To investigate in more detail which part of the DP molecule is responsible for the specificity of T-cell recognition, T-cell clones were generated against HLA-DPw3. The clones were tested for the recognition of stimulators carrying DPB1 alleles which had been defined by oligonucleotide typing and sequence analyses and which differed in a variable degree from DPB1*0301. The recognition patterns demonstrated that differences of one amino acid in polymorphic regions situated either in the beta sheets or alpha helix of the hypothetical model of the HLA class II molecule can eliminate T-cell recognition. Furthermore, sequence analyses revealed a new DPB1 allele designated DPB1*Oos.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M58608. The name DPB1*2001 has officially been assigned to the DPB*Oos allele by the WHO nomenclature Committee in March 1991. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1990b), names will be assigned as they are identified. Lists of such new names will be published in the following WHO nomenclature report.     

DNA typing forHLA-DPB1*02 and−DPB1*04 in multiple sclerosis and juvenile rheumatoid arthritis

DP gene typing using in vitro DNA amplification combined with sequence-specific oligonucleotide probes (SSOP) has recently been reported. The amplification step may be specific for theHLA-DPB locus, or it may be specific for one or a group ofHLA-DPB alleles, thus increasing the discriminatory power of the system. We report the combined use of group-specific DNA in vitro amplification followed by SSOP in typing forDPB1*02 andDPB1*04 variants. The method was used to type for these variants in 96 randomly selected, healthy Danes, in 37 patients with pauciarticular juvenile rheumatoid arthritis (PJRA); and in 38 patients with multiple sclerosis (MS). Increased frequencies of the cellularly defined HLA-DPw2 in PJRA and of HLA-DPw4 in MS have previously been reported. In the patient groups, the frequencies of theDPB1*02 andDPB1*04 variants did not differ significantly from those expected based on the cellularly defined HLA-DP types of the patients and the frequencies of theDPB1*02 andDPB1*04 variants among healthy Danes.     

DNA typing of primate major histocompatibility complex (Mhc)-DQA1 locus by PCR and dot blot hybridization.

Non-human primates (NHPs) are increasingly utilized as models to investigate different aspects of immune responses against self (autoimmunity) and foreign antigens. These animals provide valuable models for testing the efficacy of candidate vaccines against pathogens such as human immunodeficiency virus (HIV) and also fertility regulating agents (immunocontraceptives). In order to fully understand the effects of vaccination, it may be necessary to elucidate the immunogenetic background of these animals. The major histocompatibility complex (Mhc) molecules play an important role in the generation of effective immune responses. Serological techniques have been used in the identification of human leukocyte antigens (HLA) necessary for cross-matching organs and tissues for transplantation. However, the application of this technique for typing monkey Mhc alleles has been hampered by unavailability of well characterized immunological reagents. Polymerase chain reaction (PCR)-based techniques such as restriction fragment length polymorphism (RFLP) and sequence-specific oligonucleotide probe hybridization (SSOP) have been extensively used for typing HLA-DP, DQ and DR alleles. A commercially available Kit (AmpliTypeR) designed for amplification and typing of HLA DQalpha alleles is routinely used in typing DNA samples for forensic casework. In the present study, we have evaluated this kit for possible application in routine typing of primate DQA1 alleles. Genomic DNA from ten African primate species (23 individuals) was isolated from peripheral blood lymphocytes and polymorphic second exon of DQA1 locus amplified using GH26 and GH27 PCR primers. The PCR products were hybridized on a nylon membrane containing immobilized sequence-specific oligonucleotide probes. Our results show seven of the nine probes hybridizing with primate DQA1 alleles, indicating that typing of equivalent primate alleles can be accomplished at lower stringency conditions. However, it may be necessary to design additional oligonucleotides probes (based on available primate DQA1 sequences) to improve the discriminating power of this kit for use in routine typing of Old World monkey DQA1 alleles.     

HLA-DPB1 alleles in a population from South China

Using the polymerase chain reaction (PCR) and hybridization with oligonucleotide probes, we analyzed the distribution of DPB1 alleles in 99 healthy unrelated individuals from the city of Guangzhou (Canton), South China. Twelve different DPB1 alleles were found in this panel. The most common allele was DPB1*0501 (62.6%). Other major alleles detected included DPB1*02 (DPB1*0201 and DPB1*0202), DPB1*1301, DPB1*0401, and a recently described allele, designated DPB1*2101. The hybridization pattern of DPB1*2101 showed that this allele shared sequences with DPB1*0301 and DPB1*0601 in the A and F hypervariable regions, while the C, D, and E regions were identical to those of DPB1*0202. DPB1*2101 was observed in 11% of the subjects tested. It was found to be in strong linkage dis-equilibrium with DRB1*1202. In family studies, segregation of the haplotype DRB1*1202, DRB3*0301, DQA1*0601, DQB1*0301, DPB1*2101 was observed. The second exon of DPB1*2101 was sequenced from codon 8 to codon 90 and the sequence, inferred from the pattern of hybridization, was confirmed. DPB1*0301, DPB1*0402, DPB1*0101, DPB1*1401, DPB1*1901, and another recently recognized allele, now designated DPB1*2401, were detected with low frequencies. DPB1*2401 had the same hybridization pattern as DPB1*0501 except for a probe that matches codons 85–90. In this region, DPB1*2401 encoded the amino acid sequence GPMTLQ instead of EAVTLQ as in DPB1*0501.     

Typing of artiodactyl MHC-DRB genes with the help of intronic simple repeated DNA sequences

An efficient oligonucleotide typing method for the highly polymorphic MHC-DRB genes is described for artiodactyls like cattle, sheep and goat. By means of the polymerase chain reaction, the second exon of MHC-DRB is amplified as well as part of the adjacent intron containing a mixed simple repeat sequence. Using this primer combination we were able to amplify the MHC-DRB exons 2 and adjacent introns from all of the investigated 10 species of the family of Bovidae and giraffes. Therefore, the DRB genes of novel artiodactyl species can also be readily studied. Oligonucleotide probes specific for the polymorphisms of ungulate DRB genes are used with which sequences differing in at least one single base can be distinguished. Exonic polymorphism was found to be correlated with the allele lengths and the patterns of the repeat structures. Hence oligonucleotide probes specific for different simple repeats and polymorphic positions serve also for typing across species barriers. The strict correlation of sequence length and exonic polymorphism permits a preselection of specific oligonucleotides for hybridization. Thus more than 20 alleles can already be differentiated from each of the three species.     

Rapid typing of HLA-DQB1 DNA polymorphism using nonradioactive oligonucleotide probes and amplified DNA

The allelic sequence diversity at theHLA-DQBI locus has been analyzed by polymerase chain reaction (PCR) amplification and sequencing. Fifteen amino acid sequence-defined alleles (one previously unreported) and several silent nucleotide polymorphisms which subdivide these alleles have been identified. Here, we describe the specific amplification of theDQB1 second exon by several different PCR primer pairs and a simple and rapid typing procedure using a panel of 16 horseradish peroxidase (HRP)-labeled oligonucleotide probes capable of distinguishing theseDQBI alleles.     

Allele-specific hybridization using streptavidin-coated magnetic beads for species identification, S genotyping, and SNP analysis in plants

Dot-blot hybridization has been successfully used for the construction of single nucleotide polymorphism (SNP)-based linkage maps, quantitative trait locus analysis, marker-assisted selection, and the identification of species and cultivars. This method is, however, time-consuming, even for a small number of plant samples. We propose a method in which streptavidin-coated magnetic beads replace the nylon membrane for immobilization of the PCR products and are hybridized with allele-specific oligonucleotide probes and a digoxigenin-labeled oligonucleotide hybridized with the allele-specific oligonucleotide probe. After amplification of plant DNA by PCR with the biotinylated primers, those oligonucleotide probes having species-specific or allele-specific sequences were mixed together with the digoxigenin-labeled oligonucleotide and the streptavidin-coated magnetic beads at a temperature suitable for each probe. Species-specific internal transcribed spacer 1 (ITS1) sequences and allele-specific sequences of the hypervariable region I of S-locus receptor kinase (SRK) specifically detected ITS1 sequences and SRK alleles in Brassica species, respectively. SNPs were also successfully analyzed by using allele-specific oligonucleotide probes and competitive oligonucleotides. In the SNP analysis, PCR products were indirectly captured by magnetic beads. SNP alleles of eight cultivars each of Brassica rapa and Raphanus sativus were analyzed using streptavidin-coated magnetic beads. The genotyping results corresponded well with those of dot-blot-SNP analysis. Although allele-specific hybridization using streptavidin-coated magnetic beads is somewhat costly, it is easier and more rapid than dot-blot hybridization. This method is suitable for the analysis of a small number of plant samples with a large number of DNA markers.     

Highly sensitive and fast detection of Phoma tracheiphila by polymerase chain reaction

Summary A new method for the diagnosis of the plant pathogenic fungus Phoma tracheiphila has been developed. The method takes advantage of the enzymatic amplification of a specific 102 bp-long target sequence of fungal DNA by the polymerase chain reaction (PCR) using Thermus aquaticus DNA polymerase. The amplified DNA was characterized by agarose-gel electrophoresis, molecular hybridization using a synthetic oligonucleotide probe and direct sequencing. The application of the new method makes possible fast and direct detection of the pathogen in lignified plant tissues, a goal not previously achieved when a cloned probe and a dot-blot test were employed. In addition the PCR test can be used to advantage as a particularly simple and fast way of typing fungal isolates. This is achieved by submitting to DNA amplification crude homogenates of fungal mycelium and analysing the amplified DNA on an agarose mini-gel.Offprint requests to: F. Rollo     

Analysis of HLA-DP allelic sequence polymorphism using the in vitro enzymatic DNA amplification of DP-alpha and DP-beta loci

Allelic sequence variation of the HLA DP-alpha and DP-beta genes has been analyzed in a panel of 34 DP-typed cell lines. The polymorphic second exon of these genes was specifically amplified in vitro by the polymerase chain reaction method, using the thermostable DNA polymerase of Thermus, aquaticus. The analysis of M13 clones containing the amplified DP-beta sequences revealed a total of 14 allelic variants. In general, specific allelic DP-beta sequences were associated with each of the defined DPw1-w6 types, with beta allele subtypes revealed for the DPw2 and DPw4 specificities. An additional six DP-beta alleles which did not correlate with any of the T cell-defined specificities (DP "blanks") were also identified. Only the two previously characterized alleles of DP-alpha were detected. These observations suggest that the T cell-defined DP specificities are determined by polymorphic residues on the beta-chain. The sequence polymorphisms in DP-beta are clustered in a few specific regions, and can be detected using sequence-specific oligonucleotide probes and polymerase chain reaction amplified DNA in a rapid dot-blot format. This approach provides a simple and informative method of DP typing. The DP-beta sequences derived from four DP-typed celiac disease patients were compared with the distribution of DP-beta alleles in control individuals.     

Identification of RAPD markers linked to a fertility restorer gene for theOgura radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.     

Frequency of HLA-DQA1 alleles in the Japanese population.

One of the HLA class II genes, HLA-DQA1, was typed from 290 unrelated healthy Japanese using the oligonucleotide typing method. The HLA-DQA1 gene was enzymatically amplified and typed by dot-blot hybridizations with 10 sequence-specific oligonucleotide probes labeled nonradioactively. Using this method, the HLA-DQA1 genotype was theoretically classified into 36 genotypes: 8 homozygous and 28 heterozygous ones. Actually, 26 genotypes were observed in the present study, and the gene frequency of each allele was calculated. The observed numbers were in accordance with the numbers expected under the Hardy-Weinberg equilibrium. The HLA-DQA1 genotype was also determined in aged bloodstains. Since the genotype is polymorphic in the Japanese population and a very small amount of blood is required for determination, this typing is particularly useful for forensic analysis.     

Simple and efficient methods for <Emphasis Type="Italic">S</Emphasis> genotyping and <Emphasis Type="Italic">S</Emphasis> screening in genus <Emphasis Type="Italic">Brassica</Emphasis> by dot-blot analysis

In F₁ hybrid breeding of Brassica vegetables utilizing the self-incompatibility system, identification of S genotypes in breeding lines is required. In the present study, we developed S-tester lines of 87 S haplotypes, i.e., 42 S haplotypes in B. rapa and 45 S haplotypes in B. oleracea. With these materials, we established a simple, efficient, and reliable dot-blot technique for S genotyping for 40 S haplotypes of B. rapa and and 33 of B. oleracea using allele-specific oligonucleotide probes and allele-specific primer pairs designed from sequences of each SP11 allele. In this method, DNA fragments amplified using multiplex primer pairs with digoxigenin-dUTP were hybridized with dot-blotted allele-specific oligonucleotide probes with distinct signals. In addition, we developed a screening method for identification of plants harboring a particular S haplotype using a labeled allele-specific oligonucleotide probe. This method is considered to be useful for purity testing of F₁ hybrid seeds.     

Extended HLA-DPB1 polymorphism: an RNA approach for HLA-DPB1 typing

Most of the 119 human leukocyte antigen (HLA)-DPB1 alleles are defined by polymorphism in six hypervariable regions (HVRs) in exon 2 of the HLA-DPB1 gene. We investigated how DPB1 polymorphism is represented in the entire coding region. An RNA sequencing-based typing (SBT) approach was developed for the identification of HLA-DPB1 polymorphism from the 5′ untranslated region (UTR) through the 3′-UTR. B-cell lymphoblastoid cell lines, encoding 16 different DPB1 alleles, were studied. Results show additional HLA-DPB1 polymorphism in exons 1, 3, 4 and 5 and the 5′ and 3′-UTR. Four new HLA-DPB1 alleles were identified, DPB1*0502, DPB1*0602, DPB1*0802 and DPB1*0902, which have exon 2 sequences identical to other DPB1 alleles but differ in the extended region. The additional polymorphism represents two main polymorphic lineages in the DPB1 alleles. Among the HVRs in exon 2, only HVR F correlates with these two main lineages.     

Dot-blot-SNP analysis for practical plant breeding and cultivar identification in rice

We report dot-blot hybridization with allele-specific oligonucleotides for single nucleotide polymorphisms (SNPs) analysis to be applicable for practical plant breeding and cultivar identification. Competitive hybridization of a digoxigenin-labeled oligonucleotide having the sequence of a mutant allele (or a wild-type allele) together with an unlabeled oligonucleotide having the sequence of a wild-type allele (or a mutant allele) was highly effective to reduce background signals in dot-blot hybridization. All 100 tested genes (200 alleles) in rice having SNPs or insertions/deletions were detected in an allele-specific manner. Genotypes of 43 rice cultivars were identified by this technique, and eight SNP markers were found to be sufficient for distinguishing all the cultivars from each other. Dot-blot analysis was also applied to genotyping of Wx and Sd1 of F₄ plants in a conventional breeding program. Since dot-blot analysis with competitive hybridization provides a highly reliable, simple, and cost-effective technique for SNP analysis of a large number of samples, this technique is expected to realize the practical use of a novel breeding method, in which plants or breeding lines are selected by SNP analyses of many genes in a laboratory.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Microsatellites from Clarias batrachus and their polymorphism in seven additional catfish species

We isolated 18 novel microsatellite loci from the walking catfish (Clarias batrachus), and examined their cross‐amplification in seven additional catfish species from three families. Sixteen of the 18 microsatellites were polymorphic in the source species (allele number: 2–10/locus and expected heterozygosity: 0.30–0.87). Moreover, nine of these 18 primer pairs cross‐amplified specific and polymorphic products from the genome of at least six of the seven other catfish species tested. However, the success rate of cross‐species amplification varied from locus to locus, indicating that cross‐species amplification of microsatellites is locus‐dependent.     

The detection of beta-globin gene mutations in beta-thalassemia using oligonucleotide probes and amplified DNA

DNA amplification combined with the use of synthetic oligonucleotide probes has become an important tool in the identification of base substitutions. We report the use of this DNA amplification technique for the detection of mutations in beta-thalassemia. A series of oligonucleotide primers are synthesized which span the beta-globin gene; one primer is complementary to the coding strand and the other to the non-coding strand. The primers are chosen so that there is little homology with other DNA segments, especially the delta gene. Each set of primers spans an area of the gene between 100 and 300 bp, while the suspected mutation point is located between these two primers. With the use of such a primer set, the beta-globin gene region is amplified by denaturation, annealing and DNA synthesis. The amplification cycle is repeated 25-30 times, using the Klenow fragment of DNA polymerase I. The resulting amplified DNA is hybridized with normal and synthetic deoxynucleotide probes using a standard dot-blot method. We have designed a set of primers and experimental conditions which should prove useful to diagnostic centers for detection of numerous beta-thalassemia mutations.     

Novel microsatellite markers for Bactris gasipaes (Palmae)

Ten new polymorphic microsatellites were isolated and characterized in Bactris gasipaes using a microsatellite enrichment protocol and selective hybridization with oligonucleotide probes. The loci are highly polymorphic, with a mean of 14.6 alleles per locus and a mean expected heterozygosity of 0.83 among 62 individuals of the Pampa Hermosa landrace. These microsatellites will be useful for population genetic analysis and germplasm characterization for heart‐of‐palm breeding.     

Detection of mutations in PCR products from clinical samples by surface plasmon resonance

Two different strategies for scanning and screening of mutations in polymerase chain reaction (PCR) products by hybridization analysis are described, employing real-time biospecific interaction analysis (BIA) for detection. Real-time BIA was used to detect differences in hybridization responses between PCR products and different 17-mer oligonucleotide probes. For the analysis using a biosensor instrument, two different experimental formats were investigated based on immobilization of either biotinylated PCR products or oligonucleotide probes onto a sensor chip. Applied on the human tumour suppressor p53 gene, differences in hybridization levels for full-match and mismatch situations employing both formats allowed the detection of point mutations in exon 6 PCR products, derived from a breast tumour biopsy sample. In addition, a mutant sample sequence could be detected in a 50/50 background of wild type exon 6 sequence. The suitability of the different formats for obtaining a regenerable system and a high throughput of samples is discussed. © 1997 John Wiley & Sons, Ltd.     

The intranuclear organization of normal, hemizygous and excision-deficient rRNA genes during developmental amplification in Tetrahymena thermophila

In the ciliated protozoan, Tetrahymena thermophila, the diploid germinal micronucleus contains two allelic copies of the gene for ribosomal RNA (rDNA). During genesis of new somatic macronuclei the germline rDNA gene is excised by developmentally programmed chromosome breakage and preferentially amplified to ∼9,000 copies. We have studied this process by fluorescence in situ hybridization. We find that initially rDNA amplification is restricted to two separate and highly confined regions of the nucleus. Analysis of nuclei that are hemizygous for the rDNA locus reveals that each focus of hybridization is derived from a single allele of the rDNA. As rDNA amplification progresses these two foci of hybridization disperse and spread throughout the macronucleus, eventually forming ∼100–500 new nucleoli. These events are correlated with morphologically distinct developmental stages. We investigated the amplification of the C3 allele of the rDNA that confers a replication advantage over the B allele during vegetative propagation, and find no evidence for preferential amplification of the C3 early in rDNA maturation. We also show that the rmm 11 rDNA mutant allele, which is defective for developmentally programmed rDNA excision, can be amplified during the two-foci stage in mutant homozygotes and heterozygotes, but fails to amplify further and disperse into multiple nucleoli. These data indicate that amplification of the rmm 11 allele is not delayed during the initial rounds of amplification, and suggest that efficient excision is not required for this amplification to occur. We propose that rDNA amplification is a two-step process. First, the two rDNA alleles are independently amplified, while allelic copies remain closely associated. Later, copies of the rDNA disperse and are further amplified, presumably because rDNA excision has occurred, generating fully mature rDNA minichromosomes that are able to replicate to high copy number. Received: 21 February 1997; in revised form: 21 April 1997 / Accepted: 5 May 1997