Labeled Nucleoside Triphosphates with Reversibly Terminating Aminoalkoxyl Groups

Nucleoside triphosphates having a 3′-ONH₂ blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3′-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3′-ONH₂ group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3′-ONH₂ blocking group in “next generation sequencing.”     

Genotyping by apyrase-mediated allele-specific extension

This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3′-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3′-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3′-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.     

DNA sequencing by synthesis with degenerate primers

The degenerate primer-based sequencing Was developed by a synthesis method(DP-SBS)for high-throughput DNA sequencing,in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays.In this method,adifferent set of degenerate primers containing a give nnumber(n)of degenerate nucleotides at the 3'-ends were annealed to the sequenced templates that were immobilized on the solid surface.The nucleotides(n 1)on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides.The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately.The main advanmge of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension.From the present study,it is found that the DP-SBS method is reliable,simple,and cost-effective for laboratory-sequencing a large amount of short DNA fragments.     

Effects of a modified dye-labeled nucleotide spacer arm on incorporation by thermophilic DNA polymerases

The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively.     

Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA

RNA SHAPE chemistry yields quantitative, single-nucleotide resolution structural information based on the reaction of the 2'-hydroxyl group of conformationally flexible nucleotides with electrophilic SHAPE reagents. However, SHAPE technology has been limited by the requirement that sites of RNA modification be detected by primer extension. Primer extension results in loss of information at both the 5' and 3' ends of an RNA and requires multiple experimental steps. Here we describe RNase-detected SHAPE that uses a processive, 3'→5' exoribonuclease, RNase R, to detect covalent adducts in 5'-end-labeled RNA in a one-tube experiment. RNase R degrades RNA but stops quantitatively three and four nucleotides 3' of a nucleotide containing a covalent adduct at the ribose 2'-hydroxyl or the pairing face of a nucleobase, respectively. We illustrate this technology by characterizing ligand-induced folding for the aptamer domain of the Escherichia coli thiamine pyrophosphate riboswitch RNA. RNase-detected SHAPE is a facile, two-day approach that can be used to analyze diverse covalent adducts in any RNA molecule, including short RNAs not amenable to analysis by primer extension and RNAs with functionally important structures at their 5' or 3' ends.     

Effects of a Modified Dye-Labeled Nucleotide Spacer Arm on Incorporation by Thermophilic DNA Polymerases

The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively.     

Chemical primer extension: individual steps of spontaneous replication

The replication of genetic information, as we know it from today's biology, relies on template-directed, polymerase-catalyzed extension of primers. It is known that short stretches of complementary RNA can form on templates in the absence of enzymes. This account summarizes recent work on efficient enzyme-free primer extension, both with 3'-amino-terminal deoxyribonucleotide primers and with primers made of unmodified RNA. Near-quantitative primer extension with half-life times on the order of hours has been demonstrated by using azaoxybenzotriazolides of nucleotides and downstream-binding oligomers. Further, small non-nucleosidic substituents placed on the terminus of the template or the downstream-binding oligomer have been shown to increase the rate and fidelity of primer-extension reactions. Since all four templating bases (A, C, G, T/U) direct sequence-selective primer-extension steps, we feel that there is renewed hope that full, nonenzymatic replication from monomers may eventually be achieved.     

Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene

Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker. The linker assumes a bubble-like structure which itself is not involved in priming, but rather delineates the boundary between the two parts of the primer. This structure results in two primer segments with distinct annealing properties: a longer 5′-segment that initiates stable priming, and a short 3′-segment that determines target-specific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. The strength and utility of the DPO system are demonstrated here using multiplex PCR and SNP genotyping PCR.     

A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms

A new MALDI-TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3'-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis.     

Kinetic explanations for the sequence biases observed in the nonenzymatic copying of RNA templates

The identification of nonenzymatic pathways for nucleic acid replication is a key challenge in understanding the origin of life. We have previously shown that nonenzymatic RNA primer extension using 2-aminoimidazole (2AI) activated nucleotides occurs primarily through an imidazolium-bridged dinucleotide intermediate. The reactive nature and preorganized structure of the intermediate increase the efficiency of primer extension but remain insufficient to drive extensive copying of RNA templates containing all four canonical nucleotides. To understand the factors that limit RNA copying, we synthesized all ten 2AI-bridged dinucleotide intermediates and measured the kinetics of primer extension in a model system. The affinities of the ten dinucleotides for the primer/template/helper complexes vary by over 7,000-fold, consistent with nearest neighbor energetic predictions. Surprisingly, the reaction rates at saturating intermediate concentrations still vary by over 15-fold, with the most weakly binding dinucleotides exhibiting a lower maximal reaction rate. Certain noncanonical nucleotides can decrease sequence dependent differences in affinity and primer extension rate, while monomers bridged to short oligonucleotides exhibit enhanced binding and reaction rates. We suggest that more uniform binding and reactivity of imidazolium-bridged intermediates may lead to the ability to copy arbitrary template sequences under prebiotically plausible conditions.     

Rapid identification of single nucleotide polymorphisms by fluorescence-based capillary electrophoresis

We describe the application of two different fluorescence-based techniques (ddNTP primer extension and single-strand conformation polymorphism (SSCP)) to the detection of single nucleotide polymorphisms (SNPs) by capillary electrophoresis. The ddNTP primer extension technique is based on the extension, in the presence of fluorescence-labeled dideoxy nucleotides (ddNTP, terminators), of an unlabeled oligonucleotide primer that binds to the complementary template immediately adjacent to the mutant nucleotide position. Given that there are no unlabeled dNTPs, a single ddNTP is added to its 3' end, resulting in a fluorescence-labeled primer extension product which is readily separated by capillary electrophoresis. On the other hand, the non-radioisotopic version of SSCP established in this study uses fluorescent dye to label the PCR products, which are also analyzed by capillary electrophoresis. These procedures were used to identify a well-defined SNP in exon 7 of the human p53 gene in DNA samples isolated from two human cell lines (CEM and THP-1 cells). The results revealed a heterozygous single-base transition (G to A) at nucleotide position 14071 in CEM cells, proving that both fluorescence-based ddNTP primer extension and SSCP are rapid, simple, robust, specific and with no ambiguity in interpretation for the detection of well-defined SNPs.     

SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays

This study reports the development of a microarray-based allele-specific extension method for typing of single nucleotide polymorphisms (SNPs). The use of allele-specific primers has been employed previously to identify single base variations but it is acknowledged that certain mismatches are not refractory to extension. Here we have overcome this limitation by introducing apyrase, a nucleotide-degrading enzyme, to the extension reaction. We have shown previously that DNA polymerases exhibit slower reaction kinetics when extending a mismatched primer compared with a matched primer. This kinetic difference is exploited in the apyrase-mediated allele-specific extension (AMASE) assay, allowing incorporation of nucleotides when the reaction kinetics are fast but degrading the nucleotides before extension when the reaction kinetics are slow. Here we show that five homozygous variants (14% of the total number of variants) that were incorrectly scored in the absence of apyrase were correctly typed when apyrase was included in the extension reaction. AMASE was performed in situ on the oligonucleotide microarrays using fluorescent nucleotides to type 10 SNPs and two indels in 17 individuals generating approximately 200 genotypes. Cluster analysis of these data shows three distinct clusters with clear-cut boundaries. We conclude that SNP typing on oligonucleotide microarrays by AMASE is an efficient, rapid and accurate technique for large-scale genotyping.     

The structure of the human apolipoprotein C-II gene. Electron microscopic analysis of RNA:DNA hybrids, complete nucleotide sequence, and identification of 5' homologous sequences among apolipoprotein genes

Cloned human apo-C-II cDNA was used as a hybridization probe to identify the human apo-C-II gene in a genomic library constructed in our laboratory. The isolated apo-C-II DNA was studied both by electron microscopy and by direct sequence analysis. Ultrastructural morphological analysis of RNA-DNA hybrids revealed that the apo-C-II gene had complex structures because of regions of inverted complementary sequences in and around the gene forming stem-and-loop structures which interfere with the formation of stable RNA:DNA hybrids. Extensive morphological analysis revealed a minimum of 3 intervening sequences (IVS), and their lengths were measured. Direct sequence analysis of the cloned gene confirmed the presence of 3 IVS. There are 4 Alu type sequences in IVS-I. We sequenced 4340 nucleotides which include 545 nucleotides in the 5' flanking region, the entire gene which spans 3320 nucleotides, and 475 nucleotides in the 3' flanking region which also encompasses an additional Alu sequence. The 5' end of the gene was identified by primer extension and sequencing of the primer extended cDNA. Apo-C-II mRNA structure was deduced from the cDNA sequence, the primer extension experiments, and the genomic sequence. It is 494 nucleotides in length. Its sequence differs from previously published sequences in that there are 7 additional nucleotides before the polyadenylate tail. In the 5' flanking region, nucleotides -234 to -213 encompass a GC-rich region which exhibits high homology (greater than 70%) to the 5' flanking regions of the genes of all the apolipoproteins published to date, namely, apo-A-II (-497 to -471), apo-A-I (approximately -196 to -179), apo-E (-409 to -391), and apo-C-III (approximately -116 to -103). This highly conserved region might represent some evolutionarily conserved sequences from these related genes and/or might represent a region with regulatory function.     

Capped nonviral sequences at the 5' end of the mRNAs of rice hoja blanca virus RNA4.

Subgenomic RNAs of both polarities corresponding to rice hoja blanca virus (RHBV) ambisense RNA4 were detected in RHBV-infected rice tissues. Total RNA extracted from RHBV-infected and noninfected rice tissues and RNA4 purified from RHBV ribonucleoprotein particles were used as templates for primer extension studies. The RNAs extracted from RHBV-infected tissues contain a population of RNA molecules with 10 to 17 nonviral nucleotides at their 5' end. The RNA-cDNA hybrids resulting from primer extension of such RNA molecules were specifically immunoselected with anti-cap antibodies, indicating that the subgenomic RNAs are capped and probably serve as mRNAs and that the additional nucleotides at their 5' end possibly derive from host mRNAs via a cap-snatching mechanism.     

Competition between bridged dinucleotides and activated mononucleotides determines the error frequency of nonenzymatic RNA primer extension

Nonenzymatic copying of RNA templates with activated nucleotides is a useful model for studying the emergence of heredity at the origin of life. Previous experiments with defined-sequence templates have pointed to the poor fidelity of primer extension as a major problem. Here we examine the origin of mismatches during primer extension on random templates in the simultaneous presence of all four 2-aminoimidazole-activated nucleotides. Using a deep sequencing approach that reports on millions of individual template-product pairs, we are able to examine correct and incorrect polymerization as a function of sequence context. We have previously shown that the predominant pathway for primer extension involves reaction with imidazolium-bridged dinucleotides, which form spontaneously by the reaction of two mononucleotides with each other. We now show that the sequences of correctly paired products reveal patterns that are expected from the bridged dinucleotide mechanism, whereas those associated with mismatches are consistent with direct reaction of the primer with activated mononucleotides. Increasing the ratio of bridged dinucleotides to activated mononucleotides, either by using purified components or by using isocyanide-based activation chemistry, reduces the error frequency. Our results point to testable strategies for the accurate nonenzymatic copying of arbitrary RNA sequences.     

Universal and blocking primer mismatches limit the use of high‐throughput DNA sequencing for the quantitative metabarcoding of arthropods

The quantification of the biological diversity in environmental samples using high‐throughput DNA sequencing is hindered by the PCR bias caused by variable primer–template mismatches of the individual species. In some dietary studies, there is the added problem that samples are enriched with predator DNA, so often a predator‐specific blocking oligonucleotide is used to alleviate the problem. However, specific blocking oligonucleotides could coblock nontarget species to some degree. Here, we accurately estimate the extent of the PCR biases induced by universal and blocking primers on a mock community prepared with DNA of twelve species of terrestrial arthropods. We also compare universal and blocking primer biases with those induced by variable annealing temperature and number of PCR cycles. The results show that reads of all species were recovered after PCR enrichment at our control conditions (no blocking oligonucleotide, 45 °C annealing temperature and 40 cycles) and high‐throughput sequencing. They also show that the four factors considered biased the final proportions of the species to some degree. Among these factors, the number of primer–template mismatches of each species had a disproportionate effect (up to five orders of magnitude) on the amplification efficiency. In particular, the number of primer–template mismatches explained most of the variation (~3/4) in the amplification efficiency of the species. The effect of blocking oligonucleotide concentration on nontarget species relative abundance was also significant, but less important (below one order of magnitude). Considering the results reported here, the quantitative potential of the technique is limited, and only qualitative results (the species list) are reliable, at least when targeting the barcoding COI region.     

Molecular technologies in detecting sensitive and isoniazid-resistant Mycobacterium tuberculosis

Two types of techniques for detection of single nucleotide polymorphism in 315 codon of katG gene of MTB are developed. Isoniazid resistance of MTB is associated with point mutations in the mentioned codon. Two primer sets with additional competitive blocking primer containing 3'-terminal phosphate group (for elimination of unspecific amplification) allow detecting the most frequent point mutations AGC --> ACC and AGC --> AGA in 315 codon of katG gene. PCR with primer set of two primers one of which contains five LNA-monomers allows to determine an occurrence of any type from six known mutations in 315 codon of katG gene, i.e. to differentiate wild type and isoniazid-resistant MTB. Purity and structure of 17 bp long primers with LNA-modified nucleotides were characterized by time-of-flight MALDI-mass spectrometry. Duplex of 17 bp length formed by two complementary oligonucleotides with LNA-monomers was studied using melting.