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pdj-france · 1 year ago

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L'identification des victimes après une catastrophe peut offrir une fermeture aux êtres chers. AP Photo/Jae C.HongLe feu dévaste les communautés et les familles et rend compliqué l'identification des victimes. À la suite de l'incendie de forêt qui a balayé Lahaina, à Hawaï, les autorités recueillent des échantillons d'ADN auprès de proches de personnes disparues dans l'espoir que cela puisse aider à identifier ceux qui sont morts dans l'incendie. Mais dans quelle mesure l'ADN résiste-t-il dans des conditions aussi extrêmes, et quelle est la meilleure façon de récupérer l'ADN des victimes d'incendie ? Je suis un généticien anthropologue qui étudie l'ADN dégradé dans des contextes archéologiques et médico-légaux. Mon groupe de recherche applique d'anciennes méthodes d'analyse de l'ADN et médico-légales pour optimiser la récupération de l'ADN à partir d'os brûlés. Récupérer l'ADN de restes gravement brûlés afin d'identifier les victimes est un défi particulier. Analyse ADN médico-légale Dans une enquête médico-légale typique, l'ADN est extrait d'un échantillon - qu'il s'agisse de sang, de morceaux de tissus ou d'os - prélevé sur les lieux de la catastrophe ou du crime. Ce processus sépare chimiquement l'ADN des autres matériaux des cellules de l'échantillon, comme les protéines, et le purifie. Cet ADN est utilisé comme matrice pour l'analyse par réaction en chaîne par polymérase, ou PCR, une méthode qui est essentiellement le copieur Xerox de la biologie moléculaire. Même s'il n'y a que quelques cellules présentes dans l'échantillon, la PCR peut amplifier ces molécules d'ADN en milliers ou en millions de copies. Cela crée une quantité suffisante d'ADN pour les tests ultérieurs. L'analyse de l'ADN peut aider à identifier les victimes en comparant les similitudes génétiques entre les personnes. En médecine légale, l'ADN précis ciblé dans la PCR est généralement un ensemble de marqueurs hautement répétitifs appelés microsatellites, ou courtes répétitions en tandem. Les organismes d'application de la loi du monde entier utilisent des ensembles spécifiques de ces marqueurs à des fins d'identification. Aux États-Unis, les analystes médico-légaux ciblent 20 de ces répétitions d'ADN. Chaque personne a deux allèles uniques, ou choix génétiques, à chacun de ces marqueurs, et ces allèles sont téléchargés dans la base de informations Combined DNA Index System du FBI pour identifier les correspondances. L'ADN prélevé sur les proches des personnes disparues sera certainement analysé pour les marqueurs de répétition en tandem courts et leurs profils alléliques téléchargés dans l'index des proches des personnes disparues dans la base de informations. On s'attend à ce que les victimes et leurs proches biologiques partagent un pourcentage d'allèles pour ces marqueurs. Par exemple, les parents et les enfants partagent 50 % de leurs allèles, puisqu'un enfant hérite de la moitié de son ADN de chaque parent. Défi de l'ADN dégradé Dans les contextes médico-légaux, le temps entre le décès et le prélèvement d'ADN est généralement suffisamment court pour que l'ADN soit souvent encore en assez bon état, tant en termes de quantité que de qualité. Toutefois, l'ADN n'est souvent pas retrouvé dans des conditions idéales après une catastrophe. Le temps et les éléments font des ravages. Après le décès, le processus de décomposition libère des enzymes qui peuvent cliver ou endommager l'ADN, et des dommages supplémentaires se produisent au fil du temps en fonction de l'environnement dans lequel se trouve le corps. L'ADN se dégrade aussi plus rapidement dans des environnements chauds, humides et acides et plus lentement dans des environnements plus froids et plus secs qui ont un pH plus neutre ou légèrement basique. De plus, la conservation de l'ADN peut varier considérablement ont rapporté les tissus, les os et les dents récupérés. Par exemple, les chercheurs ont découvert que l'identification

par ADN des victimes des attentats du World Trade Center en 2001 était plus efficace en utilisant les os des pieds et des jambes, par rapport aux os de la tête et du torse. Les dommages à l'ADN peuvent prendre différentes formes. Les entailles et les ruptures dans l'ADN rendent l'analyse compliqué. La modification chimique de l'ADN peut entraîner des modifications de la séquence d'origine ou la rendre illisible. Cela inclut les modifications apportées aux éléments constitutifs de l'ADN appelés nucléotides qui constituent une séquence identifiable. Par exemple, l'exposition à l'eau peut provoquer une réaction chimique appelée désamination qui modifie le nucléotide cytosine de sorte qu'il semble être le nucléotide thymine durant l'analyse. Les expositions à d'autres produits chimiques ou à la lumière UV peuvent provoquer une réticulation, qui lie essentiellement l'ADN en nœuds. En conséquence, les enzymes PCR utilisées pour copier ou lire la séquence d'ADN ne peuvent pas se déplacer linéairement le long du brin d'ADN. L'exposition à des incendies intenses et prolongés peut rendre compliqué l'identification des victimes par l'analyse de l'ADN. AP Photo/Jae C.Hong Application des techniques de l'archéologie Les chercheurs rencontrent des problèmes similaires durant la manipulation de matériel génétique dégradé durant l'analyse de l'ADN de restes anciens vieux de milliers d'années. Pour relever ces défis, les généticiens médico-légaux et les chercheurs en ADN ancien comme moi emploient un certain nombre d'astuces pour optimiser la récupération de l'ADN. En premier lieu, nous avons tendance à cibler les os ou les dents denses pour l'échantillonnage, car ils sont plus imperméables à l'environnement. Nous utilisons aussi des techniques d'extraction d'ADN qui améliorent la récupération de courts fragments d'ADN. Deuxièmement, nous utilisons la PCR pour amplifier des marqueurs génétiques davantage courts, y compris des répétitions en tandem mini-courtes ou des sections du génome mitochondrial. Les mitochondries sont des structures au sein de chaque cellule qui produisent de l'énergie, et chacune a son propre ADN. L'ADN mitochondrial est transmis de la mère à l'enfant et peut être récupéré en centaines d'exemplaires dans chaque mitochondrie, ce qui facilite sa récupération et son analyse. Toutefois, l'ADN mitochondrial peut ne pas fournir suffisamment d'informations pour l'identification, car les personnes qui sont maternellement apparentées, même très éloignées, partageront la même séquence. Les chercheurs testent aussi de nouvelles méthodes d'analyse de l'ADN courantes dans le secteur de l'ADN ancien à des fins médico-légales. Par exemple, des enzymes spéciales peuvent éliminer les nucléotides chimiquement modifiés, tels que les cytosines désaminées, pour éviter une lecture erronée de la séquence d'ADN. Les chercheurs peuvent aussi employer des appâts ADN pour « pêcher » des séquences spécifiques. Cette méthode d'enrichissement ciblé permet de récupérer de très petits fragments qui peuvent être utilisés pour reconstituer la séquence génétique complète. Analyse ADN des restes brûlés Pour les victimes d'incendies, en particulier celles prises dans des incendies intenses et prolongés, l'ADN peut être très fragmenté, ce qui rend l'analyse compliqué. Les températures élevées provoquent la rupture des liaisons entre les molécules, y compris les nucléotides. Il en résulte une fragmentation et finalement une destruction de l'ADN. Car les tissus durs – les os et les dents – sont souvent tout ce qui reste après un incendie, les chercheurs médico-légaux ont étudié comment les caractéristiques des os telles que la couleur et la composition changent avec la température. Mon équipe de recherche a utilisé ces informations pour classer le niveau de brûlure auquel des échantillons d'os humains ont été soumis. En enquêtant sur la préservation de l'ADN dans ces

échantillons, nous avons découvert qu'il y a plusieurs un point important de dégradation de l'ADN quand les os atteignent des températures comprises entre 662 degrés Fahrenheit (350 degrés Celsius) et 1 022 F (550 C). À titre de comparaison, la crémation commerciale est de 1 400 à 1 600 F (760 à 871 C) pendant 30 à 120 minutes, et les incendies de véhicules atteignent généralement 1 652 degrés F (900 C) mais peuvent durer moins longtemps. Les survivants des incendies de forêt de Lahaina, qui ont débuté le 8 août 2023, traversent les conséquences. Photo AP/Rick Bowmer Notre équipe a aussi constaté que la probabilité de générer des informations répétées en tandem courtes de haute qualité ou des informations de séquence d'ADN mitochondrial, que ce soit en utilisant des techniques médico-légales ou d'ADN anciennes, diminue considérablement à des températures supérieures à 1 022 F (550 C). En somme, à mesure que la température et le temps d'exposition augmentent, la quantité d'ADN restant diminue. Cela ne conduit qu'à des profils ADN partiels, ce qui a la capacité de limiter la capacité des analystes à faire correspondre une victime à un parent avec une certitude statistique élevée ou à empêcher complètement les résultats. La preuve ADN n'est pas la seule méthode utilisée pour l'identification. Les enquêteurs combinent l'ADN avec d'autres preuves - telles que des informations dentaires, squelettiques et contextuelles - pour identifier une victime de manière concluante. Ensemble, ces informations aideront, espérons-le, à tourner la page pour les familles et les amis. Anne Stone reçoit un financement du National Institute of Justice. Source

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peertechz · 2 years ago

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Missing Persons Identification: Genetic profiling of highly charred human remains using sixteen STR loci markers

Law enforcement and Disaster Management Agencies within Governments spend substantial resources to identify human remains and dead bodies after wars, mass disasters and political unrest [1-4]. Under certain legal circumstances such as civil paternity cases and unclaimed human cadavers, biological samples may be collected from living relatives and exhumed human remains, as well as mortal remains for DNA profiling purposes [5,6]

In present-day society where socio-cultural dynamics and economic factors encourage migration in search of better living standards, relatives are always looking for missing family members.

In all these cases, DNA analysis is considered the best method of identification. Microsatellite DNA, namely Short Tandem Repeats (STRs) loci show high variability. Degraded DNA from old skeletal remains can be amplified by PCR [7].

Biological materials that are of importance in identifying exhumed remains using DNA are bone, teeth and nail due to their ability to withstand rapid decomposition. According to Vass, et al. [8] tissues such as nails, teeth and bones are more resilient to environmental factors. DNA analysis from skeletal samples has developed rapidly since its inception in the late 1980s.

The two main objectives of forensic DNA analysis are to identify the sources of biological evidence, including associations of persons through kinship, and excluding persons mistakenly linked to some evidence.

The most widely used genetic markers for forensic DNA typing in most crime laboratories are autosomal Short Tandem Repeat (STR) loci [4]. Commercially available STR kits, such as the SGM plus PCR amplification Kit, AmpFℓSTR Identifiler PCR amplification kit (Applied Biosystems, Foster City, California) or the PowerPlex 16 system (Promega, Madison, Wisconsin) make use of a set of 10–17 STR loci to provide a high level of diversity and resolution for identity testing [9]. These kits, and STR loci, have been used widely for the identification of human remains as well as in relationship testing, such as paternity testing and family reconstructions.

We report on how a highly charred human remains were genetically identified after the victim, a female, was reported missing in 2016. Intelligence gathered by Police fourteen months later resulted in the discovery of burnt and buried skeletal remains at a location about 42Km away from her home. Crime scene investigators sampled bones for forensic DNA Analysis since there was no soft tissue. Close relatives of the missing female (alleged son, and alleged mother) were invited for buccal swab sampling for comparative DNA analysis to determine the identity of the human remains.

https://www.peertechzpublications.com/articles/FST-6-117.php

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cytgen · 2 years ago

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Abstract The high-yielding Cabernet Sauvignon grape variety is susceptible to viral diseases, which may influence the agrobiological and taste characteristics of its quality. The objectives of this study are to identify a Cabernet Sauvignon variety of clonal origin from the south of Ukraine, detect the infection of plants of this variety with harmful grape viruses incorporated into the certification system of planting materials, determine the causative agent of viral diseases by biomolecular methods, and establish the nucleotide sequence of the 2CCP envelope protein gene of detected grape viruses. As a result of phytosanitary survey, some Cabernet Sauvignon grape bushes of clonal origin with symptoms of grapevine fanleaf virus (CFLV) and grapevine leaf roll-associated virus (GLRaV) have been revealed. The results of grape virus identification by the real-time reverse-transcription polymerase chain reaction (RT-PCR) have demonstrated the presence of grapevine fanleaf virus in grape plants with infection symptoms. As a result of sequencing, it has been established that the nucleotide sequence of an isolate from the Cabernet Sauvignon variety is very close to the samples from regions geographically distant from Ukraine, first of all, the United States, Iran, and France. Based on microsatellite analysis, it has been proven that specifically the Cabernet Sauvignon variety of clonal origin is infected with grapevine fanleaf virus. The obtained 2CCP envelope protein gene sequence has been deposited with the international GenBank database with no. MN072356.1.

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bisrsrch · 4 years ago

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The nanosatellite and microsatellite industry analysis by BIS Research projects the market to have a significant growth of CAGR 24.86% based on the values during the forecast period from 2020 to 2026. North America is expected to dominate the global nanosatellite and microsatellite market with an estimated share of 54.46% in 2019. North America, including the major countries such as the U.S., is the most prominent region for the nanosatellite and microsatellite market. The presence of major players and intense competition among them makes North America the most technologically advanced region. The companies in the region secure contracts from end users such as defense, commercial, and government agencies for manufacturing of their satellites.

For Sample Report, Click here: https://bisresearch.com/requestsample?id=1027&type=download

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perfectlytinyworkspace · 4 years ago

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DNA polymorphisms

DNA polymorphism (DNAPM) – segment of genomic DNA that differs from others in an individual

DNAPMs can be single nucleotide variations, small insertions or deletions, duplications / number of duplications in or of a segment

most are anonymous DNA polymorphisms which are not phenotypically reflected in any way

5000 out of millions of DNAPMs actually alter amino acid sequences in humans

context: less than 2% of human gDNA makes expressed codons out of 3E9 bp total haploid h-genome | 5% are coding sequences

also, codons can be spliced out and are degenerate anyway - so majority of human genome is non-expressed

still, non-expressed anonymous DNAPMs are experimentally useful – serve as markers for locating linked, co-inherited disease mutations

types of DNA polymorphisms

1. single nucleotide polymorphism (SNP) – difference in one sole base pair between individuals and populations

in a bi-allelic SNP, changed allele is the derived allele and unchanged allele is the ancestral allele | most SNPs thus show low heterozygosity

SNPs occur with low frequency and thus can reflect relative timing of a SNP group’s divergence from a common ancestor species or ancestral population

when using SNPs to order ancestral diversion events, careful of reversion derived mutation that seems to indicate a doubly-derived individual or population’s closer relation to the ancestral group

RFLPs are SNPs that specifically affect particular RE recognition sites, altering recog. sequence and losing the cut spot or creating new recog. sequence

2. deletion-insertion polymorphism (DIP) – difference in number of base pairs present

less frequent due to tendency to more heavily alter coded information in DNA sequence

often 1-2 bp only, resulting from DNA replication or recombination mistakes or breaks in DNA

in promoter and coding regions, DIPs cause frameshift mutations unless in multiples of 3 for trinucleotidic codon-based reading frames

3. simple sequence repeat (SSR) – aka microsatellite, difference number of tandem duplicates of a basic repeated sequence (≤ 10 bp)

SSR occurrence = most often silent and in non-coding areas

SSRs in coding regions have dire consequences on phenotype and are generally lethal or severely debilitating

initial creation of repeated sequence is infrequent, but once created the SSR causes DNAP to slip and generate more repeats during replicative synthesis → so, many SSRs = highly polymorphic on an individual or population level

stability + high polymorphism rate (high heterozygosity) = good marker for linked disease alleles (i.e., disease allele is linked to n repeat number at SSR | non-disease allele linked to n+a repeat number)

4. copy number variant (CNV) – difference in number of tandem duplicates of a larger repeated sequence (≥ 10 bp)

mostly stable and inherited rather than newly mutated in an individual | serves as a good pedigree marker for a linked disease allele in a family

CNVs increase possibility of unequal crossing over between homologs that results in larger or smaller number of repeats

genotype anonymous DNA polymorphisms to:

1. map regions in the physical genome and locate linked disease gene loci

» requirements for assessed individuals

different phenotypes in a single relevant gene

heterozygous individuals to assess recombination in

many individuals to test for recombination frequencies

» requirements for isolated DNAPM marker

1 DNAPM occurs every 1kb » can calculate number of DNAPMs available for mapping along gene via number of possible DNAPMs for mapping = gene length • 1 DNAPM / 1000 bp

must be unique and detectable in individual’s genome to work as a marker, as indicated by name

direct detection: if DNAPM is the disease-causing mutation, then DNAPM shows perfect linkage to disease gene

indirect detection: if DNAPM is not the disease-causing mutation but only closely linked to it, then DNAPM-disease allele recombination must be taken into account when assessing individual genotypes

» determining marker SNP original and variant allele sequences

use restriction fragment length polymorphisms (RFLPs)

RFLPs are relatively rare, but very detectable via probe bind, RE cut, and Southern Blot

added or removed site results in differing sizes of fragments hybridized to the probe

location of probe can indicate varied RE site location + reasons for why fragment lengths are or are not visualized

use allele-specific oligonucleotides (ASOs) in a DNA microarray

attach an ASO – oligonucleotide strand with specific SNP allele – to a fluorescence-detecting chip

fragment genomic DNA into probe-like pieces, amplify, and place on replica nylon membranes for each SNP allele

hybridize gDNA to ASO in high-stringency conditions on each membrane and measure degree of fluorescence to determine whether specific SNP allele is present

use single nucleotide primer extensions with specific primers and ddNTPs

less time-consuming than ASOs, so SNPE is a high-throughput technique, many samples at fast pace

sequence a primer for segment preceding location of SNP, so that primer ends on the base pair immediately before the SNP base

mix with sample in four different wells/tubes and add a different non 3′OH ddNTP to each so that primer is terminated after 1 base addition

if ddNTP complements allelic base at the SNP locus, the sample is visualized to be 1bp longer under gel electrophoresis

» detection of DNAPMs and associated disease alleles take place across Southern Blotting of family pedigrees

determine the following before labelling which DNAPM fragments are associated with mutant or WT disease alleles:

disease gene dominance, DNAPM linkage to disease gene, and individuals’ disease genotypes

more informative markers determine P(diseased), less informative markers assess P(non-diseased)

disease probabilities derived from less tightly-linked DNAPM markers must take linkage RFs into account

2. identify individuals for forensic purposes

» use SSR-allele combinations to identify specific individuals by their DNA fingerprint

probability of unique SSR repeat-number combination at multiple SSR loci = extremely small, so individual’s multi-SSR (13) genotype ≈ a DNA fingerprint

to determine individual’s multi-SSR (13) genotype, use PCR amplification of DNA sample with each SSR primer differentially fluorescent-labeled

using gel electrophoresis, assess fragment sizes for allele present at each color-distinct locus

» moral question: family members’ DNA can be utilized to identify criminals forensically, but the strategy places relatives under constant surveillance

#genetics #notes

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pinerstamp · 2 years ago

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J Ind Microbiol Biotechnol 21:99–114īogdanowicz SM, Mastro VC, Prasher DC, Harrison RG (1997) Microsatellite DNA variation among Asian and north American gypsy moths (Lepidoptera: Lymantriidae). Bull Entomol Res 82:151–159īlears MJ, De Grandis SA, Lee H, Trevors JT (1998) Amplified fragment length polymorphism (AFLP): a review of the procedure and its application. Insect Mol Biol 2:1–6īlack WC IV, DuTeau NM, Puterka GJ, Nechols JR, Pettorini JM (1992) Use of the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) to detect DNA polymorphisms in aphids (Homoptera: Aphididae). Insect Mol Biol 10(2):163–171īlack WC (1993) PCR with arbitrary primers: approach with care. Genetics 153:333–338īehura SK, Sahu SC, Mohan M, Nair S (2001) Wolbachia in the Asian rice gall midge, Orseolia oryzae (wood-Mason): correlation between host Mito types and infection status. Can J For Res 36:337–350īeeman RW, Brown SJ (1999) RAPD based genetic linkage maps of Tribolium castaneum. Syst Biol 54:689–693īall SL, Armstrong KF (2006) DNA barcodes for insect pest identification: a test case with tussock moths (Lepidoptera: Lymantriidae). PLoS One 12(3):e0174749īalakrishnan R (2005) Species concepts, species boundaries and species identification: a view from the tropics. Nature 393:784–786Īshfaq M, Akhtar S, Rafi MA, Mansoor S, Hebert PD (2017) Mapping global biodiversity connections with DNA barcodes: Lepidoptera of Pakistan. Mol Biochem Parasitol 61:15–24Īrnqvist G (1998) Comparative evidence for the evolution of genitalia by sexual selection. Appl Entomol Zool 49(1):159–169Īrnot DE, Rooer C, Bavoumi RAL (1993) Digital codes from hyper variable tandemly repeated DNA sequences in the Plasmodium falciparum circumsporozoite gene can genetically barcode isolates. Genetics 143:1727–1738Īrimoto M, Iwaizumi R (2013) Identification of Japanese Lymantria species (Lepidoptera: Lymantriidae) based on PCR–RFLP analysis of mitochondrial DNA. Antolin MF, Bosio CF, Cotton J, Sweeney W, Strand MR, Black WC IV (1996) Intensive linkage mapping in a wasp ( Bracon hebetor) and a mosquito ( Aedes aegypti) with single-strand conformation polymorphism: analysis of random amplified polymorphic DNA markers.

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evoldir · 2 years ago

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Fwd: Job: TexasAMU.ResAssist.ArthropodPopGenomics

Begin forwarded message: > From: [email protected] > Subject: Job: TexasAMU.ResAssist.ArthropodPopGenomics > Date: 3 August 2022 at 07:00:14 BST > To: [email protected] > > > The Vargo lab at Texas A&M University is recruiting a Research Assistant > to help with research on the population genetics and population genomics > of arthropod pests of the urban environment, including termites, ants, > cockroaches and bed bugs. The research will involve both laboratory > and field work with an emphasis on the application of molecular genetic > markers in basic and applied research in urban entomology, including DNA > and RNA extraction, PCR, microsatellite and SNP genotyping, sequencing > using Sanger and NGS platforms, and assisting in RNA seq analysis. The > successful candidate will also assist in preparing summaries of results > for reports and scientific publications, troubleshooting and modifying > molecular genetic methods as necessary to accomplish project objectives, > and organizing and managing large numbers of samples for DNA and RNA > analysis. > > Applicants should have a Bachelor's degree in Entomology, Biology or > closely related field. Knowledge and experience in basic techniques > of molecular biology, including both laboratory and analytical > skills. Preferred candidates will have an M.S. degree in Biology or > closely related field, or B.S. degree and 2 years of experience working in > a molecular biology laboratory; experience running microsatellite markers; > experience in DNA sequencing, including sequence editing and basic > phylogenetic analysis; RNA extraction and qPCR; experience working with > social insects or urban pest insects in both the field and laboratory; > experience preparing reports, writing technical publications and giving > oral presentations of research results. Working knowledge of R will be > an advantage. We are looking for candidates who are self-motivated and > who work well with others. > > The Vargo lab in the Department of Entomology at Texas A&M University > (https://ift.tt/Ti5bJkQ) focuses on basic and applied research > on urban insect pests. The group currently consists of 9 personnel > (3 Ph.D. students, two postdoc, two technicians and an administrative > assistant) and works closely with an extension entomologist. Current > projects include the invasion biology of ants and termites, immune > defenses of termites, manipulation of host behavior by parasites in ants, > and development of RNAi approaches for ant management. > > Texas A&M University is one of the largest universities in the country, > with more than 65,000 students. The metropolitan area of College > Station/Bryan has nearly 180,000 residents, and is consistently ranked > among the best places to live in the country, with an affordable cost > of living and easy access to major Texas cities, including Austin and > Houston. > > Texas A&M University is an Equal Opportunity/Affirmative > Action/Veterans/Disability Employer. > > To apply for this position, please visit: > https://ift.tt/oVvSNKc > > > For more information, please contact Ed Vargo ([email protected]). If > you need assistance in applying for this position please contact (979) > 845-2423 > > > "Edward L. Vargo"

#IFTTT #Email

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healthcare-market · 3 years ago

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In-vitro Transcription Templates Market to Flourish with an Impressive CAGR by 2030

In-Vitro Transcription Templates Market: Introduction

According to the report, the global in-vitro transcription templates market was valued at ~US$ 120 Mn in 2020 and is projected to expand at a CAGR of ~20% from 2021 to 2030. Increase in R&D funding in healthcare and biotechnology and rise in technological advancements in molecular biology are anticipated to drive the global in-vitro transcription templates market during the forecast period. Additionally, rise in prevalence of various types of cancer and infectious diseases, such as COVID-19, is expected to propel the global in-vitro transcription templates market over the next few years. Investments by key players to strengthen their position is likely to create significant opportunities in the market. For instance, in June 2020, Promega Corporation announced CE marking for the OncoMate MSI Dx Analysis System as a new in-vitro diagnostic (IVD) medical device in Europe. OncoMate MSI is a PCR-based, validated gold standard for determining microsatellite instability (MSI) status in solid tumors.

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The usage of mRNA-based personalized cancer vaccines for the treatment of cancer has increased. For instance, Moderna’s Immuno-Oncology focuses on therapeutic vaccines and intratumoral immuno-oncology therapeutics. Moderna is able to make modified, mRNA-based personalized cancer vaccines to distribute one custom-tailored medicine for one patient at a time, which is concluded through next-generation sequencing and able to recognize mutations found on a patient’s cancer cells. Hence, increase in incidence of cancer boosts usage of in-vitro transcription templates in RNA-derived vaccines and therapeutics.

North America dominated the global in-vitro transcription templates market in 2020. The trend is likely to continue during the forecast period. Well-established healthcare and life science industries, early adoption of technologically advanced products, high awareness about various infectious as well as chronic diseases, and high per capita healthcare expenditure are the major factors attributed to North America’s large market share in 2020.

Asia Pacific is projected to be a highly lucrative market for in-vitro transcription templates over the next few years. The market in the region is anticipated to expand at a high CAGR during the forecast period. The growth of the healthcare sector and the increase in the development of RNA-based vaccines and therapies in countries such as Japan, India, and China are expected to propel the market in the region during the forecast period.

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Technological Advancements to Drive In-Vitro Transcription Templates Market

The adoption of technologically advanced products is likely to drive the demand, and thereby the global market. Technological advances and modalities for targeting RNA include using CRISPR-Cas9 genome editing technology, DNA-directed RNA intervention (ddRNAi) technology, and the advancement of specific low molecular modulators for RNA or RNA-modifying enzymes. For instance, CAL-1, Calimmune's leading therapeutic agent, depicts RNA-based gene therapy using ddRNAi to suppress the CCR5 gene to regulate HIV infection and to prevent HIV-positive entities from developing AIDS. Several firms focused on the production of small-molecular RNA modulators have been set up over the past few years.

Targeting splice-variant control sequences within introns (non-coding regions of an RNA transcriptor DNA sequence within a gene) or exons (coding regions) offers opportunities to develop therapeutics. For instance, Skyhawk Therapeutics, Inc. (Waltham, Massachusetts, the U.S.) was founded with a platform to identify selective small molecule modulators of the RNA spliceosome complex that target RNA mis-splicing (exon skipping), which drives multiple diseases including neurological conditions and cancer. These emerging technologies offer significant opportunities to develop alternative strategies to target RNA for drug development.

N4 Pharma is developing Nuvec, an innovative silica nanoparticle for drug delivery with possible applications across cancer therapy and immunology. That includes enhancing the cellular uptake of novel and disruptive medicines such as mRNA and DNA vaccines or therapies.

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Infectious Diseases to Dominate In-Vitro Transcription Templates Market

In terms of disease, the global in-vitro transcription templates market has been classified into cancer, infectious diseases, lifestyle diseases, genetic diseases, and others. The infectious disease segment accounted for major share of the global market in 2020. The segment is projected to dominate the global market during the forecast period. mRNA vaccine has been studied for various diseases including CMV, Zika, and rabies. Development and launch of RNA-based vaccines are anticipated to propel the segment during the forecast period.

Vaccine to Hold Major Share of In-Vitro Transcription Templates Market

Based on treatment, the global in-vitro transcription templates market has been categorized into vaccine and therapeutic. The vaccine segment accounted for major share of the global market in 2020. For instance, the U.S. FDA issued an emergency use authorization (EUA) for Moderna’s COVID-19 vaccine for the prevention of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Clinical to Dominate In-Vitro Transcription Templates Market

Based on research stage, the global in-vitro transcription templates market has been bifurcated into exploratory and clinical. A number of RNA-based vaccines and therapies is in the pipeline and clinical stage. This is likely to augment the clinical segment over the next few years.

Pharmaceutical & Biotechnology Companies to Account for Major Share of In-Vitro Transcription Templates Market

In terms of end user, the global vitro transcription templates market has been divided into pharmaceutical & biotechnology companies, CROs & CMOs, academics & research, and others. The need of discovery of new therapeutics, vaccines, and capacity expansion leads to high adoption of in-vitro transcription templates among pharmaceutical & biotechnology manufacturers. This is projected to drive the segment during the forecast period.

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North America to Dominate In-Vitro Transcription Templates Market

In terms of region, the global in-vitro transcription templates market has been segmented into North America, Europe, Asia Pacific, Latin America, and Middle East & Africa. North America dominated the global in-vitro transcription templates market in 2020, followed by Europe. North America accounted for major share of the global in-vitro transcription templates market in 2020. The growth of the market in the region is can be attributed to increase in demand for biopharmaceuticals such as vaccines and RNA-based therapeutics, peptides for the treatment of cancer, neurological diseases, and chronic kidney diseases. Moreover, rise in prevalence of lifestyle diseases, increase in healthcare spending, and strong economy are factors responsible for North America’s dominance of the global in-vitro transcription templates market during the forecast period.

The in-vitro transcription templates market in Asia Pacific is anticipated to grow at a rapid pace during the forecast period. Increase in disposable income and purchasing power of consumers, rise in biotechnology, research institutes, and research funding by government and private bodies, expansion of healthcare infrastructure, large population base, and rise in incidence of chronic and infectious diseases are the key factors expected to augment the in-vitro transcription templates market in Asia Pacific during the forecast period.

Competition Landscape of In-Vitro Transcription Templates Market

The global in-vitro transcription templates market is fragmented in terms of number of players. Key players in the global in-vitro transcription templates market include Thermo Fisher Scientific, Inc., Promega Corporation, Agilent Technologies, Inc., New England Biolabs, Takara Bio Inc., Lucigen Corporation, Enzynomics Co. Ltd., Enzo Life Sciences, Inc. and Cytiva (Danaher)

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sciencespies · 3 years ago

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New breakthrough in research into old insect exoskeletons

https://sciencespies.com/nature/new-breakthrough-in-research-into-old-insect-exoskeletons/

New breakthrough in research into old insect exoskeletons

Research into insect species can benefit from genetic studies. However, genetic samples can often be difficult to collect in a non-invasive manner, especially when the insects are only found in a particular location or are endangered.

Some insects, such as cicadas, shed their outer hard “exoskeleton” as part of their normal growth in a process known as molting. The structures that are left behind are called exuviae. Cicada exuviae are left on tree trunks and are easy to collect. Exuviae have been used for some genetic research in the past on mitochondria, small and ubiquitous parts of the cell, but no previous studies have been able to isolate and sequence nuclear DNA from exuviae.

When only a very small DNA sample is available, the size of the sample can be increased, or “amplified,” by a process known as PCR, or polymerase chain reaction. PCR can make millions of copies of a small sample of DNA to give a large enough sample for detailed study. Now, a team from the University of Tsukuba have tested five different PCR methods to amplify the DNA isolated from exuviae for sequencing.

The team focused on sequencing microsatellites, regions of the genome where a particular DNA pattern is repeated many times. These regions are promising targets because they can be amplified from just a small amount of DNA, and the process is cost efficient. Microsatellites also show a great deal of variation and are already used in molecular ecology studies as effective genetic markers.

First, the team isolated DNA from cicada exuviae and amplified the samples using the different PCR methods. They then compared the results with those taken from adult insects, to check the quality of the samples produced. The best results were seen from the PCR reaction that used an enzyme called TaKaRa LA Taq polymerase, which resulted in a DNA sample comparable to the sample isolated from adult insects. The best results were also seen from fresh exuviae.

“Our work shows that DNA that has been isolated from cicada exuviae can be amplified by PCR, and that cicada exuviae give samples of good enough quality to allow multiple independent nuclear DNA microsatellite marker loci to be genotyped. This approach will be very useful to evaluate population genetic structure and demography of forest insect species in relation to conservation and the ecosystem under ongoing climate change,” explains senior author Yoshiaki Tsuda.

This study makes a significant contribution to insect sciences because the methods here are applicable not only to cicadas but also to any other insect species that molt to leave exuviae.

This work was supported by the Nagano Prefecture fund for promoting scientific activity and the Japan Society for the Promotion of Science KAKENHI (grant number 15K18603).

Story Source:

Materials provided by University of Tsukuba. Note: Content may be edited for style and length.

#Nature

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bloghealthcom · 3 years ago

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Short Tandem Repeats và các ứng dụng Update 08/2021

Bài viết Short Tandem Repeats và các ứng dụng Update 08/2021 được chia sẻ bởi website Blog-Health #bloghealth #suckhoe #lamdep #sinhly

Bài viết của Kỹ thuật viên Nguyễn Văn Hưng - Khối Di truyền y học, Trung tâm Công nghệ cao Vinmec

Short Tandem Repeats (STRs) là các trình tự DNA lặp lại ngắn (2- 6 nucleotides) xuất hiện phổ biến trong hệ gen của con người. Các trình tự này có tính đa hình rất cao trong tự nhiên, điều này khiến các STRs trở thành những markers di truyền rất quan trọng trong nghiên cứu bản đồ gen người và chuẩn đoán bệnh lý di truyền cũng như xác định danh tính trong lĩnh vực pháp y.

Các STRs trở nên phổ biến tại các phòng xét nghiệm pháp y bởi vì việc nhân bản và phân tích STRs chỉ cần lượng DNA rất thấp ngay cả khi ở dạng bị phân hủy việc đinh danh vẫn có thể được thực hiện thành công. Hơn nữa việc phát hiện và đánh giá sự nhiễm DNA mẫu trong các mẫu vật có thể được giải quyết nhanh với kết quả phân tích STRs. Ở Hoa Kỳ hiện nay, từ bộ 13 markers nay đã tăng lên 20 markers chính đang được sử dụng để tạo ra một cơ sở dữ liệu DNA trên toàn đất nước được gọi là The FBI Combined DNA Index System (Expaned CODIS).

CODIS và các cơ sử dữ liệu DNA tương tự đang được sử dụng thực sự thành công trong việc liên kết các hồ sơ DNA từ các tội phạm và các bằng chứng hiện trường vụ án. Kết quả định danh STRs cũng được sử dụng để hỗ trợ hàng trăm nghìn trường hợp xét nghiệm huyết thống cha con mỗi năm (1).

Các STRs trở nên phổ biến tại các phòng xét nghiệm pháp y

Với lợi ích và ứng dụng to lớn của kỹ thuật phân tích các chỉ thị STRs trên toàn thế giới vào lĩnh vực Y học, pháp y. Hiện nay, tại khối Di truyền Y học – Bệnh viện Đa khoa Quốc tế Vinmec Times City đã áp dụng kỹ thuật này vào trong một số dịch vụ xét nghiệm chẩn đoán bệnh lý di truyền để đáp ứng được các nhu cầu thực tiễn của bên lâm sàng nhằm tạo đưa ra được những kết quả có độ tin cậy và chính xác cao nhất. Một số xét nghiệm đang áp dụng kỹ thuật phân tích STRs có thể kể đến như:

Xét nghiệm chẩn đoán các bất thường lệch bội nhiễm sắc thể 13, 18 ,21 và nhiễm sắc thể giới tính (QF - PCR)

Xét nghiệm sử dụng bộ 26 STRs bao gồm cả những chỉ thỉ STR đa hình và không đa hình. Ngoài việc giúp tăng độ chính xác trong việc chẩn đoán các bất thường lệch bội nhiễm sắc thể liên quan, với lượng STRs đa hình cao xét nghiệm giúp đánh giá rất tốt sự ngoại nhiễm các nguồn DNA bên ngoài có trong mẫu bệnh phẩm được chuẩn đoán.

Xét nghiệm chẩn đoán phôi tiền làm tổ (PGT-M)

Hiện nay, xét nghiệm chẩn đoán phôi tiền làm tổ PGT-M đang được thực hiện tại Vinmec giúp chẩn đoán phôi IVF đối với 2 bệnh di truyền thường gặp là alpha thalassemia và beta thalassemia. Xét nghiệm sử dụng các STRs được liên kết với các gen Alpha và gen Beta Globin. Sự chẩn đoán tình trạng các phôi thông qua phân tích di truyền liên kết của các STRs và các gen liên quan theo sơ đồ phả hệ. Kết quả đã giúp cho các cặp vợ chồng mang những gen bệnh thalassemia có thể sinh ra những em bé hoàn toàn khỏe mạnh.

Xét nghiệm phân tích trạng thái các Microsatellite Instability (MSI) trong các khối u đặc

Xét nghiệm đánh giá sự bất ổn định vệ tinh MSI (cũng sử dụng các trình tự lặp lại tương tự STRs) thông qua việc so sánh trạng thái các chỉ thị Microsatellite giữa tế bào mô thường và tế bào mô khối u của bệnh nhân ung thư đại trực tràng (DTT). Các bệnh nhân với sự mất ổn định vệ tinh MSI cao có tiên lượng và đáp ứng với điều trị khác với các bệnh nhân có ổn định vi vệ tinh (MSS). Do vậy việc xác định MSI có ý nghĩa rất quan trọng, không chỉ để sàng lọc hội chứng Lynch mà còn giúp phân biệt giữa ung thư đại trực tràng thiếu hụt hệ thống sửa chữa ghép cặp sai với ung thư đại trực tràng ổn định vi vệ tinh (MSS), nó sẽ cung cấp các thông tin có giá trị cho tiên lượng và việc cá thể hóa trong điều trị.

Để được tư vấn trực tiếp, Quý Khách vui lòng bấm số HOTLINE hoặc đăng ký lịch trực tuyến TẠI ĐÂY. Tải ứng dụng độc quyền MyVinmec để đặt lịch nhanh hơn, theo dõi lịch tiện lợi hơn!

Tài liệu tham khảo:

Young, Brian A; Butler Gettings, Katherine; McCord, Bruce; Vallone, Peter M. (2018). Estimating Number of Contributors in Massively Parallel Sequencing Data of STR loci. Forensic Science International: Genetics, (), S1872497318303673–. doi:10.1016/j.fsigen.2018.09.007

John M. Butler (2006). Genetics and Genomics of Core Short Tandem Repeat Loci Used in Human Identity Testing. , 51(2), 253–265. doi:10.1111/j.1556-4029.2006.00046.x

source https://blog-health.com/short-tandem-repeats-va-cac-ung-dung/

#bloghealth #suckhoe #lamdep #sinhly

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duran-duran-less-official · 6 years ago

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Determining the genetic similarity of domestic and feral European honeybee (Apis mellifera) populations at Warrah Research Station.

I’d appreciate feedback on my scientific literacy, ability to explain scientific concepts to people with no background in biology/genetics, and general structure/communication.

@poorpoorpitifulme I know you have more experience writing these papers than I do, so I’d definitely appreciate your input. :)

A short primer and glossary for non-biologists before I show you the actual paper:

Microsatellites are short sequences of DNA, like only a few base pairs. They do nothing for coding proteins. Except each microsatellite repeats itself over and over again, often hundreds of times. Like “CTG” turns into “CTGCTGCTGCTGCTG...”. However, due to DNA replication errors, microsatellites often gain or lose repeats, making them handy for determining how closely related individuals and populations are. The closer the number of repeats in microsatellites, the less chance they’ve had to mutate. It’s normally prudent to study several microsats at once, because a change in just one microsat could be an outlier.

PCR stands for polymerase chain reaction, which is a very complex process I won’t go into, which involves isolating the specific region of DNA you wish to study, and essentially cloning it, so that the “signals” from any attempts to discover specific genes or sequences in that region are “amplified”, so to speak.

A Chi-squared test is basically a more complex t-test, dealing with how closely an observed set of instances matches an expected set. The only thing is, a significant chi-squared value is larger rather than smaller. How large it needs to be depends on the population size - however, the chi-squared value in this particular experiment was less than 1, which is considered small in pretty much any sample size.

A haplotype is... uh, difficult to explain. You need to understand that mitochondria also have DNA which have a different inheritance pattern (being passed down exclusively from the mother), and haplotypes refer to this pattern of inheritance. Haplotypes which are genetically distinct enough can generally be split up into subspecies or lineages. They’re related, they’ve just been geographically separated for a long time. Especially useful with bees, because they have queens from which the entire hive inherits their haplotypes.

Fit, Fst and so on are meant to be measures of population differentiation. Fst in particular is the differentiation of the subpopulation with respect to the total population.

Alright, I think that’s enough background information. If you need any more clarification of concepts and such, or want to tell me that I got the science wrong (though I’m pretty sure I have it right this time), feel free to send me a message!

Abstract

Introduced European honeybees, Apis mellifera, are considered to have a largely negative impact on native Australian wildlife. Australian A. mellifera populations are thought to have originated from different European lineages depending on whether they are recently introduced domestic bees, or feral bees which have existed as an introduced species since the 19th Century. The exception is if a feral bee colony has been directly caused by the swarming of a domestic population, in which case the two populations are likely to be comparatively similar genetically. Whether the feral colony is self-sustaining, or whether it is the result of a nearby domestic colony, affects the optimal way to reduce its environmental impact. We compared mitochondrial and microsatellite DNA from domestic and feral populations of A. mellifera at Crommelin Biological Research Station in Woy Woy, and found that there was significant genetic overlap between the two indicative of being from the same lineage, and of recent interbreeding. We suggest that this feral population has been directly caused by the domestic population, and the best course of action is to relocate the colony immediately.

Introduction

Any two populations of the same species may become more genetically distinct from each other given adequate time and separation, dividing into different lineages and subspecies. There are currently three distinct lineages of the European honey bee, Apis mellifera, in Australia – these have been designated African (A), Eastern European (C), and Western European (M) (Chapman et al. 2016; Chapman et al. 2017). There is a feral population of A. mellifera in Australia, which was introduced in 1886 (Oldroyd et al. 1997), and most likely descended from the M lineage (Oldroyd et al. 1995; Chapman et al. 2008; Chapman et al. 2016). A more recently introduced domestic population of honey bees also exists, often referred to as the commercial population, for the purposes of pollination and honey production (Hinson et al. 2015), and is most likely descended from the C lineage (Chapman et al. 2008; Oldroyd et al. 1995).

Although bees are assumed to have a positive impact on the ecosystem as an efficient pollinator (Hinson et al. 2015), this assumption has been highly contested upon closer examination (Gross and Mackay 1998; Pyke 1999). As an introduced species, they have been found to reduce the available food for native animals who subsist on nectar and pollen, by introducing competition into the ecosystem which outstrips the available resources (Oldroyd et al. 1997; Hinson et al. 2015). In such cases, the effect of the introduced species on the native species must be carefully monitored, lest the introduced species affect the overall fitness of the native ones (Gross 2001). In instances where competition for pollination does not outstrip resources, the introduced bees may pollinate adequately, and may in fact be beneficial (Gross 2001). More often, however, they pollinate inadequately compared with native ones, which may affect the overall fitness of the plant, disrupting techniques such as self-fertilization, fruit-set and seed-set, which would have been adequately enabled by native bees, birds, or other insects – this phenomenon has been observed in Grevillea barklyana and Melastoma affine (Gross and Mackay, 1998; Vaughton 1996; cited in Hinson et al. 2015). They can even disrupt the habitats of other animals, such as Calyptorhynchus banksia naso, which has been driven from its normal nesting places by competitors – the most significant of which was A. mellifera (Johnstone et al. 2013; Hinson et al. 2015).

These issues only apply to feral bee populations, however there is a constant risk of domestic populations feeding or augmenting feral ones through swarming (Chapman et al. 2008). Studies have already been conducted on feral populations and nearby domestic populations in Victoria and Western Australia, and each found genetic evidence to suggest that the feral colonies were self-sustaining (Oldroyd et al. 1997; Chapman et al. 2008). Whether a colony of feral bees is self-sustaining or not affects the best method for managing them. If a feral colony is dependent on the input of domestic escaped or swarming bees, the problem may be solved by simply moving the colony (Oldroyd et al. 1997; Pyke 1999), however this will not work for self-sustaining colonies. Therefore an analysis of the relationship between a feral colony and any nearby domestic ones is required before a solution can be sought.

There are two ways to track this relationship, and to determine whether the two populations have been interbreeding. We can compare highly mutable microsatellite markers in the DNA of two or more populations, and obtain an exact number of tandem repeats for each microsatellite in each individual using polymerase chain reaction (PCR) (Estoup et al. 1995; Solignac et al. 2003). We can also compare mitochondrial DNA (mt-DNA) among the two populations. Mt-DNA has a number of advantages over nuclear DNA, including ease of amplification, high mutability, and ability to design primers and markers (Franck et al, 1998; Galtier et al, 2009). The differences between mt-DNA samples are analysed using restriction fragment length polymorphisms (RFLPs). In particular, the COI-COII intergenic region is variable enough that it is able to distinguish the four lineages when cut (Chapman et al. 2008; Garnery et al. 1995), and the different mt-DNA lengths can be analysed by a restriction map (Franck et al. 1998; Nielsen et al. 2000). If two sets of mitochondrial or microsatellite DNA from the same species are very similar, then it is likely that they came from the same population, or two different populations which retain the ability to interbreed. If the two sets of DNA are only slightly different, this indicates that the two populations have only recently diverged, and stopped interbreeding. If the two sets of DNA are significantly different, this indicates that the two populations have been distinct for a long time, and no interbreeding has occured.

It is possible that a colony of domestic bees at the “Warrah” Crommelin Biological Research Station in Woy Woy, New South Wales, is augmenting a nearby feral population. We will sample bees from the two populations, with the objective of analyzing their mt-DNA and microsatellites, examining their phylogenetic relationship, and determining what, if any, level of interbreeding is happening between them. Since the domestic and feral bee populations originated from different lineages, unless this particular feral population was directly caused by recent swarming, we hypothesize that the two bee populations will be significantly genetically distinct.

Materials and Methods

The University of Sydney, which operates Warrah Research Station in Woy Woy, provided us with DNA stocks extracted from domestic bees. A team of approximately twenty-six people visited the national park surrounding Warrah, and identified an area known to be frequently visited by the feral bees for pollination. Using a pooter, each person captured approximately four bees, which were frozen in liquid nitrogen, and transported to a laboratory for DNA extraction. The total sample was 104 feral bees, and the same number of domestic bee stocks were used for comparison. Once in the laboratory, mitochondrial DNA was extracted from the domestic and feral bees by pulling off one leg off each bee, and grinding it down using a sterile mini pestle and 0.5mL of Chelex solution in a 1.5mL microfuge tube. The tubes were labelled to identify whether the DNA inside them came from domestic or feral bees, then incubated in a 95°C water bath for fifteen minutes. After the PCR was sufficiently prepred, the tubes were centrifuged for seven minutes at 12000xg, and DNA was taken from the top of each tube to ensure the DNA aliquots would be free of Chelex resin. The PCR was prepared in labelled 0.2mL strip tubes with 5μL of DNA template, 15μL 2.5x PCR master mix and 10μL 2.5x Primer mix, to a final concentration of 41.5 mM KCl, 167.5 mM Tris-HCl, 2.5 units Taq DNA polymerase, 1.1% Triton X-100, 0.5 mg/ml gelatin, 0.5 mM each of dATP, dCTP, dTTP, and dGTP, 6.25 mM MgCl2, and 0.4 μM each primer pair.

Following the PCR, the mitochondrial DNA was digested in labelled 1.5mL microfuge tubes using the restriciton enzyme EcoRI. To each tube was added 7μL sterile Milli-Q water, 2μL 10x EcoRI buffer, 10μL of the PCR product, and 1μL of the EcoRI enzyme. The tubes were incubated at 37°C for one hour, then stored on ice.

After the mitochondrial DNA aliquots had been added to the PCR mixtures, the leftover DNA stocks were used to extract microsatellite DNA for PCR. Each DNA sample was given two multiplexes, each of which contained a different set of primers, and hence each DNA sample was required to go through two reactions. The PCR was prepared in 0.2mL strip tubes with 5μL of 2x PCR master mix, 2.5μL of the 4x Multiplex primers, and 2.5 μL of DNA stock, to a final concentration of 41.5 mM KCl, 167.5 mM Tris-HCl, 2.5 units Taq DNA polymerase, 1.1% Triton X-100, 0.5 mg/ml gelatin, 0.5 mM each of dATP, dCTP, dTTP, and dGTP, 6.25 mM MgCl2, and 0.4 μM each primer pair.

The mt-DNA was visualized using standard gel electrophoresis at 145-160V for 45 minutes, and numerical values were assigned to the DNA. A “zero” indicated no result, with “one” indicating that the DNA had been cut during the restriction digestion, and “two” indicating that it had not been cut. These numbers were entered into a spreadsheet. The microsatellites were visualized using a 3130xl Genetic Analyzer, and an allele size report was prepared.

The mt-DNA results were analyzed using a Chi-squared test, to determine if the observed ratios of cut and uncut DNA matched the expected ratios. The microsatellite DNA results were analyzed using GenAlEx in Microsoft Excel to determine the frequency-based distribution of the population.

Results

A number of mt-DNA and microsatellite results were deemed unusable, having yielded blank data, and these were omitted from the final results. The final number of bee DNA stocks used in the mitochondrial RFLP results was 65 feral and 61 domestic. The final number of bee DNA stocks used in the microsatellite data was 93 feral and 96 domestic.

The results of the Chi-squared test on the observed and expected numbers of cut and uncut mt-DNA were not significant (Table 1) ( χ2 = 0.343951). The population assignment graph showed considerable overlap between the feral and domestic populations (Fig 1). The observed heterozygosity generally closely matched the expected heterozygosity for both populations, indicating little to no inbreeding in either population (Table 2). The average Fst values show low differentiation between the subpopulations (Table 3).

Table 2. Table of heterozygosity, f-statistics and polymorphism by population for codominant microsatellite data.

Discussion

The feral and domestic colonies of A. mellifera contain a significant amount of genetic overlap in their mitochondrial and microsatellite DNA, which is enough to suggest that the two populations are related to one another. Furthermore, the observed and expected heterozygosity levels, together with the low Fst value, suggest that there has been little to no inbreeding or differentiation in either population, suggesting that the feral one has not been isolated for very long, if at all. We must therefore reject our initial hypothesis. There are two possible causes of this relatedness. It is less likely that the feral colony of A mellifera already existed independently, and has recently been fuelled by escapees from the domestic bee colony - if that were the case, we would expect to see more genetic diversity between the feral bees and the domestic stocks, indicative of one being derived from the C haplotype, and the other from the M haplotype, even if the domestic bees had been interbreeding with the feral bees for a number of years. It is more likely that this particular feral colony of A. mellifera was caused very recently by a swarm of bees which escaped from the domestic colony, leading to a C haplotype feral colony. In this case, it is advisable that the management of the feral bee colony be carried out by moving the domestic beehive, as it is likely that the population is not self-sustaining, and in fact still dependent on escaped domestic bees for its continued survival. The only caveats to these conclusions are the bees which yielded blank data, likely due to unsuccessful PCR. A total of 58 DNA samples from the mitochondrial RFLP PCR yielded no result and had to be discarded, and a total of 19 samples from the microsatellite PCR yielded no result. Of the samples which did yield results for microsatellites, many of them had incomplete data at one or more loci. However, we do not believe this affected the overall examination of the genetic similarity between the two populations.

The impact of bees, both domestic and feral, on the environment has frequently been investigated by population biologists, and the impact is most often seen as negative (Gross and Mackay 1998; Pyke 1999; Johnstone et al. 2013). Though some colonies of feral bees have been found to be self-sustaining (Oldroyd et al. 1997; Chapman et al. 2008) and therefore in need of more careful long-term consideration to deal with, this colony appears to be an exception. Moving the beehive has often been proposed as a solution before, and in most cases, beekeepers are additionally warned to be careful with their initial placement of the hive (Gross and Mackay 1998). However, this alone may not be sufficient, as the impact of feral bees also extends to domestic bees, due to competition for pollination and the spread of disease (Pyke 1999; Gordon et al. 2014), and moving a hive frequently increases the chances that a disease will be spread (Gordon et al. 2014). Though the overlap between the two Warrah populations is such that it is unlikely for an outbreak of disease to be caused purely by the feral population, this nonetheless underscores the urgency but delicacy with which this issue must be addressed at Warrah research station, for the sake of the environment and for the original domestic colony.

The optimal course of action would be to move the domestic bee colony away from Warrah immediately, before the feral population becomes self-sustaining. It is possible that a more urbanized area may suffice, since pollination resources remain adequate, yet feral colonies appear to have difficulty establishing themselves in such areas (Hinson et al. 2015). However, additional care must be taken to protect the hives from other threats characteristic of urban areas, such as pesticides (Hinson et al. 2015). Once the hive is re-established, it would be inadvisable to move the hive again until absolutely necessary. To conclude, this move would allow the hive to continue to thrive without competition from the feral bees, and the feral bees themselves would likely die out due to a lack of sustainability, thereby reducing or nullifying the domestic bees’ negative impact on the Australian ecosystem.

References

Chapman, N.C., Lim, J., and Oldroyd, B.P. (2008), Population Genetics of Commercial and Feral Honey Bees in Western Australia Journal 1of Economic Entomology 101:2, 272-277.

Chapman, N.C., Harpur, B.A., Lim, J., Rinderer T.E., Allsopp, M.H., Zayed, A., and Oldroyd, B.P. (2016) Hybrid origins of Australian honeybees (Apis mellifera) Apidologie 47, 26-34.

Chapman, N.C., Bourgeois, A.L., Beaman, L.D., Lim, J., Harpur, B.A., Zayed, A., Allsopp, M.H., Rinderer, T.E., and Oldroyd, B.P. (2017) An abbreviated SNP panel for ancestry assignment of honeybees (Apis mellifera) Apidologie 48, 776-783.

Estoup, A., Garnery, L., Solignac, M. and Cornuet, J.-M. (1995) Microsatellite Variation in Honey Bee (Apis Mellifera L.) Populations: Hierarchical Genetic Structure and Test of the Infinite Allele and Stepwise Mutation Models Genetics 140:2, 679-695.

Franck, P., Garnery, L., Solignac, M., and Cornuet, J.-M. (1998) The Origin of West European Subspecies of Honeybees (Apis mellifera): New Insights from Microsatellite and Mitochondrial Data Evolution 52:4, 1119-1134.

Galtier, N., Nabholz, B., Glémin, S., and Hurst, G.D.D. (2009) Mitochondrial DNA as a marker of molecular diversity: a reappraisal Molecular Ecology 18, 4541-4550.

Garnery, L., Mosshine, E.H., Oldroyd, B.P., and Cornuet, J.-M. (1995) Mitochondrial DNA variation in Moroccan and Spanish honey bee populations Molecular Ecology 4, 465-471.

Gordon, R., Bresolin-Schott, N., and East, I.J. (2014) Nomadic beekeeper movements create the potential for widespread disease in the honeybee industry Australian Veterinary Journal 92:8 283-290.

Gross, C.L., and Mackay, D. (1998) Honeybees reduce fitness in the pioneer shrub Melastoma affine (Melastomataceae) Biological Conservation 86, 169-178.

Gross, C.L. (2001) The effect of introduced honeybees on native bee visitation and fruit-set in Dillwynia juniperina (Fabaceae) in a fragmented ecosystem Biological Conservation 102, 89-95.

Hinson, E.M., Duncan, M., Lim, J., Arundel, J., and Oldroyd, B.P. (2015) The density of feral honey bee (Apis mellifera) colonies in South East Australia is greater in undisturbed than in disturbed habitats Apidologie 46, 403-413.

Johnstone, R.E., Kirkby, T., and Sarti, K. (2013) The breeding biology of the Forest Red-tailed Black Cockatoo Calyptorhynchus banskii naso Gould in south-western Australia. I. Characteristics of nest trees and nest hollows Pacific Conservation Biology 19, 121-142.

Nielsen, D.I., Ebert, P.R., Page, R.E., Hunt, G.J., and Guzmán-Novoa, E. (2000) Improved Polymerase Chain Reaction-Based Mitochondrial Genotype Identification of the Africanized Honey Bee (Hymenoptera: Apidae) Annals of the Entomological Society of America 93:1, 1-6.

Oldroyd, B.P., Cornuet, J.-M., Rowe, D., Rinderer, T.E., and Crozier, R.H. (1995) Racial admixture of Apis mellifera in Tasmania, Australia: similarities and differences with natural hybrid zones in Europe Heredity 74, 315-325.

Oldroyd, B.P., Thexton, E.G., Lawler, S.H., Crozier, R.H. (1997) Population demography of Australian feral bees (Apis mellifera) Oecologia 111, 381-387.

Pyke, G.H. (1999) The introduced Honeybee Apis mellifera and the Precautionary Principle: reducing the conflict Australian Zoologist 31:1, 181-186.

Solignac, M., Vautrin, D., Loiseau, A., Mougel, F., Baudry, E., Estoup, A., Garnery, L., Haberl, M., and Cornuet, J. (2003) Five hundred and fifty microsatellite markers for the study of the honeybee (Apis mellifera L.) genome Molecular Ecology Notes 3, 307-311.

Vaughton, G. (1996) Pollination disruption by European honeybees in the Australian bird-pollinated shrub Grevillea barklyana (Proteaceae) Plant Systematics and Evolution 200, 89-100.

#biology #science #warrah

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evoldir · 2 years ago

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Fwd: Job: TexasAMU.ResAssist.ArthropodPopGenomics

#IFTTT #Email

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mbimphblog · 4 years ago

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EXPERIMENTAL INJECTION OF α-INTERFERON (CHINESE HAMSTER OVARY DERIVED) RECOMBINANT PROTEIN TO RED-CLAW CRAYFISH (Cherax quadricarinatus): THE FIRST REPORT | Asian Journal of Advances in Research

Aims: In response to viral infection, interferon (IFN) has been recognized as a natural protection for vertebrates. In later research, in Phylum Arthropoda, which are Chinese mitten crab (Ericheir sinensis) (ErS1, GenBank no. FG360214) and Pacific white shrimp (Litopenaeus vannamei), IFN regulatory factor genes (IRF)s within the Metazoan Kingdom were established (PENVA, GenBank no. AOAOR6M5F1). The goal of this study was to confirm the existence of a red claw crayfish (C. quadricarinatus) IFN pathway that also belongs to Phylum Arthropoda. Methodology: alpha-IFN, a recombinant protein derived from the Chinese hamster ovary (CHO-Derived) was subcutaneously injected into adult red-claw crayfish at different doses per body weight (BW), namely a.100 IU, b. Around 200IU, and c. Uh. 225IU. The red-accurate claw's reflection period was counted. Polymerase chain reaction (PCR) using designed Chinese mitten crab primers was performed (E. sinensis). Results: The Simple Nucleotide Local Alignment Search Tool (BLASTn) has shown very similar characteristics between our sequences and other species of crustaceans. 89 percent red-claw crayfish (C. quadricarinatus) microsatellite cqu.005 (GenBank no. AF156901)) was the highest matched value and the lowest was 66 percent Noble crayfish (Astacus astacus) microsatellite Aas8 clone (GenBank no. EU692886). 72 and 75 percent compared two of our sequences with the Chinese mitten crab (E. sinensis) microsatellite ES1919 sequence (GenBank no. DQ388785). Conclusion: In conclusion, because the built primers did not endorse the concept of an IRF as part of the red-claw crayfish IFN pathway, our analysis appears to indicate that, except maybe in the Chinese mitten crab and Pacific white shrimp, no other IRF exists in Arthropoda to date. This result supports the logical consideration that during evolution, IRFs within the Metazoan Kingdom, Phylum Arthropoda, may have somewhat diverged or been entirely lost. Please see the link :- https://mbimph.com/index.php/AJOAIR/article/view/1418

#Interferon #arthropoda #Chinese mitten crab #pacific white shrimp #red-claw crayfish

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healthcare-market · 3 years ago

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In-vitro Transcription Templates Market : Emerging Trends, Business Growth Opportunities, Major Driving Factors