Multi-frequency impedance sensing for detection and sizing of DNA fragments

AbstractElectronic biosensors for DNA detection typically utilize immobilized oligonucleotide probes on a signal transducer, which outputs an electronic signal when target molecules bind to probes. However, limitation in probe selectivity and variable levels of non-target material in complex biological samples can lead to nonspecific binding and reduced sensitivity. Here we introduce the integration of 2.8 μm paramagnetic beads with DNA fragments. We apply a custom-made microfluidic chip to detect DNA molecules bound to beads by measuring Impedance Peak Response (IPR) at multiple frequencies. Technical and analytical performance was evaluated using beads containing purified Polymerase Chain Reaction (PCR) products of different lengths (157, 300, 613 bp) with DNA concentration ranging from 0.039 amol to 7.8 fmol. Multi-frequency IPR correlated positively with DNA amounts and was used to calculate a DNA quantification score. The minimum DNA amount of a 300 bp fragment coupled on beads that could be robustly detected was 0.0039 fmol (1.54 fg or 4750 copies/bead). Additionally, our approach allowed distinguishing beads with similar molar concentration DNA fragments of different lengths. Using this impedance sensor, purified PCR products could be analyzed within ten minutes to determine DNA fragment length and quantity based on comparison to a known DNA standard.

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Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age

Genes ◽

10.3390/genes12020146 ◽

2021 ◽

Vol 12 (2) ◽

pp. 146

Author(s):

Catarina Xavier ◽

Mayra Eduardoff ◽

Barbara Bertoglio ◽

Christina Amory ◽

Cordula Berger ◽

...

Keyword(s):

Dna Extraction ◽

Ancient Dna ◽

Extraction Method ◽

Fragment Size ◽

Molecular Genetic ◽

Extraction Methods ◽

Degraded Dna ◽

Size Analysis ◽

Dna Fragments ◽

Dna Amount

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

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MESA: automated assessment of synthetic DNA fragments and simulation of DNA synthesis, storage, sequencing and PCR errors

Bioinformatics ◽

10.1093/bioinformatics/btaa140 ◽

2020 ◽

Vol 36 (11) ◽

pp. 3322-3326

Author(s):

Michael Schwarz ◽

Marius Welzel ◽

Tolganay Kabdullayeva ◽

Anke Becker ◽

Bernd Freisleben ◽

...

Keyword(s):

Dna Synthesis ◽

Web Application ◽

De Novo ◽

Supplementary Information ◽

Dna Fragments ◽

Synthetic Dna ◽

Custom Made ◽

Dna Fragment ◽

Polymerase Chain ◽

Error Probabilities

Abstract Summary The development of de novo DNA synthesis, polymerase chain reaction (PCR), DNA sequencing and molecular cloning gave researchers unprecedented control over DNA and DNA-mediated processes. To reduce the error probabilities of these techniques, DNA composition has to adhere to method-dependent restrictions. To comply with such restrictions, a synthetic DNA fragment is often adjusted manually or by using custom-made scripts. In this article, we present MESA (Mosla Error Simulator), a web application for the assessment of DNA fragments based on limitations of DNA synthesis, amplification, cloning, sequencing methods and biological restrictions of host organisms. Furthermore, MESA can be used to simulate errors during synthesis, PCR, storage and sequencing processes. Availability and implementation MESA is available at mesa.mosla.de, with the source code available at github.com/umr-ds/mesa_dna_sim. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

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A simple method for discriminating Bursaphelenchus xylophilus and B. mucronatus by species-specific polymerase chain reaction primer pairs

Nematology ◽

10.1163/1568541041217960 ◽

2004 ◽

Vol 6 (2) ◽

pp. 273-277 ◽

Cited By ~ 38

Author(s):

Koji Matsunaga ◽

Katsumi Togashi

Keyword(s):

Pcr Amplification ◽

Nematode Species ◽

Bursaphelenchus Xylophilus ◽

Polymerase Chain Reaction Primer ◽

Dna Fragments ◽

Simple Method ◽

Specific Pcr ◽

Pcr Products ◽

Specific Pcr Primer ◽

Species Specific

Abstract Two species-specific PCR primer pairs were developed for identifying the two nematode species, Bursaphelenchus xylophilus and B. mucronatus. The primer pairs were developed from the sequence of ribosomal DNA (rDNA) repeats to produce DNA fragments of different lengths by PCR amplification. The DNA fragments for B. mucronatus and B. xylophilus were 210 bp and 557 bp, respectively. When mixed, neither primer pair inhibited the PCR amplification of the other. Five isolates of B. xylophilus and four isolates of B. mucronatus showed different band profiles of PCR products between the two species, but identical profiles among isolates of the same species.

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Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E. coli and Lactobacillus

Plasmid ◽

10.1016/j.plasmid.2014.09.002 ◽

2014 ◽

Vol 76 ◽

pp. 40-46 ◽

Cited By ~ 12

Author(s):

Pinghua Cao ◽

Lei Wang ◽

Guangxian Zhou ◽

Yaoyue Wang ◽

Yulin Chen

Keyword(s):

Dna Fragments ◽

Direct Transformation ◽

E Coli ◽

Pcr Products

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A simple molecular method for rapid identification of commercially used Amblyseius and Neoseiulus species (Acari: Phytoseiidae)

Zootaxa ◽

10.11646/zootaxa.4394.2.9 ◽

2018 ◽

Vol 4394 (2) ◽

pp. 270

Author(s):

M.Y. SYROMYATNIKOV ◽

A.V. KOKINA ◽

N.A. BELYAKOVA ◽

E.G. KOZLOVA ◽

V.N. POPOV

Keyword(s):

Molecular Genetic ◽

Mite Species ◽

Rapid Identification ◽

Internal Transcribed Spacers ◽

Dna Fragments ◽

Neoseiulus Cucumeris ◽

Accurate Identification ◽

Pcr Rflp ◽

Pcr Products ◽

Its1 And Its2

Predatory mites from the Amblyseius (Neoseiulus) genus (Family Phytoseiidae, Order Parasitiformes) are widely used for protecting plants against pests, especially thrips. The differentiation of Amblyseius and Neoseiulus species by their morphological features is problematic despite the fact that they are taxonomically different genera. The Phytoseiidae family includes a lot of species that are extremely difficult to distinguish from each other. The discovery of new molecular genetic markers might considerably facilitate the express identification of commercial mite species. Despite their high morphological similarity, the three common commercial Amblyseius and Neoseiulus mite species (Neoseiulus cucumeris, Amblyseius swirskii, and Neoseiulus barkeri) differed significantly in the nucleotide sequences of DNA fragments containing the ITS1 and ITS2 internal transcribed spacers. We found that when PCR products of the amplified DNA fragments from A. swirskii, N. cucumeris, and N. barkeri were treated with a combination of AccB1I, AspLEI, and SspI endonucleases and the resulting products were separated by electrophoresis in agarose gel, the obtained picture was sufficiently specific to provide accurate identification of the analyzed mite species. The lengths of the digestion products were different enough to allow their resolution in agarose gels. The PCR-RFLP method developed by us allows the rapid and accurate identification of commercially used Amblyseius and Neoseiulus species without DNA sequencing. The combination of the three endonucleases (AccB1I, AspLEI, and SspI) and FaeI can be used for the differentiation of all other but less frequently commercially used Phytoseiidae mites.

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Development of a New Marker System for Identification ofSpirodela polyrhizaandLandoltia punctata

International Journal of Genomics ◽

10.1155/2017/5196763 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Bo Feng ◽

Yang Fang ◽

Zhibin Xu ◽

Chao Xiang ◽

Chunhong Zhou ◽

...

Keyword(s):

Capillary Electrophoresis ◽

Large Scale ◽

Pcr Amplification ◽

Identification System ◽

Dna Fragments ◽

Marker System ◽

Physiological Properties ◽

Pcr Products ◽

Plant Sciences ◽

Good For

Lemnaceae (commonly called duckweed) is an aquatic plant ideal for quantitative analysis in plant sciences. Several species of this family represent the smallest and fastest growing flowering plants. Different ecotypes of the same species vary in their biochemical and physiological properties. Thus, selecting of desirable ecotypes of a species is very important. Here, we developed a simple and rapid molecular identification system forSpirodela polyrhizaandLandoltia punctatabased on the sequence polymorphism. First, several pairs of primers were designed and three markers were selected as good for identification. After PCR amplification, DNA fragments (the combination of three PCR products) in different duckweeds were detected using capillary electrophoresis. The high-resolution capillary electrophoresis displayed high identity to the sequencing results. The combination of the PCR products containing several DNA fragments highly improved the identification frequency. These results indicate that this method is not only good for interspecies identification but also ideal for intraspecies distinguishing. Meanwhile, 11 haplotypes were found in both theS. polyrhizaandL. punctataecotypes. The results suggest that this marker system is useful for large-scale identification of duckweed and for the screening of desirable ecotypes to improve the diverse usage in duckweed utilization.

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