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Fulltext Molecular genetic approaches for the conservation of endangered orchids

Rodrigues, K. F., Yeoh, K. A., Kumar, S. V.

Buletin Persatuan Genetik Malaysia, 2006;12(1):13-14.
MyJurnal

Geographically isolated populations of endemic orchids have evolved and adapted to an existence within specifi c ecological niches. These populations are highly susceptible to anthropogenic
infl uences on their microhabitats. The primary objective of conservation programs is the restoration of endangered populations to their ecologically sustainable levels, and the fi rst stage in the process of conservation involves estimation of molecular diversity at the level of the population. The approach described in this article involves the application of RAPD, Microsatellites and Chloroplast DNA markers for the characterization of the genetic structure of Paphiopedilum rothschildianum and Phalaenopsis gigantea, two endangered and endemic orchids of Sabah. This study has isolated a total of 96 microsatellite loci in P. rothschildianum and P. gigantea, 42 specifi c primer pairs have been designed for amplifi cation of microsatellite loci and are currently being applied to screen the breeding pools. The Chloroplast DNA regions amplifi ed by the primer pairs trnH-psbA and trnL-trnF exhibit distinct polymorphisms and can be used to establish phylogenetic
relationships. The ability of microsatellite loci to cross-amplify selected varieties of orchids has been determined. The molecular markers developed will be applied to estimate population diversity
levels and to formulate long-term management strategies for the conservation of endangered species of orchids of Sabah.

Matched MeSH terms: DNA Primers; DNA, Chloroplast; Random Amplified Polymorphic DNA Technique
Complete genome sequence of Planococcus donghaensis JH1T, a pectin-degrading bacterium

See-Too WS, Chua KO, Lim YL, Chen JW, Convey P, Mohd Mohidin TB, et al.

J Biotechnol, 2017 Jun 20;252:11-14.
PMID: 28483443 DOI: 10.1016/j.jbiotec.2017.05.005

The type strain Planococcus donghaensis JH1Tis a psychrotolerant and halotolerant bacterium with starch-degrading ability. Here, we determine the carbon utilization profile of P. donghaensis JH1Tand report the first complete genome of the strain. This study revealed the strain's ability to utilize pectin and d-galacturonic acid, and identified genes responsible for degradation of the polysaccharides. The genomic information provided may serve as a fundamental resource for full exploration of the biotechnological potential of P. donghaensis JH1T.

Matched MeSH terms: DNA, Bacterial/genetics; Sequence Analysis, DNA
Long Fragment Polymerase Chain Reaction

Chua EW, Maggo S, Kennedy MA

Methods Mol Biol, 2017;1620:65-74.
PMID: 28540699 DOI: 10.1007/978-1-4939-7060-5_3

Polymerase chain reaction (PCR) is an oft-used preparatory technique in amplifying specific DNA regions for downstream analysis. The size of an amplicon was initially limited by errors in nucleotide polymerization and template deterioration during thermal cycling. A variant of PCR, designated long-range PCR, was devised to counter these drawbacks and enable the amplification of large fragments exceeding a few kb. In this chapter we describe a protocol for long-range PCR, which we have adopted to obtain products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples.

Matched MeSH terms: DNA/genetics*; DNA Primers
Sphingopyxis microcysteis sp. nov., a novel bioactive exopolysaccharides-bearing Sphingomonadaceae isolated from the Microcystis phycosphere

Zhang XL, Li GX, Ge YM, Iqbal NM, Yang X, Cui ZD, et al.

Antonie Van Leeuwenhoek, 2021 Jun;114(6):845-857.
PMID: 33770293 DOI: 10.1007/s10482-021-01563-1

During the study into the microbial biodiversity and bioactivity of the Microcystis phycosphere, a new yellow-pigmented, non-motile, rod-shaped bacterium containing polyhydroxybutyrate granules designated as strain Z10-6T was isolated from highly-toxic Microcystis aeruginosa Kützing M.TN-2. The new isolate produces active bioflocculating exopolysaccharides. Phylogenetic analysis based on 16S rRNA gene sequences indicated strain Z10-6T belongs to the genus Sphingopyxis with highest similarity to Sphingopyxis solisilvae R366T (98.86%), and the similarity to other Sphingopyxis members was less than 98.65%. However, both low values obtained by phylogenomic calculation of average nucleotide identity (ANI, 85.5%) and digital DNA-DNA hybridization (dDDH, 29.8%) separated the new species from its closest relative. The main polar lipids were sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified glycolipid and one unidentified aminophospholipid. The predominant fatty acids were summed feature 8, C17:1ω6c, summed feature 3, C16:0, C18:1ω7c 11-methyl and C14:0 2-OH. The respiratory quinone was ubiqunone-10, with spermidine as the major polyamine. The genomic DNA G + C content was 64.8 mol%. Several biosynthesis pathways encoding for potential new bacterial bioactive metabolites were found in the genome of strain Z10-6T. The polyphasic analyses clearly distinguished strain Z10-6T from its closest phylogenetic neighbors. Thus, it represents a novel species of the genus Sphingopyxis, for which the name Sphingopyxis microcysteis sp. nov. is proposed. The type strain is Z10-6T (= CCTCC AB2017276T = KCTC 62492T).

Matched MeSH terms: DNA, Bacterial/genetics; Sequence Analysis, DNA
Taxonomic note on the family Pseudonocardiaceae based on phylogenomic analysis and descriptions of Allosaccharopolyspora gen. nov. and Halosaccharopolyspora gen. nov

Teo WFA, Tan GYA, Li WJ

Int J Syst Evol Microbiol, 2021 Oct;71(10).
PMID: 34714227 DOI: 10.1099/ijsem.0.005075

The taxonomic positions of members within the family Pseudonocardiaceae were assessed based on phylogenomic trees reconstructed using core-proteome and genome blast distance phylogeny approaches. The closely clustered genome sequences from the type strains of validly published names within the family Pseudonocardiaceae were analysed using overall genome-related indices based on average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values. The family Pseudonocardiaceae consists of the type genus Pseudonocardia, as well as the genera Actinoalloteichus, Actinocrispum, Actinokineospora, Actinomycetospora, Actinophytocola, Actinopolyspora, Actinorectispora, Actinosynnema, Allokutzneria, Allosaccharopolyspora gen. nov., Amycolatopsis, Bounagaea, Crossiella, Gandjariella, Goodfellowiella, Haloactinomyces, Haloechinothrix, Halopolyspora, Halosaccharopolyspora gen. nov., Herbihabitans, Kibdelosporangium, Kutzneria, Labedaea, Lentzea, Longimycelium, Prauserella, Saccharomonospora, Saccharopolyspora, Saccharothrix, Salinifilum, Sciscionella, Streptoalloteichus, Tamaricihabitans, Thermocrispum, Thermotunica and Umezawaea. The G+C contents of the Pseudonocardiaceae genomes ranged from 66.2 to 74.6 mol% and genome sizes ranged from 3.69 to 12.28 Mbp. Based on the results of phylogenomic analysis, the names Allosaccharopolyspora coralli comb. nov., Halosaccharopolyspora lacisalsi comb. nov. and Actinoalloteichus caeruleus comb. nov. are proposed. This study revealed that Actinokineospora mzabensis is a heterotypic synonym of Actinokineospora spheciospongiae, Lentzea deserti is a heterotypic synonym of Lentzea atacamensis, Prauserella endophytica is a heterotypic synonym of Prauserella coralliicola, and Prauserella flava and Prauserella sediminis are heterotypic synonyms of Prauserella salsuginis. This study addresses the nomenclature conundrums of Actinoalloteichus cyanogriseus and Streptomyces caeruleus as well as Micropolyspora internatus and Saccharomonospora viridis.

Matched MeSH terms: DNA, Bacterial/genetics; Sequence Analysis, DNA
Molecular phylogenetic analysis of mudflat snails (Gastropoda: Euthyneura: Amphiboloidea) supports an Australasian centre of origin

Golding RE

Mol Phylogenet Evol, 2012 Apr;63(1):72-81.
PMID: 22210412 DOI: 10.1016/j.ympev.2011.12.016

Amphiboloidea is a small but widespread group of snails found exclusively, and often abundantly, in mudflat and associated salt marsh or mangrove habitat. This study uses molecular data from three loci (COI, 16S and 28S) to infer phylogenetic relationships in Amphiboloidea and examine its position in Euthyneura. All but two of the named extant species of Amphiboloidea and additional undescribed taxa from across Southeast Asia and the Arabian Gulf were sampled. In contrast to the current morphology-based classification dividing Amphiboloidea into three families, analysis of molecular data supports revision of the classification to comprise two families. Maningrididae is a monotypic family basal to Amphibolidae, which is revised to comprise three subfamilies: Amphibolinae, Phallomedusinae and Salinatorinae. Sequence divergence between Asian populations of Naranjia is relatively large and possibly indicative of species complexes divergent across the Strait of Malacca. Salinatorrosacea and Salinator burmana do not cluster with other Salinator species, and require generic reassignment. In addition, sequences were obtained from an undescribed species of Lactiforis from the Malay Peninsula. Reconstruction of ancestral distributions indicates a plesiomorphic distribution and centre of origin in Australasia, with two genera subsequently diversifying throughout Asia. Increasing the sampling density of amphiboloid taxa in a phylogenetic analysis of Euthyneura did not resolve the identity of the sister taxon to Amphibolidae, but confirmed its inclusion in Pulmonata/Panpulmonata.

Matched MeSH terms: DNA, Mitochondrial/genetics; Sequence Analysis, DNA
Fulltext Genomic DNA extraction from medicinal plants available in Malaysia using a TriOmic(TM) improved extraction kit

Mohd-Hairul AR, Sade AB, Yiap BC, Raha AR

Genet. Mol. Res., 2011;10(4):2757-64.
PMID: 22095601 DOI: 10.4238/2011.November.8.1

DNA extraction was carried out on 32 medicinal plant samples available in Malaysia using the TriOmic(TM) extraction kit. Amounts of 0.1 g flowers or young leaves were ground with liquid nitrogen, lysed at 65°C in RY1(plus) buffer and followed by RNAse treatment. Then, RY2 buffer was added to the samples and mixed completely by vortexing before removal of cell debris by centrifugation. Supernatants were transferred to fresh microcentrifuge tubes and 0.1 volume RY3 buffer was added to each of the transferred supernatant. The mixtures were applied to spin columns followed by a centrifugation step to remove buffers and other residues. Washing step was carried out twice by applying 70% ethanol to the spin columns. Genomic DNA of the samples was recovered by applying 50 μL TE buffer to the membrane of each spin column, followed by a centrifugation step at room temperature. A modification of the TriOmic(TM) extraction procedure was carried out by adding chloroform:isoamyl alcohol (24:1) steps in the extraction procedure. The genomic DNA extracted from most of the 32 samples showed an increase of total yield when chloroform:isoamyl alcohol (24:1) steps were applied in the TriOmicTM extraction procedure. This preliminary study is very important for molecular studies of medicinal plants available in Malaysia since the DNA extraction can be completed in a shorter period of time (within 1 h) compared to manual extraction, which entails applying phenol, chloroform and ethanol precipitation, and requires 1-2 days to complete.

Matched MeSH terms: DNA, Plant/isolation & purification*; DNA, Plant/chemistry
Fulltext Genetic diversity in Passiflora species assessed by morphological and its sequence analysis

Ramaiya SD, Bujang JS, Zakaria MH

ScientificWorldJournal, 2014;2014:598313.
PMID: 25050402 DOI: 10.1155/2014/598313

This study used morphological characterization and phylogenetic analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA to investigate the phylogeny of Passiflora species. The samples were collected from various regions of East Malaysia, and discriminant function analysis based on linear combinations of morphological variables was used to classify the Passiflora species. The biplots generated five distinct groups discriminated by morphological variables. The group consisted of cultivars of P. edulis with high levels of genetic similarity; in contrast, P. foetida was highly divergent from other species in the morphological biplots. The final dataset of aligned sequences from nine studied Passiflora accessions and 30 other individuals obtained from GenBank database (NCBI) yielded one most parsimonious tree with two strongly supported clades. Maximum parsimony (MP) tree showed the phylogenetic relationships within this subgenus Passiflora support the classification at the series level. The constructed phylogenic tree also confirmed the divergence of P. foetida from all other species and the closeness of wild and cultivated species. The phylogenetic relationships were consistent with results of morphological assessments. The results of this study indicate that ITS region analysis represents a useful tool for evaluating genetic diversity in Passiflora at the species level.

Matched MeSH terms: Sequence Analysis, DNA; DNA, Ribosomal Spacer*
The complete mitogenome of the marine bivalve Lutraria rhynchaena Jonas 1844 (Heterodonta: Bivalvia: Mactridae)

Gan HM, Tan MH, Thai BT, Austin CM

Mitochondrial DNA A DNA Mapp Seq Anal, 2016;27(1):335-6.
PMID: 24617474 DOI: 10.3109/19401736.2014.892104

The complete mitochondrial genome of the commercially important snout otter clam Lutraria rhynchaena was obtained from low-coverage shotgun sequencing data on the MiSeq platform. The L. rhynchaena mitogenome has 16,927 base pairs (69% A + T content) and made up of 12 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a 953 bp non-coding AT-rich region. This is the first mitogenome to be sequenced from the genus Lutraria, and the seventh to be reported for the family Mactridae.

Matched MeSH terms: DNA, Mitochondrial/analysis; DNA, Mitochondrial/genetics
Evidence of sibling species in the brown planthopper complex (Nilaparvata lugens) detected from short and long primer random amplified polymorphic DNA fingerprints

Latif MA, Soon Guan T, Mohd Yusoh O, Siraj SS

Biochem Genet, 2008 Aug;46(7-8):520-37.
PMID: 18504649 DOI: 10.1007/s10528-008-9167-5

The inheritance of 31 amplicons from short and long primer RAPD was tested for segregating ratios in two families of the brown planthopper, Nilaparvata lugens, and they were found to be inherited in a simple Mendelian fashion. These markers could now be used in population genetics studies of N. lugens. Ten populations of N. lugens were collected from five locations in Malaysia. Each location had two sympatric populations. Cluster and principal coordinate analyses based on genetic distance along with AMOVA revealed that the rice-infesting populations (with high esterase activity) at five localities clustered together as a group, and Leersia-infesting populations (with low esterase activity) at the same localities formed another distinct cluster. Two amplicons from primers OPD03 (0.65 kb) and peh#6 (1.0 kb) could be considered diagnostic bands, which were fixed in the Leersia-infesting populations. These results represent evidence of a sibling species in the N. lugens complex.

Matched MeSH terms: DNA Fingerprinting; DNA Primers; Random Amplified Polymorphic DNA Technique
Fulltext Long term Tai Chi exercise reduced DNA damage and increased lymphocyte apoptosis and proliferation in older adults

Goon JA, Noor Aini AH, Musalmah M, Yasmin Anum MY, Wan Ngah WZ

Med J Malaysia, 2008 Oct;63(4):319-24.
PMID: 19385493 MyJurnal

Effect of Tai Chi exercise on the level of DNA damage using the comet assay, lymphocyte viability and frequency of sister chromatid exchange (SCE) were determined in adults aged above 45. Tai Chi participants of 7 years (n=35), showed higher level of normal DNA and lower level of mild and severely damaged DNA as compared to the sedentary subjects (n=35). The former are suggested to have effective DNA repair mechanism as their frequency of SCE was markedly lower. Higher lymphocyte apoptosis and proliferation found in the Tai Chi participants also indicated that the exercise promotes renewal and regeneration of lymphocytes.

Matched MeSH terms: DNA Damage*; DNA Repair
Fulltext Morphometric and molecular analysis of mackerel (Rastrelliger spp) from the west coast of Peninsular Malaysia

Darlina MN, Masazurah AR, Jayasankar P, Jamsari AF, Siti AM

Genet. Mol. Res., 2011;10(3):2078-92.
PMID: 21968625 DOI: 10.4238/vol10-3gmr1249

Mackerel (Scombridae; Rastrelliger) are small commercially important pelagic fish found in tropical regions. They serve as a cheap source of animal protein and are commonly used as live bait. By using a truss morphometrics protocol and RAPD analysis, we examined morphological and genetic variation among 77 individual mackerel that were caught using long lines and gillnets at 11 locations along the west coast of Peninsular Malaysia. Nineteen morphometric traits were evaluated and genetic information was estimated using five 10-base RAPD random primers. Total DNA was extracted from muscle tissue. Morphometric discriminant function analysis revealed that two morphologically distinct groups of Rastrelliger kanagurta and a single group of R. brachysoma can be found along the west coast of Peninsular Malaysia. We also found that the head-related characters and those from the anterior part of the body of Rastrelliger spp significantly contribute to stock assessment of this population. RAPD analysis showed a trend similar to that of the morphometric analysis, suggesting a genetic component to the observed phenotypic differentiation. These data will be useful for developing conservation strategies for these species.

Matched MeSH terms: DNA/genetics; Random Amplified Polymorphic DNA Technique
Phylogeny and phylogeography of Old World fruit bats in the Cynopterus brachyotis complex

Campbell P, Schneider CJ, Adnan AM, Zubaid A, Kunz TH

Mol Phylogenet Evol, 2004 Dec;33(3):764-81.
PMID: 15522802

Taxonomic relationships within the Old World fruit bat genus, Cynopterus, have been equivocal for the better part of a century. While nomenclature has been revised multiple times on the basis of phenotypic characters, evolutionary relationships among taxa representing the entire geographic range of the genus have not been determined. We used mitochondrial DNA sequence data to infer phylogenetic relationships among the three most broadly distributed members of the genus: C. brachyotis, C. horsfieldi, and C. sphinx, and to assess whether C. brachyotis represents a single widespread species, or a complex of distinct lineages. Results clearly indicate that C. brachyotis is a complex of lineages. C. sphinx and C. horsfieldi haplotypes formed monophyletic groups nested within the C. brachyotis species complex. We identified six divergent mitochondrial lineages that are currently referred to C. brachyotis. Lineages from India, Myanmar, Sulawesi, and the Philippines are geographically well-defined, while in Malaysia two lineages, designated Sunda and Forest, are broadly sympatric and may be ecologically distinct. Demographic analyses of the Sunda and Forest lineages suggest strikingly different population histories, including a recent and rapid range expansion in the Sunda lineage, possibly associated with changes in sea levels during the Pleistocene. The resolution of the taxonomic issues raised in this study awaits combined analysis of morphometric characters and molecular data. However, since both the Indian and Malaysian Forest C. brachyotis lineages are apparently ecologically restricted to increasingly fragmented forest habitat, we suggest that reevaluation of the conservation status of populations in these regions should be an immediate goal.

Matched MeSH terms: DNA, Mitochondrial/genetics*; Sequence Analysis, DNA
Variation in pollen dispersal between years with different pollination conditions in a tropical emergent tree

Kenta T, Isagi Y, Nakagawa M, Yamashita M, Nakashizuka T

Mol Ecol, 2004 Nov;13(11):3575-84.
PMID: 15488013

We examined differences in pollen dispersal efficiency between 2 years in terms of both spatial dispersal range and genetic relatedness of pollen in a tropical emergent tree, Dipterocarpus tempehes. The species was pollinated by the giant honeybee (Apis dorsata) in a year of intensive community-level mass-flowering or general flowering (1996), but by several species of moths in a year of less-intensive general flowering (1998). We carried out paternity analysis based on six DNA microsatellite markers on a total of 277 mature trees forming four spatially distinct subpopulations in a 70 ha area, and 147 and 188 2-year-old seedlings originating from seeds produced in 1996 and 1998 (cohorts 96 and 98, respectively). Outcrossing rates (0.93 and 0.96 for cohorts 96 and 98, respectively) did not differ between years. Mean dispersal distances (222 and 192 m) were not significantly different between the 2 years but marginally more biased to long distance in 1996. The mean relatedness among cross-pollinated seedlings sharing the same mothers in cohort 96 was lower than that in cohort 98. This can be attributed to the two facts that the proportion of intersubpopulations pollen flow among cross-pollination events was marginally higher in cohort 96 (44%) than in cohort 98 (33%), and that mature trees within the same subpopulations are genetically more related to each other than those between different subpopulations. We conclude that D. tempehes maintained effective pollen dispersal in terms of outcrossing rate and pollen dispersal distance in spite of the large difference in foraging characteristics between two types of pollinators. In terms of pollen relatedness, however, a slight difference was suggested between years in the level of biparental inbreeding.

Matched MeSH terms: Sequence Analysis, DNA; DNA, Plant/analysis
Fulltext Evaluation of Seegene Anyplex II Performance for Detection of Human Papillomavirus Genotypes in Formalin-Fixed, Paraffin-Embedded Cervical Cancer Specimens

Haqshenas G, Molano M, Phillips S, Balgovind P, Garland SM, Hawkes D, et al.

Arch Pathol Lab Med, 2024 Mar 01;148(3):353-358.
PMID: 37226838 DOI: 10.5858/arpa.2022-0317-OA

CONTEXT.—: Detection of human papillomavirus (HPV) in formalin-fixed, paraffin-embedded (FFPE) tissues may identify the cause of lesions and has value for the development of new diagnostic assays and epidemiologic studies. Seegene Anyplex II assays are widely used for HPV screening, but their performance using FFPE samples has not been fully explored.
OBJECTIVE.—: To validate Anyplex II HPV HR Detection (Anyplex II, Seegene) using FFPE samples.
DESIGN.—: We used 248 stored DNA extracts from cervical cancer FFPE samples collected during 2005-2015 that tested HPV positive using the RHA kit HPV SPF10-LiPA25, v1 (SPF10, Labo Biomedical Products) HPV genotyping assay, manufacturer-validated for FFPE samples.
RESULTS.—: Of the selected 248 samples, 243 were used in our analysis. Consistent with SPF10 genotyping results, Anyplex II detected all 12 oncogenic types and had an overall HPV detection rate of 86.4% (210 of 243 samples). Anyplex II and SPF10 showed very high agreement for the detection of the 2 most important oncogenic genotypes: HPV 16 (219 of 226; 96.9%; 95% CI, 93.7-98.75) and HPV 18 (221 of 226; 97.8%; 95% CI, 94.9-99.3).
CONCLUSIONS.—: Overall results showed that both platforms produced comparable HPV genotyping results, indicating the suitability of Anyplex II for FFPE samples. The Anyplex II assay has the added convenience of being an efficient, single-well semiquantitative polymerase chain reaction assay. Further optimization of Anyplex II may enhance its performance using FFPE samples by improving the detection limit.

Matched MeSH terms: DNA, Viral/analysis; DNA, Viral/genetics
Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells

Wong YC, Ng AWR, Chen Q, Liew PS, Lee CW, Sim EUH, et al.

ACS Synth Biol, 2023 Apr 21;12(4):909-921.
PMID: 37026178 DOI: 10.1021/acssynbio.2c00580

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.

Matched MeSH terms: DNA/metabolism; DNA Replication
On-Demand Ligand-Base DNA Sensor with Electrochemical Impedance Spectroscopy

Han H, Sabani NB, Nobusawa K, Takei F, Nakatani K, Yamashita I

Anal Chem, 2023 Jul 04;95(26):9729-9733.
PMID: 37341999 DOI: 10.1021/acs.analchem.3c01126

We have developed a DNA sensor that can be finalized to detect a specific target on demand. The electrode surface was modified with 2,7-diamino-1,8-naphthyridine (DANP), a small molecule with nanomolar affinity for the cytosine bulge structure. The electrode was immersed in a solution of synthetic probe-DNA that had a cytosine bulge structure at one end and a complementary sequence to the target DNA at the other end. The strong binding between the cytosine bulge and DANP anchored the probe DNAs to the electrode surface, and the electrode became ready for target DNA sensing. The complementary sequence portion of the probe DNA can be changed as requested, allowing for the detection of a wide variety of targets. Electrochemical impedance spectroscopy (EIS) with the modified electrode detected target DNAs with a high sensitivity. The charge transfer resistance (Rct) extracted from EIS showed a logarithmic relationship with the concentration of target DNA. The limit of detection (LoD) was less than 0.01 μM. By this method, highly sensitive DNA sensors for various target sequences could be easily produced.

Matched MeSH terms: DNA/chemistry; DNA Probes
Highly sensitive silicon nanowire biosensor with novel liquid gate control for detection of specific single-stranded DNA molecules

Adam T, Hashim U

Biosens Bioelectron, 2015 May 15;67:656-61.
PMID: 25453738 DOI: 10.1016/j.bios.2014.10.005

The study demonstrates the development of a liquid-based gate-control silicon nanowire biosensor for detection of specific single-stranded DNA (ssDNA) molecules. The sensor was fabricated using conventional photolithography coupled with an inductively coupled plasma dry etching process. Prior to the application of DNA to the device, its linear response to pH was confirmed by serial dilution from pH 2 to pH 14. Then, the sensor surface was silanized and directly aminated with (3-aminopropyl) triethoxysilane to create a molecular binding chemistry for biofunctionalization. The resulting Si‒O‒Si‒ components were functionalized with receptor ssDNA, which interacted with the targeted ssDNA to create a field across the silicon nanowire and increase the current. The sensor shows selectivity for the target ssDNA in a linear range from target ssDNA concentrations of 100 pM to 25 nM. With its excellent detection capabilities, this sensor platform is promising for detection of specific biomarkers and other targeted proteins.

Matched MeSH terms: DNA, Single-Stranded/isolation & purification*; DNA, Single-Stranded/chemistry
Evolutionary origin of Ceratonova shasta and phylogeny of the marine myxosporean lineage

Fiala I, Hlavničková M, Kodádková A, Freeman MA, Bartošová-Sojková P, Atkinson SD

Mol Phylogenet Evol, 2015 May;86:75-89.
PMID: 25797924 DOI: 10.1016/j.ympev.2015.03.004

In order to clarify the phylogenetic relationships among the main marine myxosporean clades including newly established Ceratonova clade and scrutinizing their evolutionary origins, we performed large-scale phylogenetic analysis of all myxosporean species from the marine myxosporean lineage based on three gene analyses and statistical topology tests. Furthermore, we obtained new molecular data for Ceratonova shasta, C. gasterostea, eight Ceratomyxa species and one Myxodavisia species. We described five new species: Ceratomyxa ayami n. sp., C. leatherjacketi n. sp., C. synaphobranchi n. sp., C. verudaensis n. sp. and Myxodavisia bulani n. sp.; two of these formed a new, basal Ceratomyxa subclade. We identified that the Ceratomyxa clade is basal to all other marine myxosporean lineages, and Kudoa with Enteromyxum are the most recently branching clades. Topologies were least stable at the nodes connecting the marine urinary clade, the marine gall bladder clade and the Ceratonova clade. Bayesian inference analysis of SSU rDNA and the statistical tree topology tests suggested that Ceratonova is closely related to the Enteromyxum and Kudoa clades, which represent a large group of histozoic species. A close relationship between Ceratomyxa and Ceratonova was not supported, despite their similar myxospore morphologies. Overall, the site of sporulation in the vertebrate host is a more accurate predictor of phylogenetic relationships than the morphology of the myxospore.

Matched MeSH terms: DNA, Ribosomal/genetics; Sequence Analysis, DNA
Fulltext Genetic structure and preliminary findings of cryptic diversity of the Malaysian Mahseer (Tor tambroides Valenciennes: Cyprinidae) inferred from mitochondrial DNA and microsatellite analyses

Esa Y, Abdul Rahim KA

Biomed Res Int, 2013;2013:170980.
PMID: 24455674 DOI: 10.1155/2013/170980

This study examines the population genetic structure of Tor tambroides, an important freshwater fish species in Malaysia, using fifteen polymorphic microsatellite loci and sequencing of 464 base pairs of the mitochondrial cytochrome c oxidase I (COI) gene. A total of 152 mahseer samples were collected from eight populations throughout the Malaysia river system. Microsatellites results found high levels of intrapopulation variations, but mitochondrial COI results found high levels of interpopulations differentiation. The possible reasons for their discrepancies might be the varying influence of genetic drift on each marker or the small sample sizes used in most of the populations. The Kelantan population showed very low levels of genetic variations using both mitochondrial and microsatellite analyses. Phylogenetic analysis of the COI gene found a unique haplotype (ER8∗), possibly representing a cryptic lineage of T. douronensis, from the Endau-Rompin population. Nevertheless, the inclusion of nuclear microsatellite analyses could not fully resolve the genetic identity of haplotype ER8∗ in the present study. Overall, the findings showed a serious need for more comprehensive and larger scale samplings, especially in remote river systems, in combination with molecular analyses using multiple markers, in order to discover more cryptic lineages or undescribed "genetic species" of mahseer.

Matched MeSH terms: DNA, Mitochondrial/genetics*; Sequence Analysis, DNA

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