In the first reaction a pcr primer is hybridized to the target sequence. This chapter consists of a brief introduction to the general principles involved in the chemical synthesis of oligonucleotides, followed by specific methodology used for the. Specimens from hiv1infected individuals collected by 7 international laboratories, including. Rna wholemount in situ hybridisation proximity ligation. 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. The fluorescence resonance energy transfer between a fluorescent donor and a suitable fluorophore as acceptor has been applied in the past to several scientific fields. Chemical ligation enables covalent bond formation between two oligonucleotide on strands in the presence of a template on 14. Protocol for annealing oligonucleotides sigmaaldrich. The bioanalysis of oligonucleotides as therapeutics requires sensitive, specific and. Pdf dna diagnostics, the detection of specific dna sequences, will play an. Simplified paper format for detecting hiv drug resistance in. The primers are designed with either the normal or mutant nucleotides at the 3 end and a tail of different lengths to.
We have developed a pcroligonucleotide ligation assay to rapidly identify base. The dual ligation hybridization assay dla extends the specificity of the hybridization ligation assay to a specific method for the parent compound. However, the general considerations of ligasebased sequence distinction are the same, and they will be described in some detail. Dec 15, 2014 an oligonucleotide ligation assay ola designed to detect human immunodeficiency virus type1 hivdrugresistance to the nevirapine nvp selected mutations k103n, y181c, v106m and g190a was used to evaluate 200 archived dried blood spots dbs from infected infants participating in the zimbabwean early infant diagnosis eid program. Regular hiv1 viral load monitoring is the standard of care to assess antiretroviral therapy e. The desired concentration of the duplex oligonucleotide is 50 m. Our method uses the robust chemistry of the oligonucleotide ligation assay ola in conjunction with the luminex flow cytometry platform figure 1 18. Listing a study does not mean it has been evaluated by the u. To develop and validate an ultrasensitive and specific hybridizationbased enzymelinked immunosorbent assay method for quantification of two phosphorothioate oligonucleotides ps odns g39 and gti2040 in biological fluids. Oligonucleotide ligation assay ola resistance study full.
This assay was based on hybridization of analytes to the biotinlabeled capture odns followed by ligation with digoxigeninlabeled detection odn. The oligonucleotide ligation assay ola used probes that were specific for dna sequences upstream and downstream of the snp of interest, with only exact matches at the snp site allowing for the ligation of the two probes. Each signal is composed of bound fluorescent probes that appear as a distinct dot that can be. Optimization of the oligonucleotide ligation assay, a rapid. To create the flexible linker, the bglii restriction site will be destroyed during ligation of the annealed oligonucleotides figure 4 and section 11. A specific picomolar hybridizationbased elisa assay for. The method was validated with regard to mechanism, specificity, precision and accuracy. Schematic representation of a twoplex oligonucleotide gapfill ligation assay for detection of an snv. Polymerase chain reactionoligonucleotide ligation assay pcrola is a method to diagnose hereditary diseases caused by mutation not affecting restriction endonuclease sites. S1 supporting information aptamercholesterolmediated proximity ligation assay acmpla for accurate identification of exosomes xianxian zhaoab1, canjun luo c1, qiang meia, hongmin zhangd, wenqing zhanga, dongwei sue, weiling fua, yang luobf author affiliations. Oligonucleotide gapfill ligation for mutation detection.
Specific determination of oligonucleotide therapeutics by. Oligonucleotide based therapeutics are quantified with hybridization assays in biological matrices such as plasma and tissues. Institute of tropical medicine, microbiology department, nationalestraat 155, antwerp 2000, belgium. However, the higher the copy number in a genome, the more difficult. Here we present a method for the modification of the existing in situ hybridisationproximity ligation assay ishpla protocol to adapt it to the study of rna regulation rishpla. Completion of the human genome sequence and development of. 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. Schematic representation of the dual ligation immunoassay. Dual ligation hybridization assay for the specific. Upon addition of the combined mix for ligation and amplification, the samples contained 50 mm kcl, 10 mm trishcl, ph 8. Ligation between wildtypemutant and common oligonucleotides occurs when both are annealed to the complementary strand of the pcr product. Allelespecific oligonucleotide an overview sciencedirect.
Oligonucleotide ligation assay ola resistance study ola the safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Quantification of large oligonucleotides using high. This single nucleotide g is complementary to the overhang of oligonucleotide b. Sixteenplex ola genotyping reactions are carried out, and allelespecific ola products are detected on membrane arrays using radiolabeled probes. Ligation assays are based on the covalent joining of two adjacent oligonucleotide probes by a dna ligase when they are hybridized to a cdna template, usually a. Allelespecific oligonucleotide hybridization, or dot blotting, is a method for testing known mutations. A rapid multiplex assay for nucleic acidbased diagnostics. D a nickerson, r kaiser, s lappin, j stewart, l hood, and u landegren division of biology, california institute of technology, pasadena 91125. The pla technology allows the detection of protein concentrations, modifications, andor posttranslational modifications. We have developed an oligonucleotide ligation assay ola that enables us. Ligation of probes at either end of the analyte is performed via a bienzymatic reaction consisting of polynucleotide kinase and dna ligase. The ligation products can then be replicated by nucleic acid amplification through pcr using primer cipla1 and primer cipla2, while the nonspecific binding and unreacted probes remain silent.
Oligonucleotide ligation assay detects hiv drug resistance a. Oligonucleotide ligation assay ola the ola consists of two phases, a multiplex pcr amplification and a multiplex ola, in a singletube format. S1 sensitive detection of small molecules by competitive immunomagneticproximity ligation assay shuyan cheng a, feng shi a, xuecheng jiang a, luming wang a, weiqing chen b, chenggang zhu a a college of life sciences, zhejiang university, 310058, hangzhou, china. Proteinoligonucleotide conjugation kit trilink biotech. Oligonucleotide ligation assay how is oligonucleotide. This method combines polymerase chain reaction with the ligation assay. Protein detection using proximitydependent dna ligation. An oligonucleotide ligation assay ola designed to detect human immunodeficiency virus type1 hivdrugresistance to the nevirapine nvp selected mutations k103n, y181c, v106m and g190a was used to evaluate 200 archived dried blood spots dbs from infected infants participating in the zimbabwean early infant diagnosis eid program. Dual ligation qpcr for quantifying oligonucleotide therapeutics on dried blood spots a b c largely detect metabolites in addition to the parent compound. 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. An accurate method for quantifying and analyzing copy number. 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.
Design and validation of dna libraries for multiplexing. A dual ligation based hybridization assay was developed to circumvent the limitations of current assay formats. We have applied the automated pcrola procedure to diagnosis of common genetic diseases. The substrate for ligase is a bridge structure formed by hybridization of a third oligonucleotide to the oligonucleotide ends of the assay probe pair. Dehydrated oligos will have a specified quantity usually in nanomoles on the tube. Unlike other ligation based assays that require multiple steps, our protocol consists of a single tube reaction, followed by hybridization to a luminex microsphere array for detection. The padlock gap probe black hybridizes to a single stranded target sequence gray leaving a six nucleotide gap between the 5. The strategy presented here can be applied elsewhere and may be useful for other oligonucleotides. There are many unique clinical pharmacology considerations concerning the development of oligonucleotide therapeutics.
Compared to other ligation based methodologies such as multiplex ligation dependent probe amplification mlpa or the oligonucleotide ligation assay ola, where multiplexed pcr is the limiting. We call this assay multiplex oligonucleotide ligation pcr molpcr. Ola oligonucleotide ligation assay method of testing for a. Fluorescently labeled, complementary oligonucleotide probes bind to the amplified dna. Polymerase chain reaction oligonucleotide ligation assay pcrola is a method to diagnose hereditary diseases caused by mutation not affecting restriction endonuclease sites. Optimization of the oligonucleotide ligation assay, a. Sixteenplex ola genotyping reactions are carried out, and allelespecific ola products are detected on membrane. Pdf oligonucleotide ligation assay for detection of. Roosens, kathleen marchal, sophie bertrand, sigrid c. The molpcr consists of the detection of molecular markers through a ligation. Genotyping by oligonucleotide ligation assay ola sigma. Request pdf oligonucleotide ligation assay the ability of dna ligases to join nucleic acids is strongly influenced by mismatches in the ligation substrates. Techniques for studying rnaprotein interactions have lagged behind those for dnaprotein complexes as a consequence of the complexities associated with working with rna.
Applied in this format, the results of the assay are simple to interpret and can be classified as positive or. 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. In previous studies, pyrosequencing, minisequencing, realtime pcr, invader assays and other techniques have been used to detect cnvs. 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. 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. Multiplex singlemolecule dna barcoding using an oligonucleotide ligation assay. Quantification of large oligonucleotides using high resolution msms on the tripletoftm 5600 system thomas knapman1, vicki gallant1 and martyn hemsley2 1ab sciex, warrington, uk, 2covance, harrogate, uk key challenges of oligonucleotide bioanalytical assay 1. 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. The ligated probes were hybridized to spotspecific capture oligos on the nplex plates to allow for detection.
Paperbased detection of hiv1 drug resistance using. Automated dna diagnostics using an elisabased oligonucleotide ligation assay. Sensitive detection of small molecules by competitive. A multiplex oligonucleotide ligationpcr method for the. Pdf pcroligonucleotide ligation assay for detection of. Oligonucleotide ligation assay ola for the diagnosis of familial. Improvements to bead based oligonucleotide ligation snp.
Optimization of the oligonucleotide ligation assay for the. The assay probes binds to 2 different epitopes on the target protein via antibodyprotein interactions. 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. Studies show that the mechanism of action of quinolones is inhibition of the. Development and evaluation of an oligonucleotide ligation. Compared to other ligation based methodologies such as multiplex ligation dependent probe amplification mlpa or the oligonucleotide ligation assay ola, where multiplexed pcr is. This has been overcome by the proximal ligation assay pla developed by fredriksson and lundegren. A multiplexed snp panel using oligonucleotide ligation. 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. More recently, the proximity ligation assay pla was added to the toolbox of protein analytics. Simplified paper format for detecting hiv drug resistance in clinical specimens by oligonucleotide ligation plos one, dec 2019 nuttada panpradist, ingrid a. Polymerase chain reactionoligonucleotide ligation assay. Pcr ampli cation of the target dna 1 is followed by ligation of the biotinylated allelespecic.
Oligonucleotide ligation assay for detection of mutations. Fluorescently labelled oligonucleotides can be created by different types of assays, such asmultiplexpcrdirecthybridisationassay,oligoligation assay ola,allelespecificprimerextensionaspe1,and. Pcrola distinguishes between the ligation and the absence of ligation of two oligonucleotides. Guidelines for optimisation of a multiplex oligonucleotide. Pcroligonucleotide ligation assay for detection of point mutations.
Current hybridization methods do not entirely discriminate the parent compound from 5. Oligonucleotide ligation assay ola is considered to be a very useful methodology for the detection and characterization of mutations, particularly for clinical purposes. In this method dna products generated by polymerase chain reaction pcr are dotted in duplicate membranes. Representative calibration curve with the test oligonucleotide in mouse plasma.
Aside from single nucleotide polymorphisms, copy number variations cnvs are the most important factors in susceptibility to genetic disorders because they affect expression levels of genes. Article multiplex singlemolecule dna barcoding using an oligonucleotide ligation assay ivo severins,1 malwina szczepaniak,1, and chirlmin joo1, 1kavli institute of nanoscience, department of bionanoscience, delft university of technology, delft, the netherlands abstract detection of speci. Federal register evaluating the clinical pharmacology. Oligonucleotide ligation assay for detection of mutations associated with reverse transcriptase and protease inhibitor resistance in nonb subtypes and recombinant forms of. Understanding taqman chemistrybased protein assays thermo. The concentration of each oligonucleotide needs to be 2x the desired concentration of the duplex oligonucleotide. The specificity of the ligation between two oligonucleotide primers is regulated by three. Chemical ligation of oligonucleotides using an electrophilic. Oligonucleotide ligation assay for detecting mutations in the. Msds sds sheet for oligonucleotide click on the pdf link below to download oligonucleotide msds. Oligonucleotide ligation assays olas are rapid, speci. This study represents a collaborative effort to develop this assay for use in salmon genetics. Ligation of the annealed oligonucleotides a and b will create a flexible amino acid linker. Reconstitute the dehydrated oligos with sterile or rnaase, dnaase free.
The bioanalysis of oligonucleotides as therapeutics. Dna ligase catalyzes the ligation of the 3 end of a dna fragment to the 5 end of a directly adjacent dna fragment. Automated dna diagnostics using an elisabased oligonucleotide. Rapid and sensitive oligonucleotide ligation assay for. Luminex based assay development university of virginia. Oligonucleotide gapfill ligation for mutation detection and. This mechanism has been exploited in a number of assays where the ability of oligonuleotide probes to be ligated reflects the genotype of the target molecules. Oligonucleotide phosphorylation and annealing oligonucleotides oligos come in tubes dehydrated or as liquid stocks may only be available in 96well plate format. Oligonucleotide ligation assay nih aids reagent program. Preart plasmas from artnaive kenyans initiating an nnrtibased fixeddose combination art in a randomized adherence trial conducted in 2006 were retrospectively analyzed by ola for mutations conferring resistance to nnrti k103n, y181c, and g190a and lamivudine m184v. This assay has been modified and developed for nonb subtypes and recombinant strains and has been evaluated with sequencing, resulting in a more sensitive assay than sequencing. Persistently elevated viral loads indicate virologic failure vf, which warrants hiv drug resistance testing hivdrt to allow individualized regimen switches.
The oligonucleotide ligation assay ola was developed by one of us d. The oligonucleotide ligation assay ola is a point mutation assay based on the covalent joining of two adjacent, dif corresponding author. Despite hybridization ligation assays 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. Specificity of the dual ligation assay for the parent compound. Nickerson for use in human genetics research associated with the human genome project nickerson et al. Within a basic configuration, two antibodies are labeled with oligonucleotides. We report the application of a dual ligation based qpcr.
Pcr oligonucleotide ligation assay for detection of point mutations associated with quinolone resistance in streptococcus pneumoniae. Various chemical ligation reactions have been applied to nonenzymatic sequencing of ons 5, onbased diagnosis 6, biotechnology 2, 7 and nanotechnology for making functional nanoconstructs 8 11. The assay benefits from the inclusion of additional steps such as the ligase chain reaction in which the ligation reaction is reiterated with further oligonucleotides complementary to the first ligation product or rolling circle amplification technology, in which two allelespecific oligonucleotides about 80 nt in length are designed to form. 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. Mechanism of detection of the dual ligation assay, where reliance on pnk and dna ligase was tested in mouse plasma. B genes using an automated genotypingbased twostep protocol. It uses dna fragment analysis for determining the carrier status of a defined panel of cf mutations. Simultaneous pathogen detection and antibiotic resistance characterization using snpbased multiplexed oligonucleotide ligation pcr molpcr. 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. To avoid the use of multiple assays, a better option is the multiplex oligonucleotide ligation pcr molpcr, using a liquid bead suspension assay luminex xtag technology, which allows a high level of multiplexing wuyts et al. This protocol describes the oligonucleotide ligation assay ola, which uses a set of three oligonucleotides, in combination with a thermostable taq dna ligase. A rapid multiplex assay for nucleic acidbased diagnostics song et al. The celera cf gt assay is based on five major processes. Volume 115, issue 6, 18 september 2018, pages 957967.
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