Ligation of probes at either end of the analyte is performed via a bienzymatic reaction consisting of polynucleotide kinase and dna ligase. Our studies demonstrate the sensitivity, simplicity, and specificity of this genotyping system. The dual ligation hybridization assay dla extends the specificity of the hybridization ligation assay to a specific method for the parent compound. The electropherogram in which the 1078delt peak is missing. Improvement of the oligonucleotide ligation assay for. Our method uses the robust chemistry of the oligonucleotide ligation assay ola in conjunction with the luminex flow cytometry platform. Apo e binds to the ldl receptor, also termed the b,e receptor, because the receptor accepts both apo b and apo e. Representative calibration curve with the test oligonucleotide in mouse plasma. Ola oligonucleotide ligation assay method of testing. The electropherogram in which the r117h peak is missing a slight background peak is present. We have successfully applied the oligonucleotide ligation assay ola technique to screen for mutations causing familial hypercholesterolemia. The oligonucleotide ligation assay ola is a genotypic assay which has been used to identify point mutations in dna for a variety of diseases 3, 5 and to detect drug resistanceassociated mutations in hiv1 subtype b 1, 7, 8, 15.
The oligonucleotide ligation assay is a genotypic assay for the detection of resistanceassociated mutations to reverse transcriptase and protease inhibitors in human immunodeficiency virus type 1 subtype b. This report describes the detection of mutations in the pol gene of human immunodeficiency virus type 1 associated with resistance to zidovudine, didanosine, and lamivudine by genotyping by an oligonucleotide ligation assay specific codons in the pol gene amplified by pcr. Many methods have been used to detect c282y, including oligonucleotide ligation assays 1, fragment length polymorphism analyses of pcr products after restriction enzyme digestion 5, allelespecific oligonucleotide hybridization assays 2, mutagenically separated pcr assays 10, primer extension assays 11, 12, and allele refractory mutation systems, 14. Oligonucleotide ligation assay ola resistance study full. Request pdf oligonucleotide ligation assay the ability of dna ligases to join nucleic acids is strongly influenced by mismatches in the ligation substrates. Of 1763 tests performed, 5 patient samples had electropherograms with absent fluorescent signals.
With multiplex oligonucleotide ligation pcr molpcr different molecular markers can be simultaneously analysed in a single assay and high levels of multiplexing can be achieved in highthroughput format. Sixteenplex ola genotyping reactions are carried out, and allelespecific ola products are detected on membrane. Genotyping by oligonucleotide ligation assay ola sigma. Despite hybridization ligation assay s robustness, sensitivity and added specificity for the 3end of the oligonculeotide analyte, the hybridization ligation assay is not specific for the 5 end of the analyte. Dehydrated oligos will have a specified quantity usually in nanomoles on the tube. Streamlined circular proximity ligation assay provides high.
The assay is performed in 96well plate format and has an upper multiplex capability of 50 snps per well, making it suitable for a moderate number of snps in a large number of samples. Many methods have been used to detect c282y, including oligonucleotide ligation assays 1, fragment length polymorphism analyses of pcr products after restriction enzyme digestion 5, allelespecific oligonucleotide hybridization assays 2, mutagenically separated pcr assays 10, primer extension assays 11, 12, and allele refractory. However, the general considerations of ligasebased sequence distinction are. Here, we developed a highly sensitive and specific detection method that combines an advanced oligonucleotide ligation assay with multicolor singlemolecule fluorescence. A normal electropherogram pattern for no mutation detected. Read oligonucleotide ligation assay for detection of the factor v mutation arg 506 gln causing protein c resistance, thrombosis research on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Optimization of the oligonucleotide ligation assay, a. Ligation of the oligonucleotide strands was performed for 45 min at 30 c in a 1. Pretreatment hiv drug resistance pdr is increasing in subsaharan africa, driven by increasing resistance to nonnucleoside reverse transcriptase inhibitor nnrti drugs. Combined endpoint of time to death, loss to followup, or virological failure. Multiplex detection of mutations in clinical isolates of. Institute of tropical medicine, microbiology department, nationalestraat 155, antwerp 2000, belgium. Evaluation of the management of pretreatment hiv drug.
Completion of the human genome sequence and development of. A dual ligationbased hybridization assay was developed to circumvent the limitations of current assay formats. A dual ligation based hybridization assay was developed to circumvent the limitations of current assay formats. This chapter will describe two protocols for solidphase detection of reaction products in the oligonucleotide ligation assay ola, although there are several other detection schemes in use. Download fulltext pdf automated dna diagnostics using an elisabased oligonucleotide ligation assay article pdf available in proceedings of the national academy of sciences 8722. Development and evaluation of the oligonucleotide ligation. The dual ligation hybridization assay dla extends the specificity of the hybridizationligation assay to a specific method for the parent compound. Identification of cystic fibrosis variants by polymerase.
However, the general considerations of ligasebased sequence distinction are the same, and they will be described in some detail. Dual ligation hybridization assay for the specific. Here we present a rapid, sensitive and quantitative assay to measure ner activity in human cells, which we term the oligonucleotide retrieval assay ora. Development and evaluation of the oligonucleotide ligation assay ola for the detection of drug resistance mutations in. For example, a previous study revealed anomalous electropherograms in five cases screened by a pcroligonucleotide ligation assay ola, which demonstrated the phenomenon of allele drop out. The oligonucleotide ligation assay ola was developed by one of us d. Optimization of the oligonucleotide ligation assay, a rapid. Listing a study does not mean it has been evaluated by the u. Oligonucleotide ligation assay for detecting mutations in the. Circulating resistance to firstline hiv drug regimens. This protocol describes the oligonucleotide ligation assay ola, which uses a set of three oligonucleotides, in combination with a thermostable taq dna ligase enzyme, to discriminate singlenucleotide polymorphism snp alleles. Schematic representation of the dual ligation immunoassay. Oligonucleotide modification for chemical ligation biosynthesis offers several unnatural bases or functional group oligonucleotide modifications for use in chemical ligation applications.
This mechanism can be used to interrogate a snp by hybridizing two probes directly over the snp polymorphic site, whereby ligation can occur if the probes are identical to the target dna. Improvements to beadbased oligonucleotide ligation snp. The use of the oligonucleotide ligation assay ola was demonstrated by tobe et al. Despite hybridizationligation assays robustness, sensitivity and added specificity for the 3end of the oligonculeotide analyte, the hybridizationligation assay is not specific for the 5 end of the analyte. Generally, the invention relates to detecting andor analyzing nucleic acid sequences using microspherebased assays. Specificity of the dual ligation assay for the parent compound. Request pdf oligonucleotide ligation assay advances in molecular genetics and biotechnology are changing the practice of medicine. We evaluated the feasibility of the oligonucleotide ligation assay ola, a specific, sensitive, and economical ligasebased point mutation assay designed to detect hiv1 drugresistance mutations at 12 codons of hiv1 subtype b pol.
We have developed an oligonucleotide ligation assay ola that enables us to screen for highrisk individuals by testing for 19 common mutations in the ldl receptor and the apolipoprotein b genes. The method needs only four common probes to detect 15 mutational variants of the rpob gene within 12 h. Thus, the interest in apo e polymorphisms is high, both on the basis of epidemiological research and for the purpose of clarifying individual lipid disturbances or dementias. The reference method for salmonella serotyping, including slideagglutination and biochemical. Oligonucleotide ligation assay for detecting mutations in. Nickerson for use in human genetics research associated with the human genome project nickerson et al. Ola oligonucleotide ligation assay method of testing for a. We evaluated the feasibility of the oligonucleotide ligation assay ola, a specific, sensitive, and economical ligasebased point mutation assay designed to detect hiv1 drugresistance mutations at 12 codons of hiv1 subtype b pol, for potential use in resourcepoor settings. Sixteenplex ola genotyping reactions are carried out, and allelespecific ola products are detected on membrane arrays using radiolabeled probes. Oligonucleotidebased therapeutics are quantified with hybridization assays in biological matrices such as plasma and tissues. Proximity ligation assay with single and double oligonucleotide.
Streamlined circular proximity ligation assay provides. Dual hplc purification is highly recommended for postsynthetic conjugation. Current hybridization methods do not entirely discriminate the parent compound from 5. The method was validated with regard to mechanism, specificity, precision and accuracy. More specifically, the invention relates to detecting andor analyzing nucleic acid sequences using microspherebased oligonucleotide ligation multiplexed assays. A multiplex oligonucleotide ligationpcr method for the. Dna ligase catalyzes the ligation of the 3 end of a dna fragment to the 5 end of a directly adjacent dna fragment. Us20020182609a1 microsphere based oligonucleotide ligation. Assay for evaluating ribonuclease hmediated degradation of rnaantisense oligonucleotide duplexes 6.
Specific determination of oligonucleotide therapeutics by. With multiplex oligonucleotide ligationpcr molpcr different molecular markers can be simultaneously analysed in a single assay and high levels of multiplexing can be achieved in highthroughput format. Oligonucleotide phosphorylation and annealing oligonucleotides oligos come in tubes dehydrated or as liquid stocks may only be available in 96well plate format. Di and oliogonucleotide synthesis using hphosphonate chemistry 7. We have developed an oligonucleotide ligation assay ola that enables us to screen for highrisk individuals by testing for 19 common mutations in the.
Solid phase synthesis of circular oliogonucleotides 8. Salmonella is a major pathogen having a public health and economic impact in both humans and animals. In this case, the same substrates are used as in the previous assay, except that the oligonucleotide is radioactively labeled at its 5 end with 32p. Circulating resistance to firstline hiv drug regimens the. The invention relates to a method for the detection of a target nucleotide sequence in a sample based on an oligonucleotide ligation assay wherein probes are used that contain a combination of sequencebased identifiers that can identify the sample and the target sequence i. The primers are designed with either the normal or mutant nucleotides at the 3 end and a tail of different lengths to. We demonstrated that under our experimental conditions, 7nucleotide long dna barcodes. Oligonucleotide ligation assay how is oligonucleotide. Development and evaluation of an oligonucleotide ligation. Here, we describe a circular proximity ligation assay cpla, where in contrast to traditional proximity ligation assay tpla, the proximity probes are used as bridges that enable the connection of two free oligonucleotides via dual ligation events, resulting in the formation of a circle fig. Preparation of single and doubleoligonucleotide antibody. Application of a novel, rapid, and sensitive oligonucleotide. Oligonucleotide ligation assay ola resistance study ola the safety and scientific validity of this study is the responsibility of the study sponsor and investigators.
The oligonucleotide ligation assay ola is a point mutation assay based on the covalent joining of two adjacent, dif corresponding author. The primers are designed with either the normal or mutant nucleotides at the 3 end and a tail of different lengths to distinguish various pcr products based on size at the 5 end. The oligonucleotide ligation assay ola nickerson et al. We used oligonucleotide constructs containing the uvdamaged adduct, cyclobutane pyrimidine dimer cpd, to transfect human cells, and retrieved the oligonucleotides for quantification of the. Apo e is also thought to bind to a specific chylomicron remnant receptor by virtue of its structural determinants. This study represents a collaborative effort to develop this assay for use in salmon genetics. New sulfur transfer reagent in phosphorothioates oligonucleotide synthesis 5. Evaluation of the management of pretreatment hiv drug resistance by oligonucleotide ligation assay. A new approach, shortoligonucleotideligation assay on dna chip solac, is developed to detect mutations in rifampinresistant mycobacterium tuberculosis. Oligonucleotide ligation assay ola the ola consists of two phases, a multiplex pcr amplification and a multiplex ola, in a singletube format. Oligonucleotide ligation assay for detection of mutations. Detection of specific nucleic acid sequences is invaluable in biological studies such as genetic disease diagnostics and genome profiling. The present invention teaches a novel approach to detecting andor analyzing nucleic acid sequences. An oligonucleotide biotinylated at its 5end and another with a reporter group chromophore or fluorophore at its 3end are constructed to hybridize to the sequence to be detected in a template dna strand.