The dual nucleic acid amplification with dynamic light scattering strategy for ultrasensitive detection of Salmonella in milk
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Peptide nucleic acid
Reagents capable of sequence-specificrecognition of nucleic acids are becom-ing more and more important forresearch, diagnosis and therapy. Rapidprogress is being made in the eluci-dation of the human genome sequence,as well as that of other organisms,including human pathogens. As thisinformation emerges, there is an increas-ing need for new tools to probe genestructure and function and for novelreagents to allow development of gene-based diagnostics and therapeutics.Over the past few years, peptidenucleic acids (PNAs) have emerged asone of the most promising new types ofmolecules for recognition of nucleicacids. PNAs are actually neither peptidenor nucleic acid, but have a hybridstructure consisting of repeating N-(2-aminoethyl)-glycine units linked byamide bonds. The purine (A, G) andpyrimidine (T, C) nucleobases areattached to this backbone via methylenecarbonyl linkages. There are no sugar orphosphate groups. Hence, the unmodi-fied PNAs are not charged at neutralpH.Originally, PNAs were designed torecognize and bind to duplex DNA inthe major groove via Hoogsteen bond-ing to form triple helices. Although cer-tain PNA sequences can do this, it wassoon discovered that PNA oligomerscan bind to single-stranded nucleic acidsto form duplexes, either PNA–DNA orPNA–RNA, with affinity and specificitysubstantially exceeding that of compa-rable DNA or RNA oligonucleotides. In addition, triplex formation involv-ing PNAs can occur either withPNA–DNA–DNA triplexes or the morefavored PNA–DNA–PNA triplexes. Thestability of the PNA interaction withDNA is such that strand invasion ofDNA by PNAs is thermodynamicallyfavored, and can take place via eitherduplex, triplex or double duplex formation.The foregoing constitutes just a shortlist of the capabilities of PNAs. In fact,the known properties of PNAs and theirpotential applications in biomedicineare rapidly expanding as researcherscontinue to examine these fascinatingmolecules. It is therefore quite timelythat Peter Nielsen and Michael Egholm,two of the inventors of PNAs, have puttogether a book reviewing the state ofthe art in PNA research.The emphasis here is on timely. Theeditors have clearly made an effort toinclude up-to-the-minute material andto publish it quickly. As a result, thetopics include most, if not all, of therecent PNA advances, all by keyresearchers in the field. The book beginswith a useful overview by the editorsputting the development and use ofPNAs in perspective, and serving as anintroduction for those new to this area. This is followed by a series of chap-ters focusing on the fundamental chem-istry of PNA synthesis. Several of thesecover newer chemical strategies such asthe synthesis of PNA–DNA andPNA–peptide chimeras and PNA conju-gation to labeling compounds such asbiotin, fluorescein and rhodamine. Theemphasis here is on the protocols. Trueto the book’s subtitle, these chaptersread like a good manual, with substan-tial detail as well as useful discussionproviding experimental and technicalinsight. Although these sections will bemost useful to synthetic chemists, theywill also serve biologists by highlightingthe versatility of PNAs and identifyingthe possible syntheses and novel mol-ecules that can be made.Another section focuses onhybridization-based techniques, andthese chapters provide a sophisticateddiscussion of the nucleic acid-bindingproperties of the PNAs, both in a sec-tion devoted to the thermodynamics ofPNA–nucleic acid interactions as well ina series of application chapters. In thisportion of the book, a wide variety ofemerging properties and applications ofPNAs are examined. The use of PNAsfor
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Abstract A new displacement probe based on pyrrolidinyl peptide nucleic acid was designed and evaluated for DNA sequence recognition. The probe was prepared by combining an N ‐terminally fluorophore‐modified pyrrolidinyl peptide nucleic acid (Flu‐ or TMR‐acpcPNA) and a 3’‐Dabcyl‐modified DNA as a quencher. Fluorescence studies showed that the fluorophore in the acpcPNA strand was efficiently quenched by the quencher strand. After some optimisation, the fluorescence was significantly restored upon the addition of the complementary DNA target, while the fluorescence stayed at a low level with the addition of mismatched DNA. Even with double‐stranded DNA analytes, the high specificity of the PNA‐based displacement probes allowed unambiguous discrimination between complementary and single mismatched DNA targets. Furthermore, immobilisation of the probes onto agarose resin could also recognise only the complementary DNA, thereby demonstrating its potential as a practical DNA sensor.
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Target DNA fragments at 10 fM concentration (approximately 6 × 105 molecules) were detected against a DNA background simulating the noncomplementary genomic DNA present in real samples using a simple, PCR-free, optics-free approach based on electromechanical signal transduction. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is highly desired for a range of diverse applications. We previously described a potentially low-cost device for sequence-specific nucleic acid detection based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nucleic acid probe) conjugates upon hybridization with target nucleic acid. Here, we demonstrate the operation of our device with longer DNA targets, and we describe the resulting improvement in the limit of detection (LOD). We investigated the detection of DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM to 1 fM and found that the LOD decreased as DNA length increased, with 419 and 1613 nucleotide oligomers detectable down to 10 fM. In addition, no false positive responses were obtained with noncomplementary, control DNA fragments of similar length. The 1613-base DNA oligomer is similar in size to 16S rRNA, which suggests that our device may be useful for detection of pathogenic bacteria at clinically relevant concentrations based on recognition of species-specific 16S rRNA sequences.
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genomic DNA
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Abstract Nucleic acid chips are based on the method of sequencing by hybridization, where unknown DNA fragments are hybridized to complementary nucleic acid sequences that are immobilized on a solid surface in an array format. One novel approach is to use peptide nucleic acid (PNA) biosensor chips. These DNA analogue possess the ability to hybridize with complementary DNA sequences. Because the backbone of DNA contains phosphates as opposed to PNA that does not, a technique that identifies the presence of these phosphates in a molecular surface layer would allow unlabelled DNA fragments hybridized to complementary PNAs to be detected. We have successfully shown that time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) is a very useful tool for identifying hybridized DNA on PNA biosensor chips by detecting the phosphorus present in the DNA. ToF‐SIMS is also a very effective technique for studying the complexity of the immobilization and hybridization process. Copyright © 2002 John Wiley & Sons, Ltd.
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"Abstract: Peptide Nucleic Acids (PNAs) are nanostructures similar to nucleic acid molecules (synthetic DNA/RNA analogs) wherein the negatively charged backbone (sugar-phosphate) present in DNA/RNA molecules is replaced by a backbone without polyamide or peptide charge. Later, it was found that PNAs containing both purine and pyrimidine bases form highly stable duplexes with DNA and RNA. Although it is not as stable as 2PNA/DNA triplexes containing a homopyrimidine strand, it is still more stable than DNA/DNA and/or DNA/RNA duplexes. The unique characteristics of PNAs add new aspects to these nanostructures relative to conventional analogs to make them appropriate for molecular biology studies. The most important applications include the use of these nanostructures in the detection and treatment of diseases caused by threatening biological agents using the antisense/antigen technology and as genetic regulator drugs. Keywords: Peptide Nucleic Acids (PNAs), synthetic DNA analog, genetic regulator drugs, antisense-antigen technology"
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Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G 2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C 4 T 2 C 4 is an efficient template for synthesis of PNA G 4 A 2 G 4 using G 2 and A 2 units as substrates. The corresponding synthesis of PNA G 4 C 2 G 4 on DNA C 4 G 2 C 4 is less efficient. Incorporation of PNA T 2 into PNA products on DNA C 4 A 2 C 4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.
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Ribose
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Peptide nucleic acid (PNA) is a new class of DNA mimic, which is potentially useful for diagnostic and therapeutic applications due to its high affinity and specificity of binding to target nucleic acids. The goal of this work is to develop a simple and sensitive fluorimetric-based detection of DNA sequences using new fluorescent labeled PNA probes previously developed in our laboratory. The method relies on the preferential absorption of PNA·DNA hybrid (negatively charged) over unhybridized PNA (neutral) on an anion exchange solid support. This enables detection of the PNA-nucleic acid interactions, which will take place only when the PNA and DNA sequences are fully complementary directly on the solid support by fluorescence microscopy. With the high specificity of this new PNA-based technology, single mismatches in 9–13 bases target DNA sequences were readily distinguished. Based on this principle, a prototype for detection of single nucleotide polymorphisms (SNP) without the need for labeling the DNA substrates has been developed.
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Peptide nucleic acid (PNA) is a mimic of nucleic acids that is able to bind complementary oligonucleotides with high affinity and excellent selectivity. As such, the use of PNA has been proposed in numerous applications in biochemistry, medicine, and biotechnology. Sequences of pseudo-complementary PNAs containing diaminopurine (D)-2-thiouracil (S U) base pairs bind to complementary regions within double-stranded DNA targets. This type of binding is termed "double duplex invasion" and involves unwinding of the duplex accompanied by simultaneous hybridization of both DNA strands by the two pseudo-complementary PNAs. In this study, a simple method of assaying DNA strand invasion by pseudo-complementary PNAs was developed. This method is based on the incorporation of a single fluorescent cytidine analog, phenylpyrrolocytidine (PhpC), into the double-stranded DNA target such that upon invasion by PNA, the PhpC is displaced to a single-stranded region resulting in the turn-on of fluorescence emission. This fluorescent assay is applicable to studies both at equilibrium and approach-to-equilibrium (time-dependent) conditions.
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Kirsten rat sarcoma virus (KRAS) gene mutations are a type of driver mutation discovered in the 1980s, but for a long time no molecular targeted drugs were available for them. Recently, sotorasib was developed as a molecular targeted drug for KRAS mutations. It is therefore necessary to identify the characteristics of patients with KRAS mutations.This was the single-institution retrospective study. Surgically resected tumors from lung adenocarcinoma patients were collected at a single institution from June 2016 to September 2019. Peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp analysis of KRAS G12X mutations was compared with analysis by therascreen KRAS RGQ kit. The association between KRAS mutation status and patient characteristics and prognosis was assessed.Among 499 lung adenocarcinomas, KRAS mutations were evaluated in 197 cases, excluding stage IV lung cancer and tumors with epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutations. KRAS G12X mutations were detected in 59 cases (29.9%). The highest frequency by gene mutation subtype was G12V in 23 cases (39.0%), followed by G12C in 16 cases (27.1%), G12D in 12 cases (20.3%), G12S in 4 cases (6.8%) and G12A in 2 cases. For the G12C mutation, the PNA-LNA PCR clamp and therascreen methods were consistent, but for the G12D and G12S mutations, the PNA-LNA PCR clamp method showed higher detection rates. In operable tumors, G12C mutations were more frequent in males, smokers, and patients with high expression of programmed death-ligand 1 (PD-L1), and had no correlation with prognosis.By the PNA-LNA PCR clamp method, G12C mutation of surgical specimens was detected successfully. The PNA-LNA PCR clamp method is expected to be applied to the detection of druggable G12C mutations.
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