Genotyping Arabidopsis T-DNA lines v3

Mapping Intimacies ◽

10.17504/protocols.io.b2hzqb76 ◽

2021 ◽

Author(s):

Lynn Doran ◽

Steven J Burgess

Keyword(s):

Direct Pcr ◽

Genomic Locus ◽

Salk Institute ◽

Dna Oligonucleotides ◽

Genomic Location ◽

Arabidopsis Biological Resource Center ◽

Dna Insertion ◽

Primer Sets ◽

Arabidopsis Biological Resource ◽

The Uk

This protocol is used for genotyping Arabidopsis seedlings to test for the presence of a transfer DNA (T-DNA) insertion. By using two primer sets it is possible to determine whether a seedling is homozygous, heterozygous or azygous for an insertion in the predicted genomic location. To identify lines with T-DNA insertions in a gene of interest, you need the Arabidopsis Genome Identifier (AGI) number corresponding to the genomic locus (e.g. RCS1A = AT1G67090), then visit the Salk Institute T-DNA Express site to find all the mapped insertions at your locus of interest. Genotyping primers have been pre-designed for each T-DNA line, these can be retrieved from the Salk Institute T-DNA primer site, and ordered at any supplier of DNA oligonucleotides before starting the protocol. In the US T-DNA lines can be purchased from the Arabidopsis Biological Resource Center (ABRC) and in the UK and EU from the European Arabidopsis Stock Center (NASC). Recommended reading http://signal.salk.edu/tdnaprimers.2.html Setting up the PCR reaction Genotyping is performed with the Phire Plant Direct PCR Mix, this includes the polymerase, nucleotides and salts necessary for amplification. We use the “dilution protocol” which involves taking a small leaf disk and homogenizing it in dilution buffer using a gel tip (see manufacturer’s instructions for more details.)

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Genotyping Arabidopsis T-DNA lines v1

10.17504/protocols.io.bw5npg5e ◽

2021 ◽

Author(s):

Steven J Burgess

Keyword(s):

Direct Pcr ◽

Genomic Locus ◽

Salk Institute ◽

Dna Oligonucleotides ◽

Genomic Location ◽

Arabidopsis Biological Resource Center ◽

Dna Insertion ◽

Primer Sets ◽

Arabidopsis Biological Resource ◽

The Uk

This protocol is used for genotyping Arabidopsis seedlings to test for the presence of a transfer DNA (T-DNA) insertion. By using two primer sets it is possible to determine whether a seedling is homozygous, heterozygous or azygous for an insertion in the predicted genomic location. To identify lines with T-DNA insertions in a gene of interest, you need the Arabidopsis Genome Identifier (AGI) number corresponding to the genomic locus (e.g. RCS1A = AT1G67090), then visit the Salk Institute T-DNA Express site to find all the mapped insertions at your locus of interest. Genotyping primers have been pre-designed for each T-DNA line, these can be retrieved from the Salk Institute T-DNA primer site, and ordered at any supplier of DNA oligonucleotides before starting the protocol. In the US T-DNA lines can be purchased from the Arabidopsis Biological Resource Center (ABRC) and in the UK and EU from the European Arabidopsis Stock Center (NASC). Recommended reading http://signal.salk.edu/tdnaprimers.2.html Setting up the PCR reaction Genotyping is performed with the Phire Direct PCR Mix, this includes the polymerase, nucleotides and salts necessary for amplification. We use the “dilution protocol” which involves taking a small leaf disk and homogenizing it in dilution buffer using a gel tip (see manufacturer’s instructions for more details.)

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Genotyping Arabidopsis T-DNA lines v1

10.17504/protocols.io.b2huqb6w ◽

2021 ◽

Author(s):

Steven J Burgess

Keyword(s):

Direct Pcr ◽

Genomic Locus ◽

Salk Institute ◽

Dna Oligonucleotides ◽

Genomic Location ◽

Arabidopsis Biological Resource Center ◽

Dna Insertion ◽

Primer Sets ◽

Arabidopsis Biological Resource ◽

The Uk

This protocol is used for genotyping Arabidopsis seedlings to test for the presence of a transfer DNA (T-DNA) insertion. By using two primer sets it is possible to determine whether a seedling is homozygous, heterozygous or azygous for an insertion in the predicted genomic location. To identify lines with T-DNA insertions in a gene of interest, you need the Arabidopsis Genome Identifier (AGI) number corresponding to the genomic locus (e.g. RCS1A = AT1G67090), then visit the Salk Institute T-DNA Express site to find all the mapped insertions at your locus of interest. Genotyping primers have been pre-designed for each T-DNA line, these can be retrieved from the Salk Institute T-DNA primer site, and ordered at any supplier of DNA oligonucleotides before starting the protocol. In the US T-DNA lines can be purchased from the Arabidopsis Biological Resource Center (ABRC) and in the UK and EU from the European Arabidopsis Stock Center (NASC). Recommended reading http://signal.salk.edu/tdnaprimers.2.html Setting up the PCR reaction Genotyping is performed with the Phire Direct PCR Mix, this includes the polymerase, nucleotides and salts necessary for amplification. We use the “dilution protocol” which involves taking a small leaf disk and homogenizing it in dilution buffer using a gel tip (see manufacturer’s instructions for more details.)

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Design of Specific Primer Sets for the Detection of B.1.1.7, B.1.351 and P.1 SARS-CoV-2 Variants using Deep Learning

10.1101/2021.01.20.427043 ◽

2021 ◽

Author(s):

Alejandro Lopez-Rincon ◽

Carmina A. Perez-Romero ◽

Alberto Tonda ◽

Lucero Mendoza-Maldonado ◽

Eric Claassen ◽

...

Keyword(s):

Deep Learning ◽

South African ◽

Synonymous Mutations ◽

Molecular Tests ◽

Learning Techniques ◽

New Variant ◽

Primer Sets ◽

Global Initiative ◽

The Uk

ABSTRACTAs the COVID-19 pandemic persists, new SARS-CoV-2 variants with potentially dangerous features have been identified by the scientific community. Variant B.1.1.7 lineage clade GR from Global Initiative on Sharing All Influenza Data (GISAID) was first detected in the UK, and it appears to possess an increased transmissibility. At the same time, South African authorities reported variant B.1.351, that shares several mutations with B.1.1.7, and might also present high transmissibility. Even more recently, a variant labeled P.1 with 17 non-synonymous mutations was detected in Brazil. In such a situation, it is paramount to rapidly develop specific molecular tests to uniquely identify, contain, and study new variants. Using a completely automated pipeline built around deep learning techniques, we design primer sets specific to variant B.1.1.7, B.1.351, and P.1, respectively. Starting from sequences openly available in the GISAID repository, our pipeline was able to deliver the primer sets in just under 16 hours for each case study. In-silico tests show that the sequences in the primer sets present high accuracy and do not appear in samples from different viruses, nor in other coronaviruses or SARS-CoV-2 variants. The presented methodology can be exploited to swiftly obtain primer sets for each independent new variant, that can later be a part of a multiplexed approach for the initial diagnosis of COVID-19 patients. Furthermore, since our approach delivers primers able to differentiate between variants, it can be used as a second step of a diagnosis in cases already positive to COVID-19, to identify individuals carrying variants with potentially threatening features.

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A Rapid and Low-Cost protocol for the detection of B.1.1.7 lineage of SARS-CoV-2 by using SYBR Green-Based RT-qPCR

10.1101/2021.01.27.21250048 ◽

2021 ◽

Author(s):

Fadi Abdel Sater ◽

Mahmoud Younes ◽

Hassan Nassar ◽

Paul Nguewa ◽

Kassem Hamze

Keyword(s):

Low Cost ◽

False Negative ◽

Sybr Green ◽

Reproductive Number ◽

Rt Pcr ◽

Negative Results ◽

False Negative Results ◽

New Variant ◽

Primer Sets ◽

The Uk

AbstractBackgroundThe new SARS-CoV-2 variant VUI (202012/01), identified recently in the United Kingdom (UK), exhibits a higher transmissibility rate compared to other variants, and a reproductive number 0.4 higher. In the UK, scientists were able to identify the increase of this new variant through the rise of false negative results for the spike (S) target using a three-target RT-PCR assay (TaqPath kit).MethodsTo control and study the current coronavirus pandemic, it is important to develop a rapid and low-cost molecular test to identify the aforementioned variant. In this work, we designed primer sets specific to SARS-CoV-2 variant VUI (202012/01) to be used by SYBR Green-based RT-PCR. These primers were specifically designed to confirm the deletion mutations Δ69/Δ70 in the spike and the Δ106/Δ107/Δ108 in the NSP6 gene. We studied 20 samples from positive patients, 16 samples displayed an S-negative profile (negative for S target and positive for N and ORF1ab targets) and four samples with S, N and ORF1ab positive profile.ResultsOur results emphasized that all S-negative samples harbored the mutations Δ69/Δ70 and Δ106/Δ107/Δ108. This protocol could be used as a second test to confirm the diagnosis in patients who were already positive to COVID-19 but showed false negative results for S-gene.ConclusionsThis technique may allow to identify patients carrying the VUI (202012/01) variant or a closely related variant, in case of shortage in sequencing.

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A seed resource for screening functionally redundant genes and isolation of new mutants impaired in CO2 and ABA responses

10.1101/415976 ◽

2018 ◽

Author(s):

Felix Hausera ◽

Paulo H. O. Ceciliatoa ◽

Yi-Chen Lin ◽

DanDan Guo ◽

JD Gregerson ◽

...

Keyword(s):

Genetic Screens ◽

Resource Center ◽

New Genes ◽

Redundant Genes ◽

Forward Genetic Screens ◽

Genes Encoding ◽

Arabidopsis Biological Resource Center ◽

Redundant Gene ◽

Aba Insensitive ◽

Arabidopsis Biological Resource

AbstractThe identification of homologous genes with functional overlap in forward genetic screens is severely limited. Here we report the generation of over 14,000 amiRNA-expressing plants that enable screens of the functionally redundant gene space in Arabidopsis. A protocol is developed here for isolating robust and reproducible amiRNA-mutants. Examples of validation approaches and essential controls are presented for two new amiRNA mutants that exhibit genetically redundant phenotypes and circumvent double mutant lethality. In a forward genetic screen for abscisic acid (ABA)-mediated inhibition of seed germination, amiRNAs that target combinations of known redundant ABA receptor and SnRK2 kinase genes were rapidly isolated, providing a strong proof of principle for this approach. A new ABA insensitive amiRNA line is isolated, which targets three genes encoding avirulence-induced gene2-like (AIG2) genes. A thermal imaging screen for plants with impaired stomatal opening in response to low CO2 exposure led here to isolation of a new amiRNA targeting two essential proteasomal subunits, PAB1 and PAB2. The seed library of 14,000 T2 amiRNA lines generated here provides a new platform for forward genetic screens and is being made available to the Arabidopsis Biological Resource Center (ABRC) and optimized procedures for amiRNA screening and controls are described.HighlightThe generation of over 14,000 amiRNA-expressing plants is reported that are being made publicly available enabling screens of redundant genes in Arabidopsis. Identification of known and new genes is reported.

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Following Phenotypes: An Exploration of Mendelian Genetics Using Arabidopsis Plants

The American Biology Teacher ◽

10.1525/abt.2018.80.4.291 ◽

2018 ◽

Vol 80 (4) ◽

pp. 291-300

Author(s):

Courtney G. Price ◽

Emma M. Knee ◽

Julie A. Miller ◽

Diana Shin ◽

James Mann ◽

...

Keyword(s):

School Level ◽

High School Level ◽

Resource Center ◽

Mendelian Genetics ◽

Hands On ◽

Two Generations ◽

Hands On Learning ◽

Arabidopsis Biological Resource Center ◽

Mutant Phenotypes ◽

Arabidopsis Biological Resource

Arabidopsis thaliana, a model system for plant research, serves as the ideal organism for teaching a variety of basic genetic concepts including inheritance, genetic variation, segregation, and dominant and recessive traits. Rapid advances in the field of genetics make understanding foundational concepts, such as Mendel's laws, ever more important to today's biology student. Coupling these concepts with hands-on learning experiences better engages students and deepens their understanding of the topic. In our article, we present a teaching module from the Arabidopsis Biological Resource Center as a tool to engage students in lab inquiry exploring Mendelian genetics. This includes a series of protocols and assignments that guide students through growing two generations of Arabidopsis, making detailed observations of mutant phenotypes, and determining the inheritance of specific traits, thus providing a hands-on component to help teach genetics at the middle and high school level.

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Arabidopsis Biological Resource Center

Plant Molecular Biology Reporter ◽

10.1007/bf02668747 ◽

1994 ◽

Vol 12 (3) ◽

pp. 242-244 ◽

Cited By ~ 2

Author(s):

Randy Scholl ◽

Mary Anderson

Keyword(s):

Biological Resource ◽

Resource Center ◽

Arabidopsis Biological Resource Center ◽

Arabidopsis Biological Resource

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Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9

Bioscience Reports ◽

10.1042/bsr20203595 ◽

2021 ◽

Vol 41 (1) ◽

Author(s):

Chun Hang Lau ◽

Edward L. Bolt

Keyword(s):

Protein Complexes ◽

Dna Integration ◽

Synthetic Dna ◽

Methanosarcina Mazei ◽

Dna Oligonucleotides ◽

Double Stranded Dna ◽

Dna Insertion ◽

Dna Capture ◽

Target Molecules

Abstract CRISPR systems build adaptive immunity against mobile genetic elements by DNA capture and integration catalysed by Cas1–Cas2 protein complexes. Recent studies suggested that CRISPR repeats and adaptation module originated from a novel type of DNA transposons called casposons. Casposons encode a Cas1 homologue called casposase that alone integrates into target molecules single and double-stranded DNA containing terminal inverted repeats (TIRs) from casposons. A recent study showed Methanosarcina mazei casposase is able to integrate random DNA oligonucleotides, followed up in this work using Acidoprofundum boonei casposase, from which we also observe promiscuous substrate integration. Here we first show that the substrate flexibility of Acidoprofundum boonei casposase extends to random integration of DNA without TIRs, including integration of a functional gene. We then used this to investigate targeting of the casposase-catalysed DNA integration reactions to specific DNA sites that would allow insertion of defined DNA payloads. Casposase–Cas9 fusions were engineered that were catalytically proficient in vitro and generated RNA-guided DNA integration products from short synthetic DNA or a gene, with or without TIRs. However, DNA integration could still occur unguided due to the competing background activity of the casposase moiety. Expression of Casposase-dCas9 in Escherichia coli cells effectively targeted chromosomal and plasmid lacZ revealed by reduced β-galactosidase activity but DNA integration was not detected. The promiscuous substrate integration properties of casposases make them potential DNA insertion tools. The Casposase–dCas9 fusion protein may serves as a prototype for development in genetic editing for DNA insertion that is independent of homology-directed DNA repair.

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