A new era of genomics for plant science research has opened due the complete genome sequencing projects of Arabidopsis thaliana and rice. The sequence information available in public database has highlighted the need to develop genome scale reverse genetic strategies for functional analysis (Till et al., 2003). As most of the phenotypes are obscure, the forward genetics can hardly meet the demand of a high throughput and large-scale survey of gene functions. Targeting Induced Local Lesions in Genome TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identity point mutations in regions of interest (McCallum et al., 2000). This strategy works with a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms in genes of interest. A newly developed general reverse genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It allows the rapid and inexpensive detection of induced point mutations in populations of physically or chemically mutagenized individuals. To create an induced population with the use of physical/chemical mutagens is the first prerequisite for TILLING approach. Most of the plant species are compatible with this technique due to their self-fertilized nature and the seeds produced by these plants can be stored for long periods of time (Borevitz et al., 2003). The seeds are treated with mutagens and raised to harvest M1 plants, which are consequently, self-fertilized to raise the M2 population. DNA extracted from M2 plants is used in mutational screening (Colbert et al., 2001). To avoid mixing of the same mutation only one M2 plant from each M1 is used for DNA extraction (Till et al., 2007). The M3 seeds produce by selfing the M2 progeny can be well preserved for long term storage. Ethyl methane sulfonate (EMS) has been extensively used as a chemical mutagen in TILLING studies in plants to generate mutant populations, although other mutagens can be effective. EMS produces transitional mutations (G/C, A/T) by alkylating G residues which pairs with T instead of the conservative base pairing with C (Nagy et al., 2003). It is a constructive approach for users to attempt a range of chemical mutagens to assess the lethality and sterility on germinal tissue before creating large mutant populations.
Marker assisted breeding of biotic stress resistance in Rice Senthil Natesan
A marker is a DNA sequence which serves as a signpost/flag post
linked to the trait/gene of interest and is co-inherited along with
the trait
Presence of specific allele of marker = Presence of specific allele of target gene based on the concept the MAS practiced -R.M. Sundaram
Directorate Rice of Research, Hydrabad , July 3rd 2009, CPMB&B, TNAU presentation
Targeted Induced Local Lesions IN Genome. Mutations (Single base pair substitution) are created by traditionally used chemical mutagens. Identify SNPs and / or INDELS in a gene / genes of interest from a mutagenized population.
Multiple inbred founder lines are inter-mated for several generations prior to creating inbred lines, resulting in a diverse population whose genomes are fine scale mosaics of contributions from all founders.
A new era of genomics for plant science research has opened due the complete genome sequencing projects of Arabidopsis thaliana and rice. The sequence information available in public database has highlighted the need to develop genome scale reverse genetic strategies for functional analysis (Till et al., 2003). As most of the phenotypes are obscure, the forward genetics can hardly meet the demand of a high throughput and large-scale survey of gene functions. Targeting Induced Local Lesions in Genome TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identity point mutations in regions of interest (McCallum et al., 2000). This strategy works with a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms in genes of interest. A newly developed general reverse genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It allows the rapid and inexpensive detection of induced point mutations in populations of physically or chemically mutagenized individuals. To create an induced population with the use of physical/chemical mutagens is the first prerequisite for TILLING approach. Most of the plant species are compatible with this technique due to their self-fertilized nature and the seeds produced by these plants can be stored for long periods of time (Borevitz et al., 2003). The seeds are treated with mutagens and raised to harvest M1 plants, which are consequently, self-fertilized to raise the M2 population. DNA extracted from M2 plants is used in mutational screening (Colbert et al., 2001). To avoid mixing of the same mutation only one M2 plant from each M1 is used for DNA extraction (Till et al., 2007). The M3 seeds produce by selfing the M2 progeny can be well preserved for long term storage. Ethyl methane sulfonate (EMS) has been extensively used as a chemical mutagen in TILLING studies in plants to generate mutant populations, although other mutagens can be effective. EMS produces transitional mutations (G/C, A/T) by alkylating G residues which pairs with T instead of the conservative base pairing with C (Nagy et al., 2003). It is a constructive approach for users to attempt a range of chemical mutagens to assess the lethality and sterility on germinal tissue before creating large mutant populations.
Marker assisted breeding of biotic stress resistance in Rice Senthil Natesan
A marker is a DNA sequence which serves as a signpost/flag post
linked to the trait/gene of interest and is co-inherited along with
the trait
Presence of specific allele of marker = Presence of specific allele of target gene based on the concept the MAS practiced -R.M. Sundaram
Directorate Rice of Research, Hydrabad , July 3rd 2009, CPMB&B, TNAU presentation
Targeted Induced Local Lesions IN Genome. Mutations (Single base pair substitution) are created by traditionally used chemical mutagens. Identify SNPs and / or INDELS in a gene / genes of interest from a mutagenized population.
Multiple inbred founder lines are inter-mated for several generations prior to creating inbred lines, resulting in a diverse population whose genomes are fine scale mosaics of contributions from all founders.
Genomics, proteomics and metabolomics are the three core omics technologies, which respectively deal with the analysis of genome, proteome and metabolome of cells and tissues of an organism.
Genotyping by Sequencing is a robust,fast and cheap approach for high throughput marker discovery.It has applications in crop improvement programs by enhancing identification of superior genotypes.
Within the last twenty years, molecular biology has revolutionized conventional breeding techniques in all areas. Biochemical and Molecular techniques have shortened the duration of breeding programs from years to months, weeks, or eliminated the need for them all together. The use of molecular markers in conventional breeding techniques has also improved the accuracy of crosses and allowed breeders to produce strains with combined traits that were impossible before the advent of DNA technology
Transgenes may be used to produce GMS which is dominant to fertility.
In these cases it is essential to develop effective fertility restoration systems for hybrid seed production.
An effective restoration system is available in at least one case, Barnase/Barstar system
Recombinant DNA techniques have made it possible to engineer new systems of male sterility by disturbing any or number of developmental steps specifically required for the production of functional pollen within the microspore or for the development of any somatic tissues .
The presentation was done as part of the course STAT 504 titled Quantitative Genetics in Second Semester of MSc. Agricultural Statistics at Agricultural College, Bapatla under ANGRAU, Andhra Pradesh
A concise and well fabricated presentation the current techniques used for plant genome editing including CRISPER/cas9 system, TALENS, TELES, ZINC FINGER NUCLEASES(ZFN), HEJ (homologous endjoing) and many other high throughout techniques along references.
Genomics, proteomics and metabolomics are the three core omics technologies, which respectively deal with the analysis of genome, proteome and metabolome of cells and tissues of an organism.
Genotyping by Sequencing is a robust,fast and cheap approach for high throughput marker discovery.It has applications in crop improvement programs by enhancing identification of superior genotypes.
Within the last twenty years, molecular biology has revolutionized conventional breeding techniques in all areas. Biochemical and Molecular techniques have shortened the duration of breeding programs from years to months, weeks, or eliminated the need for them all together. The use of molecular markers in conventional breeding techniques has also improved the accuracy of crosses and allowed breeders to produce strains with combined traits that were impossible before the advent of DNA technology
Transgenes may be used to produce GMS which is dominant to fertility.
In these cases it is essential to develop effective fertility restoration systems for hybrid seed production.
An effective restoration system is available in at least one case, Barnase/Barstar system
Recombinant DNA techniques have made it possible to engineer new systems of male sterility by disturbing any or number of developmental steps specifically required for the production of functional pollen within the microspore or for the development of any somatic tissues .
The presentation was done as part of the course STAT 504 titled Quantitative Genetics in Second Semester of MSc. Agricultural Statistics at Agricultural College, Bapatla under ANGRAU, Andhra Pradesh
A concise and well fabricated presentation the current techniques used for plant genome editing including CRISPER/cas9 system, TALENS, TELES, ZINC FINGER NUCLEASES(ZFN), HEJ (homologous endjoing) and many other high throughout techniques along references.
Single Nucleotide Polymorphism Genotyping Using Kompetitive Allele Specific ...MANGLAM ARYA
Single Nucleotide Polymorphism
Single nucleotide polymorphism (SNP) refers to a single base change in a DNA sequence
SNP: Commonly biallelic
Two types(Based on presence in genome)
Synonymus
Non-synonymus
SNPs have largely replaced simple sequence repeats (SSRs)
Advantage of using SNPs
Low assay cost
High genomic abundance
Locus specificity
co-dominant inheritance
Simple documentation
Potential for high-throughput Analysis
Relatively low genotyping error rates
SNP genotyping platforms
BeadXpressTM,GoldenGateTM and Infinium from Illumina
GeneChipTM and GenFlexTM Tag array from Affimetrix
SNaPshotTM and TaqManTM from the Applied Biosystems
SNPWaveTM from KeyGene
iPLEX GoldTM Assay and Mass-RRAYTM from Sequonome
Variables to be considered
Throughput
Data turnaround
Time
Ease of use
Performance (sensitivity, reliability, reproducibility, and accuracy),
Flexibility (genotyping few samples with many snps or many samples with few snps),
Number of markers generated per run (uniplex versus multiplex assay capability)
Assay development requirements and genotyping cost per sample or data point.
KASP
KBioscience Competitive Allele-Specific PCR
Homogenous, Fluorescence-based genotyping technology, based on
Allele-specific oligo extension (primer)
Fluorescence resonance energy transfer
KASP Applications
Genotyping a wide range of species for various purposes.
KASP for Quality analysis, QTL mapping, MARS, and allele mining
Quality Control Analysis
QC analysis should be done for two reasons by genotyping the parents and F1s with the same subset of SNPs, in order to
confirm if F1s contains true-to-type alleles from their parents
check the genetic purity of the inbred parents.
F1s with true-to-type parental alleles for at least 90 % of the SNPs that were polymorphic between the parents should be advanced, while those with less than 10 % nonparental alleles should be discarded.
QTL Mapping
QTL mapping identifies a subset of markers that are significantly associated with one or more QTL influencing the expression of the trait of interest.
1) Select or develop a bi-parental mapping population.
2) Phenotype the population for a trait under greenhouse or field conditions.
3) Choose a molecular marking system – genotype parents of the mapping population and F1s with large numbers of markers, then select 200-400 markers exhibiting polymorphism between the parents.
4) Choose a genotyping approach, then generate molecular data for polymorphic markers
5) Identify the molecular markers associated with major QTL using statistical programs.
Large-scale allele mining
Allele mining is a promising approach to dissecting naturally occurring allelic variation at candidate genes controlling key agronomic traits.
KASP platform at CIMMYT has been used for the systematic mining of large germplasm collections for specific functional polymorphisms.
SNPs or small indels that
Digital PCR for soybean GMO detection on the OpenArray Platform: a case study...Thermo Fisher Scientific
The detection and quantification of the GMO present in certified seed lots, and in food products, is essential to fulfill regulatory traceability requirements and downstream food labeling.
In this collaboration with DNA LandMarks (BASF), Life Technologies designed TaqMan® assays for GMO and WT soybean DNA detection and validated assay specificity with target-specific gDNA using dPCR concentration range and saturated gDNA concentration.
Life Technologies offers a range of real-time and digital PCR solutions to address your throughput, accuracy, and detection sensitivity needs with high confidence.
Creating Better Gene-Edited Cell Lines with the FAST-HDR SystemInsideScientific
Cell lines are the core of biological research. Scientists need cell lines for drug development, basic biology research, safety testing, and biologic therapeutic production. Since the 1980s, genetic manipulation has allowed researchers to tailor cell lines to the experiment or production purpose. Over time, the requirements for these cell lies have risen. In many cases, the cells require multiple genetic edits and must produce data that passes FDA. Moreover, the current funding environment often requires rapid delivery of these cells so scientists can produce data to support further budget and/or investment. This is particularly acute for knock-in cell lines. Current technologies may take months to complete a cell line, allow a limited number of edits, and often have off-target effects that are not suitable for FDA filings. ExpressCells uses its patented FAST-HDR plasmid--along with CRISPR, to address these problems. The FAST-HDR process can precisely knock-in multiple genes (while supporting other types of genetic modifications), ensure precise placement of these edits, and deliver them months faster than competing technologies.
This webinar will discuss the basis of the FAST-HDR technology and illustrate several uses. The first part is a presentation by Oscar Perez-Leal, MD, the inventor of the technology. Oscar will discuss the problems he faced as a researcher and how FAST-HDR was designed to address them. He will outline the details of the technology, the history of its development, and several examples where he used FAST-HDR. The second part is a conversation with Jon Weidanz, PhD. Jon will outline the challenges he faced at AbeXXa and how he selected a FAST-HDR custom cell line for his project. He'll outline the learnings from using this cell line, some of which were unexpected, but valuable to future development.
By attending this program, attendees will:
- Understand the current challenges in creating custom gene-edited cell lines
- Know the technology underlying the FAST-HDR gene-editing system, including its use with CRISPR
- Be able to describe the advantages of the FAST-HDR system
- Learn about several case studies using gene-edited cell lines
DNA sequence analysis and genotyping of biological samples using innovative instrumentation, such as next-generation sequencing (NGS) platforms, is often limited by the small amount of sample available. The REPLI-g Single Cell Kit is specially designed to uniformly amplify genomic DNA from single cells (1 to <1000 bacterial or tumor cells) or purified genomic DNA with complete genome coverage. Additional protocols are also available for use with fresh or dried blood or fresh or frozen tissue. Dedicated buffers and reagents undergo a unique, controlled decontamination procedure to avoid amplification of contaminating DNA, ensuring highly reliable results every time. Accurate amplification of genomes with negligible sequence bias and no genomic drop-outs is achieved with innovative Multiple Displacement Amplification (MDA) technology. In contrast to PCR-based WGA technologies, high fidelity rates are increased up to 1000-fold, avoiding costly false positive or negative results.
Step by Step, from Liquid Biopsy to a Genomic Biomarker: Liquid Biopsy Series...QIAGEN
Liquid biopsies enable us to monitor the evolution of genetic aberrations in primary tumors as they shed the tumor cells into the circulation. The limitation is the ability to detect these low frequency genetic aberrations in a consistent manner to understand short- and long-term implications and how this information will be used in the clinic. This slidedeck will cover the challenges and solutions associated with multiple steps as one starts with liquid biopsy and move towards finding a new biomarker.
Achieve Complete Coverage of the SARS-CoV-2 GenomeCamille Cappello
Utilize multiple overlapping amplicons in a single tube, using a rapid, 2-hour workflow to prepare ready-to-sequence libraries. The PCR1+PCR2 workflow generates robust libraries even from low input quantities of DNA that may be subsequently quantified and normalized with conventional methods such as Qubit® or Agilent Bioanalyzer, or optionally using the included Swift Normalase reagents.
Provides coverage of >99% of the SARS-CoV-2 genome from limited viral titers
Identity testing by NGS as a means of risk mitigation for viral gene therapiesMerck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/3RijkHC
Detailed description:
Imagine you’ve just completed a manufacturing run for your viral vector. Identity testing is performed to confirm the vector sequence. But when the results come back the data reveals unexpected sequence variants! With an appropriate risk mitigation testing strategy, this situation can be prevented.
The situation described above is not hypothetical, and happens more that you think, costing valuable time and resources.
Investigatory testing has shown that sequence variants present in starting materials (e.g. plasmids) are likely to make their way to the final product. Adequate identification of low-level variants with an appropriately sensitive method is critical in ensuring the quality of the final product. A risk-based testing strategy, in the context of identity, for viral vector manufacturing will be presented, focusing on key testing points. NGS assays for identity and variant detection will be highlighted due to their extremely sensitive nature compared to traditional approaches.
In this webinar, we'll explore:
• Regulatory requirements for identity testing
• NGS applications for identity testing as compared to traditional methods
• A case study on the impact of not establishing a proper risk-based testing strategy
Presented by: Bradley Hasson, Director of Lab Operations for NGS Services
Identity testing by NGS as a means of risk mitigation for viral gene therapiesMilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3RijkHC
Detailed description:
Imagine you’ve just completed a manufacturing run for your viral vector. Identity testing is performed to confirm the vector sequence. But when the results come back the data reveals unexpected sequence variants! With an appropriate risk mitigation testing strategy, this situation can be prevented.
The situation described above is not hypothetical, and happens more that you think, costing valuable time and resources.
Investigatory testing has shown that sequence variants present in starting materials (e.g. plasmids) are likely to make their way to the final product. Adequate identification of low-level variants with an appropriately sensitive method is critical in ensuring the quality of the final product. A risk-based testing strategy, in the context of identity, for viral vector manufacturing will be presented, focusing on key testing points. NGS assays for identity and variant detection will be highlighted due to their extremely sensitive nature compared to traditional approaches.
In this webinar, we'll explore:
• Regulatory requirements for identity testing
• NGS applications for identity testing as compared to traditional methods
• A case study on the impact of not establishing a proper risk-based testing strategy
Presented by: Bradley Hasson, Director of Lab Operations for NGS Services
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
Introduction to AI for Nonprofits with Tapp NetworkTechSoup
Dive into the world of AI! Experts Jon Hill and Tareq Monaur will guide you through AI's role in enhancing nonprofit websites and basic marketing strategies, making it easy to understand and apply.
1.4 modern child centered education - mahatma gandhi-2.pptx
High throughput genotyping
1. High throughput SNP genotyping
Cosentino Cristian, PhD
Genomics and Bioinformatics unit
Filarete Foundation – Milan
cosentia@gmail.com
2. Illumina genotyping
• Illumina BeadArray technology
• Simultaneous genotyping of thousand to milion of loci
• Entire genome accessible to analysis
• Low amount of gDNA required (250 – 750 ng)
• Automation of the protocol
• Randomly assembled arrays (decoding process)
3. High throughput
The laboratory is divided in:
1. WetLab: working on biological samples
(Biotechnologists, Technicians)
• Pre-PCR area
• Post-PCR area
2. DryLab: working on data analysis
(Bioinformaticians, IT systemists, Clinicians)
[WetLab] Samples management for Genotyping:
1. Sample validation
2. Sample preparation
3. Genotyping
4. RawData evaluation
4. Genotyping protocols
GoldenGate:
96 samples/array and thousand of SNPs simultaneously assayed; completely cutomizable
Infinium:
2-12 samples/chip and hundred of thousand of SNPs simultaneously assayed; customizable
up to 20-30.000 ca. SNPs per sample
9. Sample preparation
DNA arrival DNA extraction
Sample
validation Move vials into 96
well plates
Sample
preparation
DNA quantification
Genotyping
DNA dilution
RawData
evaluation
Quality control on gel
GWA analysis
Move to new fullfilled
plate
10. High throughput sample management
Tecan Liquid handler
DNA extraction
DNA arrival
Move vials into 96
Sample
well plates
validation
Sample
DNA quantification
preparation
Genotyping DNA dilution
RawData
evaluation Quality control on gel
GWA analysis
Move to new fullfilled Usage:
plate Vial-to-plate and Plate-to-plate sample movement
Normalization of the sample concentration
Advantages:
1. Cost-saving (30’ x 2 operators vs 20’ x 1 operator/plate)
2. Tracking of the sample movement (personal responsability)
11. High throughput sample management
Multi-sample spectrophotometer
DNA extraction
DNA arrival
Move vials into 96
Sample
well plates
validation
Sample
DNA quantification
preparation
Genotyping DNA dilution
RawData
evaluation Quality control on gel
GWA analysis Usage:
Move to new fullfilled
plate Nucleic acid quantification
Advantages:
1. 16 samples/run assayed
2. Small sample volume used
3. Quantification in standard routine
4. Connection to the liquid handler normalization software
12. High throughput sample management
Pre-casted agarose gel
DNA extraction
DNA arrival
Move vials into 96
Sample
well plates
validation
Sample
DNA quantification
preparation
Genotyping DNA dilution
RawData
evaluation Quality control on gel
GWA analysis Usage:
Move to new fullfilled
plate Screening of the DNA integrity
Advantages:
1. 48 samples/run assayed in 20’
2. Standarsd screening conditions
3. System copatible with TECAN liquid handler
4. Barcode ID for easy data collection
14. Single base extension
DNA arrival
Single base extension
Sample Markers: ddA, ddT, ddC, ddG
validation
Sample
preparation
Genotyping
RawData
evaluation
GWA analysis
