scholarly journals DNA as a Recyclable Natural Polymer

2021 ◽  
Author(s):  
Weina Liu ◽  
Simone Giaveri ◽  
Daniel Ortiz ◽  
Francesco Stellacci
Keyword(s):  
Amino Acids ◽  
Circular Economy ◽  
Natural Polymer ◽  
Chain Reaction ◽  
One Pot ◽  
Target Dna ◽  
Phosphorylation Reaction ◽  
Dna Strands ◽  
Polymerase Chain ◽  
Living Organisms

Nature has the ability of circularly re-using its components to produce molecules and materials it needs. An example is the ability of most living organisms of digesting proteins they feed off into amino acids and then using such amino acids in the ribosomal synthesis of new proteins. Recently, we have shown that such recycling of proteins can be reproduced outside living organisms. The key feature of proteins that allows for this type of recycling is their being sequence-defined polymers. Arguably, The most famous sequence-defined polymer in Nature is DNA. Here we show that it is possible starting from sheared calf-DNA to obtain all the four nucleotides as monophosphate-nucleotides (dNMPs). These dNMPs were phosphorylated in a one-pot, multi-enzymes, phosphorylation reaction to generate triphosphate-nucleotides (dNTPs). Finally, we used the dNTPs so achieved (with a global yield of ~60%) as reagents for PCR (polymerase chain reaction) and quantitative PCR (qPCR) to produce target DNA strands. This approach is an efficient, convenient, and environmentally friendly way to produce dNTPs and DNA through recycling according to the paradigm of circular economy.

Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

2006 ◽  
Vol 52 (5) ◽  
pp. 451-461 ◽  
Author(s):  
S S Hynes ◽  
O Chaudhry ◽  
M A Providenti ◽  
M L Smith
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Environmental Persistence ◽  
Fungal Strains ◽  
Target Dna ◽  
Polymerase Chain

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (~14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.Key words: Canada domestic substances list (DSL), Canadian Environmental Protection Act (CEPA), genetically modified organisms (GMO), quantitative polymerase chain reaction (qPCR).

scholarly journals Evaluation of Resistance to Rhabdocline Needlecast in Douglas Fir Variety Shuswap, with Quantitative Polymerase Chain Reaction

2010 ◽  
Vol 100 (4) ◽  
pp. 337-344 ◽  
Author(s):  
M. Catal ◽  
G. C. Adams ◽  
D. W. Fulbright
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Douglas Fir ◽  
Chain Reaction ◽  
Disease Symptoms ◽  
Seed Sources ◽  
Release Times ◽  
Spore Release ◽  
Target Dna ◽  
Polymerase Chain

A quantitative polymerase chain reaction assay was developed that could detect DNA of Rhabdocline pseudotsugae and R. oblonga among DNA of Douglas fir needles to a limit as low as three copies of target DNA. Differential infection rates of two varieties (seed sources) of Douglas fir interplanted in a field were studied in relation to staggered bud breaks. Infection of Douglas fir var. San Isabel corresponded to ascospore release times for Rhabdocline spp., whereas infection of var. Shuswap Lake did not occur throughout the spore release period during 2 years of study, despite abundant inoculum and adequate moisture during bud break. Rhabdocline spp. DNA was never detected in Shuswap Lake and disease symptoms were not observed in any year. We provide evidence that Shuswap Lake is resistant and probably immune to Rhabdocline spp. infection and Rhabdocline needlecast under Michigan conditions.

scholarly journals Performance of a Commercial Polymerase Chain Reaction Test for EndocervicalChlamydia trachomatisInfection in a University Hospital Population

1998 ◽  
Vol 6 (5) ◽  
pp. 224-229
Author(s):  
C. H. Livengood III ◽  
K. A. Boggess ◽  
J. W. Wrenn ◽  
A. P. Murtha
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Test ◽  
Laboratory Evaluation ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Predictive Values ◽  
Target Dna ◽  
Wide Range ◽  
Polymerase Chain ◽  
Pcr Test

Objectives:To examine the accuracy of a commercial polymerase chain reaction (PCR) test (Amplicor CTR, Roche Diagnostic Systems, Branchburg NJ) for identification of endocervical chlamydial infections through both laboratory evaluation and among a diverse teaching hospital patient population.Methods:Testing of reliable threshold inocula and reproducibility were carried out using laboratory stock organisms. Paired endocervical samples from patients with a wide range of indications were tested by PCR and an established culture procedure, and discrepant pairs were further analyzed to determine true results.Results:Laboratory evaluation suggested that one copy of target DNA from a viable organism consistently yielded a positive result, and test reproducibility was very good, with an overall coefficient of variation of 15%. Compared to true results in 1,588 paired clinical samples from 1,489 women with a 10% prevalence of infection, the PCR test and culture yielded respective sensitivities of 87.4% and 78.0%, and negative predictive values of 98.6% and 97.6%. Specificity and positive predictive value for both tests were 100%. Cost per specimen was nearly identical at $18.84 and $18.88 respectively. Polymerase inhibitors and organisms lacking target DNA were not found in false-negative PCR samples.Conclusion:This commercial PCR test is accurate, cost-competitive, and much faster than culture for diagnosis of endocervical chlamydia infections in our population of intermediate prevalence of chlamydial infection.

scholarly journals HIV-1 Preintegration Complexes Preferentially Integrate into Longer Target DNA Molecules in Solution as Detected by a Sensitive, Polymerase Chain Reaction-based Integration Assay

2001 ◽  
Vol 276 (50) ◽  
pp. 46946-46952 ◽  
Author(s):  
Alexei Brooun ◽  
Douglas D. Richman ◽  
Richard S. Kornbluth
Keyword(s):  
Polymerase Chain Reaction ◽  
Host Cell ◽  
Polymerase Chain Reaction Amplification ◽  
Chain Reaction ◽  
Preintegration Complex ◽  
Target Dna ◽  
Host Cell Factors ◽  
A Cell ◽  
Polymerase Chain ◽  
Exonuclease Digestion

After entering a cell and undergoing reverse transcription, the retroviral genome is contained in a preintegration complex (PIC) that mediates its integration into host cell DNA. PICs have been shown to prefer torsionally strained DNA, but the effect of target DNA length has not been previously examined. In this report, concatemerization of a repeating 105-base pair unit was used to vary target DNA length independently from basic DNA sequence, while maintaining both PICs and target DNAs in solution. Integration junctions were quantified by real-time fluorescence-monitored polymerase chain reaction amplification using primers in the viral long terminal repeat and the target DNA. Unreacted target DNA severely inhibited the post-reaction polymerase chain reaction detection step, requiring its removal using λ exonuclease digestion. Integration into a 32-unit concatemer of target DNA was markedly more efficient than integration into a monomeric unit, indicating that longer target DNA was preferred. This substrate was used to construct a simple, robust, and adaptable assay that can serve as a method for studying the host cell factors that enhance PIC integration, and as a drug discovery platform for integration inhibitors active against PICs.

scholarly journals ANALISIS MOLEKULER FILOGENETIK DAN STRUKTUR ANTIGENIC VIRUS AVIAN INFLUENZA SUBTIPE H5N1 ISOLAT LAMPUNG TAHUN 2008-2013

2015 ◽  
Vol 9 (1) ◽  
Author(s):  
Eko Agus Srihanto ◽  
Widya Asmara ◽  
Michael Haryadi Wibowo
Keyword(s):  
Amino Acids ◽  
Avian Influenza ◽  
Animal Health ◽  
Antigenic Site ◽  
Multiple Alignment ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Phylogenic Tree ◽  
Tree Analysis ◽  
Polymerase Chain

Penelitian ini bertujuan melakukan karakterisasi molekuler antigenic site terhadap isolat virus avian influenza (AI) Balai Penyidikan dan Pengujian Veteriner (BPPV) Regional III Lampung dari tahun 2008-2013. Amplifikasi RNA dilakukan dengan teknik reverse transcription polymerase chain reaction (RT-PCR) menggunakan 4 pasang primer referens dari Australian Animal Health Laboratory (AAHL) Geelong Australia (HA10, HA20, dan HA30) dan dilanjutkan dengan proses pengurutan. Analisis hasil pengurutan dengan menggunakan perangkat lunak MEGA versi 5.05 yang meliputi multiple alignment, deductive amino acids prediction, dan phylogenic tree analysis diperoleh hasil perbedaan genetik antar isolat Lampung dari tahun 2003-2013 ditemukan berkisar 1,1-9,1% dengan tingkat homologi mencapai 90,9-98,9%. Variasi genetik ditemukan adanya substitusi pada posisi 53 (R53K), 126 (D126E), 136 (P136), 138 (H138Q, dan H138L), 140 (R140K, R140S, dan R140N), 141 (S141P), dan 189 (K189R). Berdasarkan analisis filogenic tree isolat Lampung tahun 2008-2011 termasuk ke dalam clade 2.1.3. Analisis filogenik isolat AI tahun 2012-2013 yang menginfeksi unggas air mempunyai homologi sekitar 98,5-99,1% dibandingkan dengan isolat AI yang menginfeksi unggas air asal Jawa dan termasuk ke dalam clade 2.3.2.1.

Polymerase chain reaction (PCR) v1

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Plasmid Construction ◽  
Target Dna ◽  
Dna Fragment ◽  
Polymerase Chain

This protocol is used to amplify target DNA fragment for plasmid construction or other use.

Polymerase chain reaction (PCR) v2

2021 ◽  
Author(s):  
Shuning Guo
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Plasmid Construction ◽  
Target Dna ◽  
Dna Fragment ◽  
Polymerase Chain

This protocol is used to amplify target DNA fragment for plasmid construction or other use.

The genes encoding granule-bound starch synthases at the waxy loci of the A, B, and D progenitors of common wheat

Genome ◽  
2000 ◽  
Vol 43 (2) ◽  
pp. 264-272 ◽  
Author(s):  
Liuling Yan ◽  
Mrinal Bhave ◽  
Robert Fairclough ◽  
Christine Konik ◽  
Sadequr Rahman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Polymerase Chain Reaction ◽  
Amino Acid ◽  
Common Wheat ◽  
Sequence Variation ◽  
Waxy Gene ◽  
Chain Reaction ◽  
Genes Encoding ◽  
Polymerase Chain ◽  
Granule Bound Starch Synthase

Three genes encoding granule-bound starch synthase (wx-TmA, wx-TsB, and wx-TtD) have been isolated from Triticum monococcum (AA), and Triticum speltoides (BB), by the polymerase chain reaction (PCR) approach, and from Triticum tauschii (DD), by screening a genomic DNA library. Multiple sequence alignment indicated that the wx-TmA, wx-TsB, and wx-TtD genes had the same extron and (or) intron structure as the previously reported waxy gene from barley. The lengths of the three wx-TmA, wx-TsB, and wx-TtD genes were 2834 bp, 2826 bp, and 2893 bp, respectively, each covering 31 bp in the untranslated leader and the entire coding region consisting of 11 exons and 10 introns. The three genes had identical lengths of exons, except exon1, and shared over 95% identity with each other within the exon regions. The majority of introns were significantly variable in length and sequence, differing mainly in length (1-57 bp) as a result of insertion and (or) deletion events. The deduced amino acid sequence from these three genes indicated that the mature WX-TMA, -TSB, and -TTD proteins contained the same number of amino acids, but differed in predicted molecular weight and isoelectric point (pI) due to amino acid substitutions (13-18). The predicted physical characteristics of the WX proteins matched the respective proteins in wheat very closely, but the match was not perfect. Furthermore the exon5 sequences of the wx-TmA, wx-TsB, and wx-TtD genes were different from a cDNA encoding a waxy gene of common wheat previously reported. The striking difference was that an insertion of 11 amino acids occurred in the cDNA sequence that could not be observed in the exons of the A, B, and D genes. It was noted, however, that the 3prime end of intron4 of these genes could account for the additional 11 amino acids. The sequence information from the available waxy genes identified the intron4-exon5-intron5 region as being diagnostic for sequence variation in waxy. The sequence variation in the waxy genes provides the basis for primer design to distinguish the respective genes in common wheat, and its progenitors, using PCR. Key words: Angiosperms, Poaceae, Triticeae, Triticum monococcum, Triticium speltoides, Triticum tauschii, granule-bound starch synthase, polymerase chain reaction (PCR), molecular evolution.

Polymerase Chain Reaction–Mediated Characterization of Molds Belonging to the Aspergillus flavus Group and Detection of Aspergillus parasiticus in Peanut Kernels by a Multiplex Polymerase Chain Reaction

2002 ◽  
Vol 65 (5) ◽  
pp. 840-844 ◽  
Author(s):  
RUEY-SHYANG CHEN ◽  
JWU-GUH TSAY ◽  
YU-FEN HUANG ◽  
ROBIN Y.-Y. CHIOU
Keyword(s):  
Polymerase Chain Reaction ◽  
Aspergillus Flavus ◽  
Aspergillus Parasiticus ◽  
Regulatory Protein ◽  
Aflatoxin Production ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Target Dna ◽  
Polymerase Chain ◽  
Aspergillus Flavus Group

The Aspergillus flavus group covers species of A. flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds. Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition. Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated. In this study, 19 strains of the A. flavus group, including A. flavus, A. parasiticus, A. oryzae, A. sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis. Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection. Fifteen strains were shown to possess the four target DNA fragments. With regard to aflatoxi-genicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin. When peanut kernels were artificially contaminated with A. parasiticus and A. niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca. 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected. The target DNA fragments were detected in the kernels infected with A. parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A. niger.

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