Multiplexed SNP genotyping using the Qbead system: a quantum dot-encoded microsphere-based assay

We have developed a new method using the Qbead system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral 'barcodes' are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein-protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications.     

Multiplexed SNP genotyping using the Qbead™ system: a quantum dot-encoded microsphere-based assay

We have developed a new method using the Qbead™ system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot™ semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral ‘barcodes’ are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein–protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications.     

Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films

Allele-specific hybridization to a DNA microarray can be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose mutations in the human beta-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding melting point temperature ( approximately 20 degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments conducted using a target DNA specific for the TC tag of the immobilized probes showed that the spotting and hybridization procedure had a variance of 20%, indicating that signal differences as low as twofold could be detected between perfect match and mismatch. Together, our results show that the use of microarrays of TC-tagged probes that have been immobilized on agarose films grafted onto glass is a robust and inexpensive genotyping method.     

Cationic oligonucleotides can mediate specific inhibition of gene expression in Xenopus oocytes.

Base-specific hydrogen bonding between an oligonucleotide and the purines in the major groove of a DNA duplex provide an approach to selective inhibition of gene expression. Oligonucleotide-mediated triplex formation in vivo may be enhanced by a number of different chemical modifications. We have previously described an in vitro analysis of triplex formation using oligonucleotides containing internucleoside phosphate linkages modified with the cation N , N -diethyl-ethylenediamine (DEED). When compared with unmodified oligonucleotides of identical base composition, DEED-modified oligonucleotides were better able to form DNA triplexes under conditions that approximate the pH, magnesium and potassium levels found in vivo . Here we report the ability of DEED-modified oligonucleotides to inhibit the expression of plasmid DNA injected into Xenopus oocytes. Inhibition is specific to plasmids containing a triplex formation target and sensitive to sequence alteration in the triplex forming target site. Inhibition of gene expression was nearly complete when oligonucleotide and plasmid were mixed together prior to injection. Inhibition was partial when oligonucleotide was injected first and not evident when plasmid was injected and allowed to form chromatin prior to oligonucleotide injection. Thus, access to DNA is a determining factor in effective triplex inhibition of gene expression.     

Automated parallel synthesis of 5′-triphosphate oligonucleotides and preparation of chemically modified 5′-triphosphate small interfering RNA

A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5′-triphosphate and 5′-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5′-triphosphates and 5′-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5′-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5′-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5′-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform.     

LNA-modified oligonucleotides effectively drive intramolecular-stable hairpin to intermolecular-duplex state

Sequence-specific hybridization of antisense and antigene agent to the target nucleic acid is an important therapeutic strategy to modulate gene expression. However, efficiency of such agents falls due to inherent intramolecular-secondary-structures present in the target that pose competition to intermolecular hybridization by complementary antisense/antigene agent. Performance of these agents can be improved by employing structurally modified complementary oligonucleotides that efficiently hybridize to the target and force it to transit from an intramolecular-structured-state to an intermolecular-duplex state. In this study, the potential of variably substituted locked nucleic acid-modified oligonucleotides (8mer) to hybridize and disrupt highly stable, secondary structure of nucleic acid has been biophysically characterized and compared with the conventionally used unmodified DNA oligonucleotides. The target here is a stem-loop hairpin oligonucleotide-a structure commonly present in most structured-nucleic acids and known to exhibit an array of biological functions. Using fluorescence-based studies and EMSA we prove that LNA-modified oligonucleotides hybridize to the target hairpin with higher binding affinity even at lower concentration and subsequently, force it to assume a duplex conformation. LNA-modified oligonucleotides may thus, prove as potential therapeutic candidates to manipulate gene expression by disruption of biologically relevant nucleic acid secondary structure.     

Locked nucleic acid: a potent nucleic acid analog in therapeutics and biotechnology

Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing very high affinity and excellent specificity toward complementary DNA and RNA, and LNA oligonucleotides have been applied as antisense molecules both in vitro and in vivo. In this review, we briefly describe the basic physiochemical properties of LNA and some of the difficulties that may be encountered when applying LNA technology. The central part of the review focuses on the use of LNA molecules in regulation of gene expression, including delivery to cells, stability, unspecific effects, toxicity, pharmacokinetics, and design of LNA oligonucleotides. The last part evaluates LNA as a diagnostic tool in genotyping.     

Peptide conjugates for chromosomal gene targeting by triplex-forming oligonucleotides

Triplex-forming oligonucleotides (TFOs) are DNA-binding molecules, which offer the potential to selectively modulate gene expression. However, the biological activity of TFOs as potential antigene compounds has been limited by cellular uptake. Here, we investigate the effect of cell-penetrating peptides on the biological activity of TFOs as measured in an assay for gene-targeted mutagenesis. Using the transport peptide derived from the third helix of the homeodomain of antennapedia (Antp), we tested TFO–peptide conjugates compared with unmodified TFOs. TFOs covalently linked to Antp resulted in a 20-fold increase in mutation frequency when compared with ‘naked’ oligonucleotides. There was no increase above background in mutation frequency when Antp by itself was added to the cells or when Antp was linked to mixed or scrambled sequence control oligonucleotides. In addition, the TFO–peptide conjugates increased the mutation frequency of the target gene, and not the control gene, in a dose-responsive manner. Confocal microscopy using labeled oligonucleotides indicated increased cellular uptake of TFOs when linked to Antp, consistent with the gene-targeting data. These results suggest that peptide conjugation may enhance intranuclear delivery of reagents designed to bind to chromosomal DNA.     

Enzymatic methylation of cytosine in DNA is prevented by adjacent O6-methylguanine residues

The effect of O6-alkylation of guanine residues on the enzymic methylation of cytosine has been studied using synthetic oligonucleotides in which all guanines in cytosine-guanine sequences at potentially methylatable sites are replaced by O6-methylguanine. In contrast with the unmodified forms, which showed high acceptance activity for methyl-3H-labeled groups from S-adenosyl-L-[methyl-3H]methionine in the presence of DNA methylase, the modified oligonucleotides were not substrates for the enzyme either in the single-stranded or annealed forms. In view of the importance of cytosine methylation in the down-regulation of certain genes, the potential to affect gene expression by this mechanism may be a contributory factor in the toxic and carcinogenic effects of chemical methylating agents.     

Study of the base discrimination ability of DNA and 2'-O-methylated RNA oligomers containing 2-thiouracil bases towards complementary RNA or DNA strands and their application to single base mismatch detection

Precise detection of target DNA and RNA sequences using chemically modified oligonucleotides is of crucial importance in gene analysis and gene silence. The hybridisation and base discrimination abilities of oligonucleotides containing 2'-O-methyl-2-thiouridine (s(2)Um) in homo- and hetero-duplexes composed of DNA and RNA strands have been studied in detail. When s(2)Um was incorporated into RNA or DNA strands, the hybridisation and base discrimination abilities of the modified RNA or DNA oligomers towards the complementary RNA strands were superior to those of the corresponding unmodified oligomers. On the other hand, their base discrimination abilities towards complementary DNA strands were almost the same as those of the unmodified ones. The base discrimination abilities of 2-thiouracil base-containing oligonucleotide probes on slide glass plates were also studied. These modified probes exhibited efficient detection of mismatched base pairing.     

Effects of β1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro

Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the β1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [³H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate.     

Simultaneous analysis of multiple polymorphic loci using amplified sequence polymorphisms (ASPs)

In this paper we present a systematic approach to gene mapping and genotyping based on the simultaneous analysis of multiple amplified sequence polymorphisms (ASPs). These genetic markers measure variation in DNA sequences which have been amplified by a polymerase and/or a ligase. The amplified sequence lengths are determined by appropriate choice of oligonucleotides used in the amplification reaction. We describe three classes of ASPs: restriction site polymorphisms, sequence length polymorphisms, and DNA base pair changes not associated with restriction sites. Simultaneous analysis of multiple ASPs using a modified automated DNA sequencing apparatus should be possible because amplification with oligonucleotides provides control over the fragment lengths generated. Development of an automated ASP technology is therefore the next logical step for efficient gene mapping and genotyping of individuals. With this technology, one gel would be sufficient to indicate the most probable locations of a gene and a second gel would permit the selection of the correct location while simultaneously providing a fine structure map.     

Construction of microarrays for genotyping of DQA using unmodified 45-mer oligonucleotide

The human leukocyte antigen (HLA) class II system is strongly connected to immunological response and its compatibility between tissues is critical in transplantation. The simple robust typing analyses of HLA genes are extremely important. In this paper, we developed an approach based on microarray technology for genotyping of DQA gene. The microarrays were constructed with a total 31 unmodified 45-mer oligonucleotide. The second exon of DQA gene was amplified, and allowed to hybridize with the array. DQA genotypes were assigned by quantitative analysis of the hybridization results. The arrays were evaluated by DQA genotyping of nine reference samples and 120 clinical samples. The results demonstrate that the genotyping accuracy/concordance achieved 97.5% compared with the direct DNA sequencing. Although our methods did not perform high-resolution genotyping, it could be an alternative for serological typing in routine medical practice.     

Immobilization of oligonucleotides on glass surface using an efficient heterobifunctional reagent through maleimide-thiol combination chemistry

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl) cyclohexanamide (TPMC), was developed for the immobilization of thiol-modified oligonucleotides on an unmodified glass surface. The heterobifunctionality of the reagent was used for the construction of a DNA microarray in which the triethoxysilyl functionality has specificity toward a glass surface, whereas the maleimide functionality has thiol-modified oligonucleotides via a stable thioether linkage. Immobilization of DNA was achieved by two alternative approaches. In the first approach, the reagent TPMC was treated with oligonucleotides to get triethoxysilyl-oligonucleotide conjugate, which was then covalently attached via specific triethoxysilyl functionality to an unmodified glass surface. In the second approach, the reagent was first covalently linked with an unmodified glass surface to get maleimide functionality on a glass surface, which was then used for the immobilization of oligonucleotides via a stable thioether linkage. The applicability of the reagent was explored by hybridization studies with the fluorescein-labeled complementary DNA strand and in mismatch discrimination.     

Oligonucleotide probes containing polylysine residues for fabrication of DNA chips on various solid surfaces

Various materials, such as glass, plastic, metals, etc., are utilized for preparing DNA chips. In each particular case special approaches are used for immobilization of different oligonucleotide derivatives on the solid supports. We describe a general technique for DNA chips preparation on various unmodified surfaces using one type of oligonucleotide derivative, polylysine-oligonucleotide conjugates (PL-oligo). A long polyamine spacer in the PL-oligo conjugates provides a durable irreversible non-covalent immobilization onto a variety of solid supports and enough distance between oligonucleotides and the surface. The resulting DNA chips were shown to be useful for the detection of PCR DNA fragments and to be sensitive to single nucleotide discrepancies. They represent a promising instrument for revealing genetic diseases, genotyping viruses and bacteria, and for displaying their drug-resistant strains.     

Drosophila microarray platforms.

The advent of high throughput microarrays and the complete sequencing of the Drosophila melanogaster genome have enabled global gene expression analysis in this powerful genetic model organism. Currently, researchers are using three main Drosophila array platform types, with elements composed of cDNA amplicons, oligonucleotides (short and long) or genomic amplicons. This paper provides a broad overview of these platforms.     

Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo

We have studied mutagenic specificities of DNA lesions in vivo in yeast CYC1 oligonucleotide transformation assay. We introduced two lesions into oligonucleotides. One was a nucleoside analog, 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one 2'-deoxyriboside (dP), which is highly mutagenic to bacteria. It is supposed to be a miscoding, but otherwise good template for DNA polymerases. The other lesion was the TT pyrimidine(6-4)pyrimidone photoproduct, one of the typical UV lesions, which blocks DNA replication. These oligonucleotides were used to transform yeast cyc1 mutants with ochre nonsense mutation to Cyc1+. As expected from its templating properties in vitro, the transforming activity of dP-containing oligonucleotides was similar to those of unmodified oligonucleotides. Results indicated that dP may direct incorporation of guanine and adenine at a ratio of 1:20 or more in vivo. An oligonucleotide containing the photoproduct showed the transforming activity of as low as 3-5% of that of the corresponding unmodified oligonucleotide. This bypass absolutely required REV1 gene. The sequence analysis of the transformants has shown that the lesion was read as TT and TC at a ratio of 3:7, indicating its high mutagenic potential.