Detection and genetic analysis of β-thalassemia mutations by competitive oligopriming

A new approach for the detection of β-thalassemia mutations has been applied, based on competitive oligonucleotide priming (COP) of in vitro DNA amplification at the mutation site. This method allows genotyping of the template DNA, through differential labeling of the allele-specific competitive oligoprimers and biotinylation of the common reverse primer. The system provides a basis for rapid, simple, and reliable detection of the numerous known β-thalassemia mutations, revealing the precise nature of the mismatch in each case, and thereby facilitating the molecular genetic analysis of the disease. © 1995 Wiley-Liss, Inc.     

Single nucleotide polymorphisms detection based on DNA microarray technology: HLA as a model

The significance of DNA variations among individuals, including single nucleotide polymorphisms (SNPs) and/or genome nucleotide mutations as well as to their detection by using new technology, will improve and facilitate the knowledge of each gene sequence. Microarray may provide information about thousands of gene simultaneously, leading to a more rapid and accurate genotyping. In this view, we developed a new methodology as an example for the detection of SNPs based on DNA microarray, using a panel of HLA alleles representative of loci A, B, DRB1. A panel of 180 oligonucleotide probes was selected to identify polymorphic positions located in exons 2 and 3 of HLA-A and B, and in exon 2 of HLA-DRB1 locus. Each oligonucleotide sequence was designed with a nucleotide mismatch located in the same position as the center of the hybridization sequence. Hybridization experiments were carried out with genomic probes constructed with an asymmetric PCR strategy. The amplified DNAs were obtained from bone marrow cells of donors previously typed for transplant. The results obtained were showing that the method was reliable thus providing a feasible technique both for HLA typing and for the investigation of other regions of genetic and clinical interest including polymorphisms correlated with different autoimmune diseases.     

Detection of single nucleotide polymorphisms in the mannose-binding lectin gene using minor groove binder-DNA probes

Structural point mutations in exon 1 at codons 52, 54 and 57 and a promotor polymorphism at -221 bp of the mannose-binding lectin (MBL) gene are associated with increased susceptibility to various infectious diseases. We developed a genotyping method based on the 5' nuclease (TaqMan) assay in combination with the use of minor-groove-binder (MGB) probes in screening for these mutations/polymorphisms. In contrast to conventional probes, MGB probes have a short length and can be used for detection of mutations that are in close proximity to each other, as is the case for the structural mutations in exon 1 of the MBL gene. Results obtained with the 5' nuclease assay using MGB probes were identical with results obtained with classical techniques such as restriction fragment length polymorphism, allele-specific PCR, and sequencing. In conclusion, the 5' nuclease assay using MGB probes is useful for large-scale screening of point mutations/polymorphisms, even when they are in close proximity.     

PAP: detection of ultra rare mutations depends on P* oligonucleotides: "sleeping beauties" awakened by the kiss of pyrophosphorolysis

Pyrophosphorolysis-activated polymerization (PAP) was initially developed to enhance the specificity of allele-specific PCR for detection of known mutations in the presence of a great excess of wild-type allele. The high specificity of PAP derives from the serial coupling of activation of a 3' blocked pyrophosphorolysis-activable oligonucleotide (P(*)) with extension of the unblocked, activated P(*). In theory, PAP can detect a copy of a single base mutation present in 3x10(11) copies of the wild-type allele. In practice, the selectivity of detection is limited by polymerase extension errors, a bypass reaction, from the unblocked oligonucleotide annealed to the opposing strand. Bi-directional PAP allele-specific amplification (Bi-PAP-A) is a derivative of PAP that uses two opposing pyrophosphorolysis activable oligonucleotides (P(*)) with one nucleotide overlap at their 3' termini. This eliminates the problematic bypass reaction. The selectivity of Bi-PAP-A was examined using lambda phage DNA as a model system. Bi-PAP-A selectively detected two copies of a rare mutated allele in the presence of at least 2x10(9) copies of the wild-type lambda phage DNA. We then applied Bi-PAP-A to direct detection of spontaneous somatic mutations in the lacI transgene in BigBlue transgenic mice at a frequency as low as 3x10(-9). A 370-fold variation in the frequency of a specific somatic mutation among different mouse samples was found, implying hyper-Poisson variance and clonal expansion of mutation occurring during early development. Bi-PAP-A is a simple, rapid, and general method capable of automation and particularly suited to detection of ultra rare mutations. We also show that P(*) oligonucleotides have the novel and unexpected property of high specificity to mismatches with the template throughout lengths of the P(*). Thus, PAP also can form the basis of microarray-based scanning or resequencing methods to detect virtually all mutations.     

Allele-specific competitive blocker PCR: A one-step method with applicability to pool screening

We have developed a novel one-step pool screening PCR procedure which is based on the principles of amplification refractory mutation system (ARMS) and competitive oligonuleotide priming (COP) PCR. In addition to the usual primers, this approach uses two allele-specific competitive oligonucle-otides, one of which is 3′-end labeled with a dideoxynucleotide and blocks amplification of the wild-type allele. An allele-specific product is generated only in the presence of the mutation. The introduction of an allele-specific competitive blocker oligonucleotide improves the specificity and robustness of ARMS-PCR. Further its sensitivity is dramatically increased, which allows detection of one mutant allele in a large excess of wild-type-bearing genomic DNA by electrophoresis in an ethidium bromide- -stained agarose gel (up to 1 in 10⁴ alleles). This makes the method ideal for nonradioactive pool screening. The successful application of the method has been demonstrated for four different point mutations, two in the apolipoprotein B gene (R3500Q, R3531C) which result in familial defective apolipoprotein B-100, one in the CFTR gene (R1162X), and one in the gene for lipoprotein lipase (G188E). © 1995 Wiley-Liss, Inc.     

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Simple two-color array-based approach for mutation detection

The ability to analyze multiple polymorphic/mutation sites rapidly and accurately is pivotal in all areas of genetic analysis. We have applied single nucleotide primer extension (SNE) for detection of multiple point mutations in a micro-array format using two-color, fluorescent dye-tagged dideoxynucleoside triphosphate terminators (ddNTPs). The oligonucleotide primer ending one nucleotide short of the mutation site being probed is bound to the slide and single-base extended in place with two different Cy5/Cy3 dye-tagged terminators using solution-phase, locus-specific, single-stranded complementary templates generated by PCR from genomic DNA. The composite fluorescence produced contains peaks of distinct wave lengths corresponding to each Cy dye-tagged terminator incorporated, resulting in a fluorescent 'fingerprint' for each DNA target. DNA polymerase-catalyzed incorporation of Cy dye-tagged dideoxynucleoside triphosphates was dependent on the particular dyes, the specific ddNTP, the DNA target concentration, sequence of the template, on-slide temperature cycling and washing conditions. Results from analysis of mutations in the human hemochromatosis and connexin 26 genes show that this approach has several advantages over existing methods and is simple, rapid, robust, cost effective and accurate with potential applications in many areas of genetic analysis.     

Genetic determinants and polymorphisms specific for human-adapted serovars of Salmonella enterica that cause enteric fever

Salmonella enterica serovars Typhi, Paratyphi A, and Sendai are human-adapted pathogens that cause typhoid (enteric) fever. The acute prevalence in some global regions and the disease severity of typhoidal Salmonella have necessitated the development of rapid and specific detection tests. Most of the methodologies currently used to detect serovar Typhi do not identify serovars Paratyphi A or Sendai. To assist in this aim, comparative sequence analyses were performed at the loci of core bacterial genetic determinants and Salmonella pathogenicity island 2 genes encoded by clinically significant S. enterica serovars. Genetic polymorphisms specific for serovar Typhi (at trpS), as well as polymorphisms unique to human-adapted typhoidal serovars (at sseC and sseF), were observed. Furthermore, entire coding sequences unique to human-adapted typhoidal Salmonella strains (i.e., serovar-specific genetic loci rather than polymorphisms) were observed in publicly available comparative genomic DNA microarray data sets. These polymorphisms and loci were developed into real-time PCR, standard PCR, and liquid microsphere suspension array-based molecular protocols and tested for with a panel of clinical and reference subspecies I S. enterica strains. A proportion of the nontyphoidal Salmonella strains hybridized with the allele-specific oligonucleotide probes for sseC and sseF; but the trpS allele was unique to serovar Typhi (with a singular serovar Paratyphi B strain as an exception), and the coding sequences STY4220 and STY4221 were unique among serovars Typhi, Paratyphi A, and Sendai. These determinants provided phylogenetic data on the genetic relatedness of serovars Typhi, Paratyphi A, and Sendai; and the protocols developed might allow the rapid identification of these Salmonella serovars that cause enteric fever.     

Oligonucleotide-mediated gene repair at DNA level: the potential applications for gene therapy

Mutations in gene sequence can cause many genetic disorders, and researchers have attempted to develop treatments or cures at the DNA level for these diseases. Several strategies including triple-helix-forming oligonucleotides (TFOs), chimeric RNA/DNA oligonucleotide (RDO), and short single-stranded oligodeoxynucleotide (ODN) have been used to correct the dysfunctional genes in situ in the chromosome. Experimental data from cells and animal models suggest that all these strategies can repair the mutations in situ at DNA level. More effective structures of oligonucleotide, efficient delivery systems, and gene correction efficiency should be improved. Development of these strategies holds great potentials for treatments of genetic defects and other disorders.     

A Homogeneous, Ligase-Mediated DNA Diagnostic Test

Single-nucleotide variations are the most widely distributed genetic markers in the human genome. A subset of these variations, the substitution mutations, are responsible for most genetic disorders. As single nucleotide polymorphism (SNP) markers are being developed for molecular diagnosis of genetic disorders and large-scale population studies for genetic analysis of complex traits, a simple, sensitive, and specific test for single nucleotide changes is highly desirable. In this report we describe the development of a homogeneous DNA detection method that requires no further manipulations after the initial reaction is set up. This assay, named dye-labeled oligonucleotide ligation (DOL), combines the PCR and the oligonucleotide ligation reaction in a two-stage thermal cycling sequence with fluorescence resonance energy transfer (FRET) detection monitored in real time. Because FRET occurs only when the donor and acceptor dyes are in close proximity, one can infer the genotype or mutational status of a DNA sample by monitoring the specific ligation of dye-labeled oligonucleotide probes. We have successfully applied the DOL assay to genotype 10 SNPs or mutations. By designing the PCR primers and ligation probes in a consistent manner, multiple assays can be done under the same thermal cycling conditions. The standardized design and execution of the DOL assay means that it can be automated for high-throughput genotyping in large-scale population studies.     

Diagnosis of sickle cell anemia and beta-thalassemia with enzymatically amplified DNA and nonradioactive allele-specific oligonucleotide probes

We have developed a simple and rapid nonradioactive method for detecting genetic variation and have applied it to the diagnosis of sickle cell anemia and beta-thalassemia. The procedure involves the selective amplification of a segment of the human beta-globin gene with oligonucleotide primers and a thermostable DNA polymerase, followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes covalently labeled with horseradish peroxidase. The hybridized probes were detected with a simple colorimetric assay. We demonstrated the usefulness of this method in a retrospective analysis of two pregnancies at risk for beta-thalassemia and one at risk for sickle cell anemia, as well as in an analysis of nine DNA samples simulating three family sets.     

Dual-allele dipstick assay for genotyping single nucleotide polymorphisms by primer extension reaction

We have developed a dry-reagent dipstick test for simultaneous visual detection of two alleles in single nucleotide polymorphisms (SNPs). The strip comprises two test zones and a control zone. Oligonucleotide-functionalized gold nanoparticles are used as reporters. PCR-amplified DNA that spans the interrogated sequence is subjected to primer extension (PEXT) reactions using allele-specific primers. Digoxigenin-dUTP and biotin-dUTP are incorporated in the extended fragments. The primers contain an oligo(dA) segment at the 5' end. The PEXT products are applied to the sample area of the strip, which is then immersed in the appropriate buffer. As the buffer migrates along the strip by capillary action, the extension products of the two alleles are captured at the test zones from immobilized anti-digoxigenin and streptavidin, whereas the oligo(dA) segment of the primers hybridizes with oligo(dT) strands attached to gold nanoparticles, thus generating characteristic red lines. The excess nanoparticles are captured from immobilized oligo(dA) strands at the control zone of the strip. The test was applied to the genotyping of two SNPs of the Toll-like receptor 4 gene (Asp299Gly and Thr399Ile), one SNP of CYP2C19 gene (CYP2C19(*)3) and one SNP of the TPMT gene (TPMT(*)2). Contrary to most genotyping methods, the dipstick test does not require costly specialized equipment for detection of PEXT products. The PCR product is pipetted directly into the PEXT reaction mixture without prior purification. The high sensitivity of the strip allows completion of PEXT reaction in three cycles only (7 min). The visual detection of both alleles is complete in 15 min.     

Mutational analysis using oligonucleotide microarrays

The development of inexpensive high throughput methods to identify individual DNA sequence differences is important to the future growth of medical genetics. This has become increasingly apparent as epidemiologists, pathologists, and clinical geneticists focus more attention on the molecular basis of complex multifactorial diseases. Such undertakings will rely upon genetic maps based upon newly discovered, common, single nucleotide polymorphisms. Furthermore, candidate gene approaches used in identifying disease associated genes necessitate screening large sequence blocks for changes tracking with the disease state. Even after such genes are isolated, large scale mutational analyses will often be needed for risk assessment studies to define the likely medical consequences of carrying a mutated gene. This review concentrates on the use of oligonucleotide arrays for hybridisation based comparative sequence analysis. Technological advances within the past decade have made it possible to apply this technology to many different aspects of medical genetics. These applications range from the detection and scoring of single nucleotide polymorphisms to mutational analysis of large genes. Although we discuss published scientific reports, unpublished work from the private sector could also significantly affect the future of this technology.     

Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay

Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.     

Mutation detection by TaqMan-allele specific amplification: application to molecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency

We have devised an allele-specific amplification method with a TaqMan fluorogenic probe (TaqMan-ASA) for the detection of point mutations. Pairwise PCR amplification using two sets of allele-specific primers in the presence of a TaqMan probe was monitored in real time with a fluorescence detector. Difference in amplification efficiency between the two PCR reactions was determined by "threshold" cycles to differentiate mutant and normal alleles without post-PCR processing. The method measured the efficiency of amplification rather than the presence or absence of end-point PCR products, therefore allowing greater flexibility in designing allele-specific primers and an ample technical margin for allelic discrimination. We applied the TaqMan-ASA method to detect a prevalent 727G>T mutation in Japanese patients with glycogen storage disease type Ia and a common 985A>G mutation in Caucasian patients with medium-chain acyl-CoA dehydrogenase deficiency. The method can be automated and may be applicable to the DNA diagnosis of various genetic diseases.     

Determination of beta2-adrenergic receptor (ADRB2) haplotypes by a multiplexed polymerase chain reaction assay

The beta2-adrenergic receptor (B2AR or ADRB2) is the target of beta2-agonist drugs used for bronchodilation in asthma and other respiratory diseases. The gene for this receptor (ADRB2) contains numerous single nucleotide polymorphisms (SNPs) some of which may be of pharmacogenetic relevance, although a consistent picture of genotype-phenotype relationships has yet to emerge. Recently, 12 distinct haplotypes of ADRB2were described along with preliminary evidence that certain haplotypes, rather than specific SNPs, determine differential response to the beta-agonist drug albuterol. In order to further evaluate the role of ADRB2haplotypes as pharmacogenetic determinants, simple and accurate methods for haplotyping clinical samples are required. To this end we have developed a multiplexed, allele-specific PCR assay that interrogates six ADRB2 SNPs in a manner that permits rapid and accurate assignment of ADRB2 haplotype pairs. This assay will facilitate studies of ADRB2haplotypes in phenotypes such as patient responses to beta2-agonists, bronchial hyper-responsiveness, and cardiovascular conditions.     

Antimicrobial agent resistance in mycobacteria: molecular genetic insights.

The primary theme emerging from molecular genetic work conducted with Mycobacterium tuberculosis and several other mycobacterial species is that resistance is commonly associated with simple nucleotide alterations in target chromosomal genes rather than with acquisition of new genetic elements encoding antibiotic-altering enzymes. Mutations in an 81-bp region of the gene (rpoB) encoding the beta subunit of RNA polymerase account for rifampin resistance in 96% of M. tuberculosis and many Mycobacterium leprae isolates. Streptomycin resistance in about one-half of M. tuberculosis isolates is associated with missense mutations in the rpsL gene coding for ribosomal protein S12 or nucleotide substitutions in the 16S rRNA gene (rrs). Mutations in the katG gene resulting in catalase-peroxidase amino acid alterations nad nucleotide substitutions in the presumed regulatory region of the inhA locus are repeatedly associated with isoniazid-resistant M. tuberculosis isolates. A majority of fluoroquinolone-resistant M. tuberculosis isolates have amino acid substitutions in a region of the DNA gyrase A subunit homologous to a conserved fluoroquinolone resistance-determining region. Multidrug-resistant isolates of M. tuberculosis arise as a consequence of sequential accumulation of mutations conferring resistance to single therapeutic agents. Molecular strategies show considerable promise for rapid detection of mutations associated with antimicrobial resistance. These approaches are now amenable to utilization in an appropriately equipped clinical microbiology laboratory.     

A robotics-assisted procedure for large scale cystic fibrosis mutation analysis

We describe a convenient, efficient, semiautomated protocol for assaying large numbers of DNA samples for over 20 mutations causing cystic fibrosis. The protocol uses the following: (1) a programmable robotic workstation to perform rapid pipetting and dot-blotting operations, (2) an allele-specific oligonucleotide hybridization in a single water bath without correcting for G + C content of oligonucleotides, and (3) a combinatorial system that allows direct determination of the genotype for more frequent mutations. We have used this system routinely for 16 months for carrier detection and for diagnosis of cystic fibrosis. The method can be readily applied to any combination of allele-specific oligonucleotide assays whether for multiple alleles at one locus or for a few alleles at multiple loci. © 1994 Wiley-Liss, Inc.