Rapid HLA-DQB1 genotyping for four alleles in the assessment of risk for IDDM in the Finnish population. The Childhood Diabetes in Finland (DiMe) Study Group.

OBJECTIVE: To study the effectiveness of MHC genotyping in the assessment of risk for IDDM based on the identification of alleles that are significantly associated with risk for IDDM (DQB1 *0302 and *0201) and protection from it (DQB1 *0602/*0603 and *0301). RESEARCH DESIGN AND METHODS: A long series of 649 index cases of IDDM, together with their healthy siblings and 756 healthy blood donors, was collected in Finland. The samples were analyzed using a large-scale assay procedure that was developed for rapid screening purposes. The method utilizes time-resolved fluorometry to detect the hybridization of lanthanide-labeled allele-specific oligonucleotide probes with amplified gene product. RESULTS: A total of 61.9% of IDDM index cases had high risk (DQB1 *0201/*0302) or moderate risk (DQB1 *0302/x [x meaning DQB1 *0302 or a nondefined allele]) genotypes compared with 14.3% of the reference population. In patients and control subjects, the frequencies of low risk genotypes were 28.0 and 22.1%, respectively, and those of decreased risk genotypes, 10.0 and 63.6%. The relative risk of a *0201/*0302 genotype was 53.5 (31.1-92.8) compared with the decreased risk genotypes (63.6% of controls). The graded risk estimation was equally efficient in assessing the risk of IDDM in siblings of child with IDDM. CONCLUSION: The near-automatic typing procedure developed is attractive for large-scale screening projects, such as diabetes prevention and intervention trials.

The frequency of an inactivating point mutation (566C-->T) of the human follicle-stimulating hormone receptor gene in four populations using allele-specific hybridization and time-resolved fluorometry.

We have described previously in the Finnish population an inactivating point mutation (566C-->T) in the human FSH receptor (FSHR) gene. In women, this mutation causes hypergonadotropic ovarian failure with arrest of follicular maturation and infertility, whereas in men, there is variable suppression of spermatogenesis, but no absolute infertility. To determine whether the same FSHR mutation occurs in other populations, its frequency was determined in Finland, Switzerland, Denmark, and the Chinese population of Singapore. The mutation was screened for using genomic DNA extracted from whole blood or dried blood spots. Exon 7 of the FSHR gene was first amplified using a pair of biotinylated primers. The PCR products were then immobilized on streptavidin-coated microtitration wells and hybridized using short allele-specific oligonucleotide probes labeled with europium. Time-resolved fluorometry was used for europium signal detection. To test the reliability of this method, 40 isolated DNA samples and 35 dried blood spot samples were blindly tested for the 566C-->T FSHR mutation. The analyses yielded identical results with denaturing gradient gel electrophoresis and allele-specific restriction enzyme digestion of the same samples, thus demonstrating the reliability of the tested method. Automation of this procedure allows the screening of large numbers of samples, which was subsequently carried out to investigate the frequency of the 566C-->T mutation in the study populations. A total of 4981 samples from the above-mentioned 4 countries were analyzed. The frequency of the 566C-->T mutation was 0.96% for all Finnish samples (n=1976), with a strong enrichment of the mutant allele in the northeastern part of the country. Only 1 mutation carrier was identified in the samples from Switzerland (n=1162), whereas none was found in samples from Denmark (n=1094) and the Singapore Chinese (n=540). These results suggest that the 566C-->T mutation of the FSHR gene is enriched in Finland, but is uncommon in other populations.

Detection of a point mutation using short oligonucleotide probes in allele-specific hybridization.

Two nonradioactive and simple procedures were developed to detect the A985G point mutation that causes medium-chain acyl-CoA deficiency. In both of these assays, short oligonucleotide probes were used in allele-specific hybridization combined with DNA amplification. The lower limit for a useful probe was found to be between 9 and 12 base pairs. Time-resolved fluorometry was utilized as the label technology and microtitration plates as the solid support. In one of the assay formats, probes labeled with europium and samarium chelates were used to simultaneously detect the mutant and normal alleles from the same hybridization reaction. In addition, the discrimination efficiency of different probes was characterized by cross-reactivity determinations and by measuring affinities of the probes towards fully complementary as well as towards mismatch-forming target oligonucleotides. All of the 80 coded patient samples analyzed were correctly typed in both of the assay formats used.

Triple-label hybridization assay for type-1 diabetes-related HLA alleles.

We describe a method for the detection of two type 1 (insulin-dependent) diabetes susceptibility (*0201, *0302) alleles and two protective (*0301, *0602/0603) alleles of the HLA-DQB1 gene on the human major histocompatibility complex (MHC). The test is based on DNA amplification with PCR followed by simultaneous, allele-specific triple-label hybridization performed in microtitration wells. In the hybridization, very short allele-specific oligonucleotides labeled with europium (Eu), terbium (Tb) or samarium (Sm) are used. The labeled probes could be detected using time-resolved fluorometry with sensitivities of 1 x 10(7), 3 x 10(8) and 3 x 10(8) molecules, respectively. Cross-reactions were not found among samples containing 14 common DQB1 alleles. To test the utility of the developed assay, 100 DNA and 14 dried blood spot samples with known DQB1 alleles were analyzed. A 100% agreement with the reference method was reached. Thus, this triple-label hybridization assay proved to be suitable even for detection of a large number of samples.

Detection of Philadelphia chromosome using PCR and europium-labeled DNA probes.

More than 95% of the patients with chronic myelogenous leukemia (CML) carry translocations between protooncogene abl of chromosome 9 and bcr gene of chromosome 22, resulting in the Philadelphia chromosome (Ph1). After allogeneic bone marrow transplantation (BMT) it is important to detect possible residual malignant cells in CML patients. A new sensitive hybridization method combined with polymerase chain reaction (PCR), based on the detection of the europium (Eu3+) label by time-resolved fluorescence, was applied for the detection of Ph1 chromosome. Total RNA from 10(6) peripheral blood leukocytes was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction. After cDNA synthesis by reverse transcriptase, the PCR amplification (30 cycles) was carried out. In the detection phase two oligonucleotide probes were used in the hybridization reaction, one biotinylated (bcr gene, exon 2) and one (abl gene) labeled with Eu3+. The hybrids were collected in a streptavidin-coated microtitration well and the bound Eu3+ was measured in a time-resolved fluorometer. To assess the sensitivity of the method, different numbers of CML cell line K562 cells were mixed with 10(5) apparently normal human leukocytes. Five K562 cells/10(5) leukocytes could be detected. Six patients with CML confirmed by clinical and cytogenetic criteria were studied. Three of the patients underwent an allogeneic BTM 6-18 months before the investigation and all of them were Ph1-negative. The other three patients who were nontransplanted were positive as expected.

Simultaneous detection of two cystic fibrosis alleles using dual-label time-resolved fluorometry.

A simple dual-label hybridization test for normal and mutant cystic fibrosis (CF) alleles is described. The assay is based on time-resolved fluorometry (TRF), which allows the simultaneous detection of DNA probes labelled with different lanthanides from one hybridization reaction. DNA was liberated from dried blood disks, normally used in neonatal screening programmes, by boiling in alkaline solution. A 138 bp region including the site of deletion, delta F-508, which is present on about 70% of cystic fibrosis chromosomes, was amplified using the polymerase chain reaction (PCR). The presence or absence of normal and mutant alleles was then determined in a solution hybridization using allele specific oligonucleotide probes labelled either with europium (Eu) or with samarium (Sm) chelates. A common biotinylated probe was used for binding the hybrids onto microtitration wells coated with streptavidin. Some 5 x 10(7) molecules of the normal allele (Eu) and 5 x 10(8) molecules of the mutant allele (Sm) could be detected simultaneously in a single hybridization reaction. The assay was simple to perform and made it possible to reduce the number of hybridizations needed to interpret the sample as being normal, carrier or mutant with regard to the mutation, delta F-508.

Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: hybridization assays on polymer particles obtained by direct solid phase assembly of the oligonucleotide probes.

Oligodeoxyribonucleotides were assembled by conventional phosphoramidite chemistry on uniformly sized (50 microns) porous glycidyl methacrylate/ethylene dimethacrylate (SINTEF) and compact polystyrene (Dynosphere) particles, the aminoalkyl side chains of which were further derivatized with DMTrO-acetyl groups. The linker was completely resistant toward ammonolytic deprotection of the base moieties. The quality of oligonucleotides was assessed by repeating the synthesis on the same particles derivatized with a cleavable ester linker. The ability of the oligonucleotide-coated particles to bind complementary sequences via hybridization was examined by following the attachment of oligonucleotides bearing a photoluminescent europium(III) chelate to the particles. The fluorescence emission was measured directly on a single particle. The effects of the following factors on the kinetics and efficiency of hybridization were studied: number of particles in a given volume of the assay solution, loading of oligonucleotide on the particle, concentration of the target oligonucleotide in solution, length of the hybridizing sequence, presence of noncomplementary sequences, and ionic strength. The fluorescence signal measured on a single particle after hybridization was observed to be proportional to the concentration of the target oligonucleotide in solution over a concentration range of 5 orders of magnitude.

Simple triple-label detection of seven cystic fibrosis mutations by time-resolved fluorometry.

We describe a simple hybridization assay performed in microtitration wells with use of DNA probes labeled with three different lanthanide chelates for detection of seven mutations that cause cystic fibrosis. The assay is based on DNA amplification of four fragments containing the mutations (delta F508, G1717-->A, G542X, R553X, 3905 insertion T, W1282X, and N1303K) by PCR, followed by hybridization with short, allele-specific oligonucleotide probes labeled with europium, terbium, or samarium chelates. Because the technology makes it possible to hybridize three DNA probes simultaneously in one reaction, all 14 mutation-related alleles were detected in a total of five reaction wells. Blood spot specimens, obtained from children with cystic fibrosis, their parents, and their siblings, have been assayed, and for all the probes the positive signal-to-noise ratios are > 10. Solution hybridization utilizing triple-label time-resolved fluorometry combined with PCR is a suitable procedure for large-scale screening and automation.

The use of europium (Eu3+) labelled primers in PCR amplification of specific target DNA.

The polymerase chain reaction (PCR) has many potential applications in the field of DNA probe diagnostics. Here we describe a method that utilizes PCR and time-resolved fluorometry (TRF) for the detection of specific target DNA. First the DNA segment to be detected is amplified according to standard procedures. Then a pair of europium (Eu3+) and biotin-labelled primers nested within the amplified fragment is incorporated in a few additional PCR cycles. Thus amplified DNA fragments are generated that contain an affinity label (biotin) and a detectable label (europium). The doubly-labelled amplified DNA fragments are collected onto streptavidin coated microtitration strips and the bound Eu3+ is measured in a time-resolved fluorometer. We show here the application of this method to the detection of HIV-1 DNA. As few as five copies of HIV-1 DNA could readily be detected using this assay. The method described here is sensitive, rapid and easy to employ. In addition it lends itself to automation.

C-terminal truncations of a thermostable Bacillus stearothermophilus alpha-amylase.

A series of truncated proteins from a thermostable Bacillus stearothermophilus alpha-amylase was prepared to study the importance of the extension in the C-terminus compared with other liquefying Bacillus alpha-amylases. The mutations introducing new translation termination sites shortened the 515 amino acid residue-long wild type enzyme by 17, 32, 47, 73 or 93 residues. The longer the truncation, the lower the specific activity of the enzyme. Only the two longest mutant proteins were active: the specific activity of the 498 residue variant was 97% and protein 483 was 36% that of the parental enzyme. The Km values of starch hydrolysis changed from 1.09 for wild type enzyme to 0.35 and 0.21 for mutants 498 and 483, respectively, indicating altered substrate binding. The mutant enzymes had almost identical pH and temperature optima with the wild type amylase, but enhanced thermal stability and altered end product profile. The consequences of the truncation to the structure and function of the enzymes were explored with molecular modeling. The liquefying amylases seem to require approximately 480 residues to be active, whereas the C-terminal end of B.stearothermophilus amylase is required for increased activity.

Detection of mutation delta F508 in the cystic fibrosis gene using allele-specific PCR primers and time-resolved fluorometry.

A method to detect the main cystic fibrosis (CF) mutation delta F508 from dried blood spots, whole blood, or saliva using the polymerase chain reaction (PCR) and time-resolved fluorometry (TRF) is described. Samples are treated by boiling in mild alkaline solution, after which two allele-specific PCR reactions are performed. Allele-specific primers and a common biotinylated primer are used in the amplification reactions. To detect the PCR product, an europium-labeled oligonucleotide, complementary to the biotinylated strand of the PCR product, is used in a solution hybridization. Hybridization is done in streptavidin-coated microtitration wells, making the detection easy to perform. After a washing step, the bound label is detected using a time-resolved fluorometer. To analyze function of the assay, 20 dried blood spot samples were tested. PCR amplification of the deletion region combined with gel retardation assay was used as a control method. In the initial testing, 2 samples giving discrepant results in the two assays were found. In addition, 17 samples from known CF patients together with 6 normal control samples were analyzed. Among these patient samples, 10 homozygotes and 6 carriers for mutation delta F508 were found.

Detection of amplified HTLV-I/-II viral sequences using time-resolved fluorometry.

Since its discovery, the polymerase chain reaction (PCR) has been used for different purposes in the field of DNA research. We tested the PCR for the diagnosis of HTLV-I/-II infections. PCR was used to amplify 141- and 149-base pair regions from the HTLV-I and HTLV-II virus genomes, respectively. The annealing temperature in the PCR amplification was optimized using 20% polyacrylamide gels and silver staining. Even a slight change (3 degrees C) in the annealing temperature had an effect on the specificity of the reaction. The PCR products were detected with biotin and Eu-labeled oligonucleotide probes in a solution hybridization format. The linearity of the assay was tested with serial dilutions of purified chromosomal DNA containing integrated HTLV-II sequences. The linearity was found to be dependent on the number of cycles used in the PCR amplification. The best linearity, at a target level of a few copies, was achieved using a low number of cycles. The specificity of the assay was tested using HTLV-I and HTLV-II-infected lymphocytes from the cell lines Hut102 and MO480, respectively. No cross reactivity between these analytes was observed.

Population screening for the common G985 mutation causing medium-chain acyl-CoA dehydrogenase deficiency with Eu-labeled oligonucleotides and the DELFIA system.

We have screened 10171 neonatal blood spots from the Trent and West Midlands regions of the UK for the common G985 mutation to more accurately establish the incidence of medium-chain acyl coenzyme (Co)A dehydrogenase (MCAD) deficiency. We have used a technique involving PCR and Eu-labeled allele-specific oligonucleotides detected by using time-resolved fluorometry on the dissociation-enhanced fluorescence immunoassay (DELFIA) system for the detection of the G985 mutation. We have also evaluated the feasibility of neonatal screening with this technique. We identified 158 G985 heterozygotes and no G985 homozygotes. The calculated incidence of MCAD deficiency in the population studied (all mutations, assuming 90% of MCAD mutations are G985) is 1 in 13426 (95% confidence limits 1 in 10070-1 in 18791). At the optimum cutoff criteria, the technique has a sensitivity of 97.5%, specificity of 99.6%, and positive predictive value of 80.2%. We conclude that this study confirms that MCAD deficiency is a common inherited metabolic disease and is a candidate for neonatal screening. The methodology used is robust and suitable for large-scale population studies such as this. The technique is also potentially suitable for screening.

Quantitative reverse transcription-PCR assay with an internal standard for the detection of prostate-specific antigen mRNA.

BACKGROUND: Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. METHODS: The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates. RESULTS: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 10(6) copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 +/- 200 to 44 100 +/- 4900 (mean +/- SD) in the samples, corresponding to approximately 1-100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 +/- 100 and 2000 +/- 900 (mean +/- SD) PSA mRNA copies in 5 mL of blood for the 2 males]. CONCLUSIONS: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.

Time-resolved fluorometry in the genetic diagnosis of familial defective apolipoprotein B-100.

A novel technique for screening point mutations has been developed for diagnosis of familial defective apolipoprotein (apo) B-100 (FDB). In FDB, an amino acid exchange occurs at position 3500 in apoB-100 due to a point mutation. Polymerase chain reaction (PCR) was performed on the appropriate region of the apoB gene, and the PCR products were hybridized in solution with europium-labeled oligonucleotides, complementary to either the wildtype or the mutant genome. The presence or absence of the apoB-3500 mutation was monitored by time-resolved fluorescence of the europium chelate. The method allows a larger number of samples to be processed simultaneously, and the detection system displays a high level of sensitivity without the hazards connected to the use of radioactivity. When 127 Swedish patients, clinically diagnosed as suffering from heterozygous familial hypercholesterolemia, were screened for the presence of the apoB-3500 mutation, two patients, unrelated to each other, were found to be heterozygotes. These patients are the first reported cases of FDB from Sweden, and the frequency rate observed among hypercholesterolemic patients, 1.6%, is in accordance with the figures reported for several other patient population in Europe and the United States.

Detection of human immunodeficiency virus type 1 by using the polymerase chain reaction and a time-resolved fluorescence-based hybridization assay.

The polymerase chain reaction (PCR) has many potential applications in the field of nucleic acid diagnostics. In particular, it has been successfully applied to the detection of pathogens present in low copy numbers such as the human immunodeficiency virus type 1. Here we describe a time-resolved fluorescence-based hybridization assay which, combined with the PCR, offers an extremely sensitive method for the detection of nucleic acids. In this assay format, the PCR is run by standard procedures and the subsequent hybridization reaction is carried out in solution by using two oligonucleotide probes, one biotinylated and one labeled with europium (Eu3+). The sandwich hybrids are then collected onto a streptavidin-coated microtitration well, and the bound Eu3+ is measured in a time-resolved fluorometer. This assay is rapid, user friendly, and quantitative and lends itself to automation. The application of this assay to the detection of human immunodeficiency virus type 1 is described.

Time-resolved fluorometry in the diagnosis of Leber hereditary optic neuroretinopathy.

We have applied time-resolved fluorometry (TRF) to construct a DNA hybridization assay for the diagnosis of Leber hereditary optic neuroretinopathy (LHON). A rapid and reliable detection of the most prevalent mitochondrial DNA (mtDNA) point mutation associated with LHON is demonstrated. In addition, the TRF-method can be used in the quantification of heteroplasmy, a phenomenon commonly present in mtDNA mutations. The assay includes PCR amplification of a fragment encompassing the mutation site followed by hybridization reactions with allele-specific europium (Eu)-labelled oligonucleotide probes. A time-resolved fluorometer is used to measure the bound label. The TRF assay was successfully used to demonstrate the ND4/11778 mutation in patient samples. For quantification of heteroplasmy, synthetic target oligonucleotide mixtures with known ratios of wild-type and mutated sequences were used as standards to control the hybridization step. The assay allowed the detection of heteroplasmy ranging from 5 to 95%. This was also shown in a family with several heteroplasmic members.

Robust nonradioactive oligonucleotide ligation assay to detect a common point mutation in the CYP2D6 gene causing abnormal drug metabolism.

A new nonradioactive oligonucleotide ligation assay for the detection of a common point mutation in the CYP2D6 gene is presented. The assay takes advantage of simultaneous time-resolved fluorescence measurements of lanthanide-labeled probes and the specificity of T4-DNA ligase in combination with the polymerase chain reaction. This strategy makes it possible to perform the assay using both the wild-type-specific and mutant-specific probes simultaneously, securing an internal control in each reaction. We show that the allele-specific ligation part of the assay can be performed with great accuracy over a wide range of temperatures, salt concentrations, and T4-DNA ligase concentrations. This eliminates the risk of false-positive or false-negative reactions due to variations in these factors. Because the assay is simple to perform, is very reliable, and can be partly automated, we conclude that it is well-suited for analysis in a routine laboratory.

Europium-labeled oligonucleotide hybridization probes: preparation and properties.

A chemical method for labeling of oligonucleotide probes with europium chelates is presented. A modified deoxycytidine phosphoramidite is used to introduce multiple reactive amino groups to the oligonucleotide during the synthesis phase. Upon deprotection and purification of the modified oligonucleotide, an isothiocyanate derivative of a stable Eu chelate is reacted with the primary amino groups. The labeling technology enables the coupling of a high number of Eu chelates to a single probe. The melting temperatures and hybridization efficiencies of the oligonucleotides are not significantly altered by the labeling process. However, hybridization kinetics of the oligonucleotides are affected by the introduction of multiple modified deoxycytidine residues. In a solid-phase hybridization assay up to 10(7) target molecules can be detected.