Genetic Analysis Solutions for Plant Sciences

Plant Agriculture Biotechnology: Empower Your Plant Science ResearchFull-spectrum genetic analysis tools and beyond

2 6/28/2012 | Life Technologies™ Corporation

Outline• The challenge: feeding and fueling the world

• Life Technologies mission and our portfolio plant agricultural biotechnology products

• Key applications for plant researchers and supporting data

Plant genome and transcriptome sequencing, and SNP discovery

Plant resequencing and SNP genotyping

QTL mapping and marker-assisted selection

GMO detection and screening

Plant genetic engineering

Plant gene expression

DNA and RNA isolation

• Summary

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/agricultural-biotechnology/plant-genome-sequencing.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/agricultural-biotechnology/plant-gene-expression-analysis.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/agricultural-biotechnology/plant-genotyping.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/agricultural-biotechnology/plant-genetic-engineering.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/agricultural-biotechnology/gmo-testing-detection.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/agricultural-biotechnology/qtl-mapping-marker-assisted-selection.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/agricultural-biotechnology/plant-DNA-RNA-isolation.html

3 6/28/2012 | Life Technologies™ Corporationhttp://documents.wfp.org/stellent/groups/public/documents/communications/wfp229328.pdf

http://documents.wfp.org/stellent/groups/public/documents/communications/wfp229328.pdf

http://documents.wfp.org/stellent/groups/public/documents/communications/wfp229328.pdf


Global challenges—population, food, and land • World population expected to reach 8 billion by 2025*

− 20% of the world’s population is not receiving the minimum food required for a healthy life

− Global demand for food will increase three times in the next 15 years

− Pests destroy 1/3 of the food produced globally ($30 billion spent on pesticides)

− 1.2 billion people globally exist in poverty, with earnings < $1/day

• Recent notable climate events− Increase in intensity of natural disasters and extreme weather events

− Rising sea levels, contamination of water and agricultural land

− Changes in rainfall patterns and water shortage

> Hotter climates contributed to lower wheat (–5.5%) and corn (–3.8%) yields**

*Agricultural Biotechnology: A Global Strategic Business Report. Global Industry Analysts, Inc. 10/2010.**Agriculture: A Global Industry Outlook. Global Industry Analysts, Inc. 01/2012


Plant biotechnology offers many solutions

• Higher crop yield per acre

• Resistance to insect pests, diseases, droughts, and salt

• Lower production costs and lower environmental impact

• Foods with improved nutrient profiles

• Renewable energy sources

• New applications: high-value chemicals, plastics, vaccines, phytoremediation, etc.


Life Technologies mission: to empower your plant science research

• Providing innovative platforms optimized for each step of plant biotechnology workflows, from tools that help elucidate the genetic makeup of plants to DNA manipulation, gene and protein expression, cell imaging, and copy number variation

• Offering the widest technology selection, with the highest quality at every budget to help address challenges like food production, land conservation, and biodiversity protection


Key applications in plant biotechnology

Desired phenotypesHigher yields

Pest and environmental stress tolerance

Healthier diets

More efficient fertilizer use

Biofuels

Nutraceuticals

QTL mapping & marker-assisted selection

Plant de novo genome

sequencing

Plant genotyping

Plant gene expression

analysis


GMO testing & detection

http://products.invitrogen.com/ivgn/product/12322012?ICID=search-product

Plant Genome & Transcriptome Sequencing


Plant genome and transcriptome sequencing

• Plant genome de novo sequencing: sequencing and assembling a plant genome without any reference genome sequence. Next-generation sequencing technologies allow researchers to move beyond model organisms and gain an understanding of all plant genomes—a critical step in unraveling the complexity of plants.

• Plant transcriptome sequencing: study the gene expression profiling at the whole transcriptome level; also used to reduce the complexity of a large, complex, and polyploid genome before sequencing entire genomes



Life Technologies solutions for plant genome and transcriptome sequencing

de novo genome

sequencing

<1 Gb

BAC clones

de novo genome

sequencing

>1 Gb

Transcriptomesequencing

Small genome region

sequencing

Sequence confirmation



Ion PGM™ Sequencer: the fastest in the world

Key plant genomics applications

• Plant de novo sequencing for genomes <1 Gb

• Plant transcriptome sequencing

• Genotyping by sequencing

Key features

• Speed: 1.5 hour runs

• Scalability: 10 Mb to 1 Gb

• Simplicity: automated workflows, benchtop convenience

• Affordable



Achieved in 2011

• 100-fold scaling and 200 bp kits, 525-base perfect reads achieved

• Breakthrough Ion AmpliSeq™ Designer, microbial, and RNA-Seq apps

• 5,000 member Ion Community

2012 Roadmap

• 2 x 200 paired-end kit, 400 bp kits

• Custom and fixed Ion AmpliSeq™ Panels

• FDA submission and CE-IVD certification

The promise of semiconductor sequencing First 100-fold scaling delivered and more

Ion 314™ Chip

Ion 316™ Chip

Ion 318™ Chip*



Sedum album—small genome and facultative CAM plant

• Sedum album, white stonecrop− 142 Mb by flow cytometry

− 2n = 34

− Hart, 1991

• C3-CAM photosynthesis switching− Facultative crassulacean acid metabolism (CAM) plant

− Under well-watered conditions, fixes carbon through C3 photosynthesis (light)

− During drought, switches to CAM and fixes carbon at night (dark)

Courtesy of Dr. Todd Michael, Monsanto, PAG Conference 2012, San Diego, USA



Genome and transcriptome analysis on theIon PGM™ Sequencer in <20 days*

Day 1Buy plant

Day 2–3Extract DNA and make Ion library

Day 4–820 Ion runs

Day 9–10Assemble genome

Day 11–13Extract RNA, sequencing libraries

Day 15–17Transcriptomeanalysis

*Twenty noncontiguous days.

Courtesy of Dr. Todd Michael, Monsanto, PAG Conference 2012, San Diego, USA with minor modification



Genome assembly, gene calling, and annotation

• Error-correct raw reads (SAET)

• Remove any remaining adaptors

• Assemble using CLC

• Annotate using SNAP (against Arabidopsis) to improve the assembly

Avg stDev Min Max Range Median L50 (bp) N50 #Seq #Base

1,205 1,111 501 32,123 31,622 822 1,400 23,085 101,283 121,999,640




drought_onlydrought_up_sigwater_up_sigwater_only

Transcriptome profiling: 1,183 genes significantly differentially expressed

Drought_only: genes only expressed under drought condition, not detected under waterconditionDrought _up_sig: genes significantly up-regulated under drought conditionWater_up_sig: genes significantly up-regulated under water conditionWater_only: genes only expressed under water condition, not detected under drought condition

Courtesy of Dr. Todd Michael, Monsanto, PAG Conference 2012, San Diego, USA with minor modification



Conclusions of the Monsanto Corporation Sedum album sequencing project

• Ion PGM™ System provides a low-cost and robust platform for genome and transcriptome discovery

• Sedum album genome is small; similar to the size of Arabidopsis

• Drought reduces cell wall gene expression

• FRIGIDA is up-regulated suggesting flowering suppression




De novo assembly of a 5 Mb algae genome using an extra-long read sequencing protocol

Clancy et al. PAG Conference 2012, San Diego, USA

Leptolyngbya sp. strain BL 0902 gDNA

Sheared into ~400 bp fragments by acoustic disruption

Sequencing library prep:end-repairing sheared DNA, ligating Ion adaptors, sizing ~480 bp library fragments

Target enrichment with modifications to enable extra-long templating (400 bp)

Ion PGM™ Sequencing: Ion 316™ Chips

de novo assembly and data analysis

Conclusion: The >350 bp read protocol yielded a contig N50>16,000 bp (largest size = 105,000 bp). The long read length enabled de novo assembly of this 5 Mb genome in a single day.



Coming soon—the Ion Proton™ Sequencer The benchtop genome center

• Supports Ion Proton™ I and Proton™ II chips: for any plant genomes− Proton™ I chip : 165 million

wells, up to 10 Gb data

− Proton™ II chips: 660 million wells, up to 20x coverage of human size genome

• State-of-the art electronics to support highest throughput

The content provided herein may relate to products that have not been officially released and is subject to change without notice



Unprecedented scale increases every 6 months


Plant Resequencing & SNP Genotyping


Plant genotyping and SNP discovery

• SNP (single nucleotide polymorphism): A small variation in DNA sequences of a genome. These variations can be used to track inheritance in families or species.

• Plant SNP discovery: discover SNPs associated with desired traits to improve or enhance certain characteristics such as higher yield or better stress tolerance.

• SNP discovery and trait association study strategies− SNP microarray arrays: identify informative SNPs in a collection of known SNPs

− Targeted resequencing: discover known SNPs and identify informative SNPs



Life Technologies SNP genotyping solutionsResearch projects Best

approachLife Technologies

platformsWhy

Compare sequences of several crop variants to discover functional SNPs

Genotyping by sequencing

(GBS)

Ion Proton™ and PGM™ • Speed, cost, scalability, simplicity, ease of use

Map up to 10 SNPs in different regions of the genome

Fragment analysis

3500 Genetic Analyzer • Multiplexing capability: up to 10 SNPs per reaction

• Gold standard• Accuracy, low cost• Simple workflow

Map SNPs in a small region of the genome

Sanger sequencing

Confirm putative SNP and develop SNP assays

Real-time PCR QuantStudio™ 12K Flex

• Easy and fast workflow• Gold standard• High call rate and accuracy• Formats for different

project sizes (No. of SNPs xNo. of samples)



Targeted resequencing on the Ion PGM™ Sequencer

• The fastest sequencing runs and overall workflow

• Flexible solutions regardless of the size of the amplicon or target region

• Scalable for resequencing project needs, whether running single samples or multiplexing



Genotyping by sequencing (GBS) in plants• NGS has greatly increased SNP discovery in crop plant species such as rice, maize, soybean,

sorghum, and even in wheat’s predecessor, Aegilops tauschii

• GBS using next-generation sequencing technologies is becoming increasingly important:

• It is cost-effective

• It offers utility with complex genomes and those without a reference sequence

• GBS is a good approach for:

• Marker discovery

• Linkage mapping of QTL in a biparental cross

• Fine-mapping QTL

• Bulked segregant analysis (BSA)

• Genome-wide association studies (GWAS)

• NAM-GWAS

• Improving reference genome assembly



Advantages of genotyping by sequencing

• Faster, simpler protocol than traditional restriction-site-associated DNA (RAD) method or full de novo sequencing

• Allows de novo marker (SNP) discovery, even in the absence of a reference genome

• High accuracy of SNP calling

• Low cost

• Low amounts of input DNA needed

• Simplified computational analysis



Genotyping by Sequencing Strategies and Workflows

• For unknown SNPs (discovery): restriction enzyme digestion is employed to reduce complexity

• Known SNPs (screening): multiplexing PCR primers are designed and barcoded to screen hundreds of SNPs in one sequencing run



GBS on Ion PGM™ Sequencer: a case study on barley

• Drs. Nils Stein (IPK, Germany) and Jesse Poland (USDA-ARS2, Manhattan, KS, USA) partnered with Life Technologies to develop a protocol for GBS in barley using two restriction enzymes

• The challenge: large, complex genome without complete sequence available to date (~5.5 Gb, diploid)

• The goal: develop barley plants with improved traits (e.g., drought tolerance, higher yield)

− Discovery of high-density molecular markers is required for better understanding of genetics of complex traits for breeding

− Approach: genome-wide association (GWAS) studies and genomic selection



GBS approach for barley: RE and Ion PGM™ Sequencer• Restriction enzyme digestion of the genomic DNA to reduce complexity

− GBS targets the genomic sequence flanking restriction enzyme sites

• GBS is similar to RAD (restriction-site associated DNA) tagging but has greatly simplified library construction that:

− Requires less DNA and avoids random shearing

− Is completed in two steps followed by PCR of the pooled library

• For barley, the original GBS protocol [1] was extended to a two-restriction-enzyme approach [2]

• Completed a GBS feasibility study using the Ion PGM™ Sequencer

[1] Elshire et al. (2011) A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species. PLoS ONE 6(5):e19379. doi:10.1371/journal.pone.0019379.

[2] Poland et al. (2012) Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach. PLoS ONE 7(2):e32253. doi:10.1371/journal.pone.0032253.



Barley‘s GBS using the restriction enzyme approach

Simplified workflow chart for GBS library preparation using two restriction enzymes for barley

1. Plant gDNA cleavage using PstI and MspI for desired restriction fragments

2. Ligation of specific and common adapters

3. Fragment preamplification followed by NGS on Ion PGM™ Sequencer

1

2

3



Barley GBS using Ion PGM™ Sequencer

• Feasibility study

• 4 barley samples; 2 parental, 2 F1-hybrids

• Sample prep using custom protocol

• Individual library preparation (previous slide)

• Multiplexed sequencing (barcodes)

− Ion PGM™ Sequencer, Ion 316 ™ Chip, 200 bp sequencing

• 2-day protocol

− Day 1: Library prep, template prep (Ion OneTouch™ System)

− Day 2: Enrichment (Ion OneTouch™ ES), Ion PGM™ Sequencing



Sequencing and SNP results in barley

• ~200 Mb Q20 sequence

− Approx. 500 k restriction fragments sequenced at 200 bp per sample

− 1-fold base coverage achieved in this study

• Good sample separation through barcodes

− >90% barcodes separated

− Barcode sequence followed by exact match to restriction site

• Roughly 5,000 SNPs per sample called

− SNP agreement >99.5% between Ion PGM™ Sequencer and Illumina® HiSeq® System (NGS platform previously used by collaborator)

− Customer statement: “Concordance is as high as between runs on our platform”

• Technical feasibility acknowledged



Data analysis

Two independent approaches used

• KSU: TASSEL pipeline

• Life Technologies − Mapping/Alignment

> Torrent Suite Software v2.1; TMAP (Torrent Mapping alignment program)

> Input is SFF file format, output is SAMtools BAM file format

− SNP calling

> SAMtools* mpileup (http://samtools.sourceforge.net/mpileup.shtml)

> Output is ‘variant call format’ (VCF)

*The Sequence Alignment/Map (SAM) format and SAMtools: bioinformatics.oxfordjournals.org/content/early/2009/06/08/bioinformatics.btp352



Conclusions and outlook

• Promising results led to an extended study (phase 2; in progress)

• Design:

− Increased sample number (two 24-plex pools)

− Increased coverage for higher SNP-counts per sample

> Ion 318™ Chips, 200 bp sequencing

− Comparison of Life Technologies sample prep solutions with customer protocol

• Data to be compared to Illumina® HiSeq® results

• Ion semiconductor sequencing has huge potential for large GBS studies:

− High SNP calling accuracy

− Highly competitive cost per sample

− Unmatched sequencing workflow speed Learn morelifetechnologies.com/agbiolifetechnologies.com/gbs


http://www.lifetechnologies.com/agbio

http://www.lifetechnologies.com/gbs

QTL Mapping & Marker-Assisted Selection


A

Gene of interest

A Marker linked to gene

X

A

Cycles of breeding

A A A

A

A

X Eliminateindividualswithout marker

New variety

Courtesy of Jochum Wiersma, U. Minnesota Extension

• Useful in early generations• Ability to select for recessive alleles • Fast and cost-effective

Marker-assisted selection (MAS)



* number of SNPs or other markers in a design/panel** number of samples screened using a design/panel

Life Technologies solutions for QTL mapping and marker-assisted selection

Number of samples in the project**

Number of SNPs in the project*

10

100

500

5,000

10 100 1,000 5,000

TaqMan® Assays, HRM, SSRs•Fast and Easy workflow•Gold standard with high call rate and accuracy (TaqMan®)•Very low cost (HRM)

Genotyping by sequencing (GBS) on

Ion PGM™ System•Low cost for total project•Fast and easy workflow•Flexibility•Affordability

500

TaqMan® Assays on QuantStudio™ OpenArray®

•Low cost•Proven TaqMan® chemistry•Streamlined workflow•Reduction in consumables and steps

TaqMan® Assays on Douglas Array Tape™•High throughput•Automated workflow•Low cost•Proven TaqMan® chemistry



Life Technologies solutions for QTL mapping and marker-assisted selection



Examples of publications using TaqMan® SNP and SSR genotyping assays for MAS



Designing TaqMan® assays for crops with unknown genomes: canola example

• Canola (Brassica napus): a tetraploid crop converged from diploid Brassica rapa and Brassica oleracea

• Canola genome has not been sequenced

• Assay design pipeline predicted propensity to cross hybridize to nontarget loci in the genome by mapping the assays to B. rapa and B. oleacera

• Genome cross-hybridization analysis increased the success rate by 30%

www.nrc-cnrc.gc.ca/eng/news/pbi/2011/08/28/brassica.html

Brzoska, et al. Plant and Animal Genomics. San Diego, CA.2011.

http://www.nrc-cnrc.gc.ca/eng/news/pbi/2011/08/28/brassica.html





TaqMan® OpenArray® on QuantStudio™12K Flex Real-Time PCR System• Fast and simple workflow: 4 hours from DNA to genotyping call

• High sample throughput: screen up to 256 SNPs across >1,500 samples (>70k data points) in one day without the use of robotics

• Low cost per data point

Wrong, et al. Maize Genetics Conference, Portland, OR. 2012

Assays Samples

16 144

32 96

64 48

128 24

192 16

256 2

OpenArray® flexible formats



High-throughput SSR genotyping by HRM

• The goal: develop an accurate SSR genotyping approach using HRM that is compatible with high-throughput breeding programs in Jatropha curcas

• Subtropical plant that produces a high-quality oil for biodiesel, renewable jet fuel, or specialty products

• Low genetic variation in geographic regions outside Central America (where the plant originated)

• HRM assays (MeltDoctor™ HRM Master Mix) and ViiA™ 7 Real-Time PCR System were used to discover remarkable genetic diversity in the SG Biofuels germplasm collection

• Results

− High allelic polymorphism of SSR26 in the SG Biofuels germplasm collection

− Among 380 unique accessions, 9 alleles and 24 different genotypes were detected by HRM and confirmed by sequencing

Download the application note here

http://www.sgfuel.com/index.php

http://www.invitrogen.com/etc/medialib/files/Cloning/PDFs.Par.12187.File.dat/CO24629_HRM-Analysis-Real-Time-PCR-LifeTechnologies.pdf


GMO Detection & Screening


Genetically modified crops: facts and regulations• Genetically modified (GM) crops: first cultivated on a commercial scale in 1996;

major GM crops include soybeans, corn, alfalfa, canola, and cotton

• GM crops are grown on 1/10 of total cultivated land globally (170 million acres) and are expected to expand to 20 million farmers in 40 nations by 2015

• GMO testing in seed, grain, and processed food and their ingredients is required in many countries− Amount of GM ingredients that can be present in a food product without being labeled as

“GM” is 0.5% in EU and 5% in Japan

Agricultural Biotechnology - A Global strategic business report. Global Industry Analysts, Inc. 10/2010Map (2008 view): sustainablelinfield.edublogs.org/files/2011/05/Picture-1-1y5b0mr.png

http://sustainablelinfield.edublogs.org/files/2011/05/Picture-1-1y5b0mr.png

http://sustainablelinfield.edublogs.org/files/2011/05/Picture-1-1y5b0mr.png



Technologies for GMO testing and detection

• Digital PCR on QuantStudio™ 12K Real-Time PCR System for:

− Contamination or GMO detection

− Rare mutation detection

• TaqMan® Real-time PCR assays

• Dedicated TaqMan® GMO kits:

− TaqMan® GMO Maize 35S Detection

− TaqMan® GMO Soy 35S Detection



GMO detection of maize endogenous reference genes using TaqMan® Real-Time PCR Assays

• Research goal: select the best TaqMan® Assay and real-time PCR condition to detect GMO maize

• Approach: − Five TaqMan® real-time PCR assays targeting adh1 and hmg genes were designed and

tested using different amplification profiles on the 7900HT system

− Equal amounts of DNA from 7 EU-certified maize flours were pooled

− Both pooled and single DNAs were serially diluted 8 times

• Conclusion: TaqMan® Real-Time PCR assay targeting maize endogenous reference genes can be used for the quantification of transgenic events in Zea mays

PATERNO` et al. J. Agric. Food Chem. 2009, 57, 11086–11091

Relative standard deviation (RSD)% over the concentration rangeAssay met4 (hmg) demonstrated the best regression parameters and a higher repeatability over the dilution rage



Contaminant detection in soybean by dPCR on the OpenArray® System

• 6 allele-specific TaqMan® SNP Assays were designed and validated for distinguishing soybean strains

• Achieved a detection sensitivity of 1:10,000 contaminant variety B in variety A soybean seed DNA background

• The dPCR approach is an ideal solution for GMO testing and other AgBio solutions

Webster et al. Plant and Animal Genomics, San Diego, 2012

Spike-in simulation of seed contaminationDigital PCR workflow


Other Products & ApplicationsPlant Genetic EngineeringGene Expression AnalysisPlant DNA and RNA Isolation Products


Plant genetic engineering as a tool for plant research

A basic primer on biotechnology. Dr. Peel, NDSU Extension, October 2011.

Plant transformation and regeneration of transgenic plants is a key approach for plant research:

− Understanding gene expression and regulation

− Decipher metabolic and signal pathways

− Developing plants with new characteristics

What are the challenges?

− Manipulation of DNA elements

− Effective plant transformation and tissue culture techniques

− Achieve desirable gene expression levels and ultimately, the desired plant phenotype

http://www.ag.ndsu.edu/pubs/plantsci/crops/a1219w.htm



Plant genetic engineering solutions

If you want to do your own cloning

• Plant DNA cloning and site-directed mutagenesis kits

• Competent E.coli cells and transformation

• Custom DNA oligonucleotides

• PCR enzymes and thermal cyclers

If you want us to do the cloning for you

• Cloning services

• GeneArt® gene synthesis services

Plant transformation

• Agrobacterium tumefaciens LBA4404 competent cells

• Antibiotics: carbenicillin, kanamycin, hygromycin B

Genome engineering

• GeneArt® Precision TALs

GeneArt® Chlamydomonas Engineering Kits

NEW

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/cloning-methods/plant-dna-cloning.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Transformation/Trans-Misc/Learn_More_About_Choosing_Competent_Cells.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Product-Types/Primers-Oligos-Nucleotides/invitrogen-custom-dna-oligos.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/PCR.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Services/custom-services/Molecular-Biology-Services/Cloning-Services.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/gene-synthesis.html

http://products.invitrogen.com/ivgn/product/18313015?ICID=search-product

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/gene-synthesis/geneart-precision-tals.html



Custom DNA binding proteins for precision DNA targeting

What are they used for?

• Gene targeting (Fok1 nuclease pair)

• Silencing

• Incorporation of exogenous DNA

• Activation (activator vp16 or vp64)

• Increasing the expression level of endogenous gene isoforms

• Effector domain targeting (MCS vector)

• Target any locus in the genome with the effectordomain of your choice with our multiple cloning site vector

GeneArt® Precision TALs—our new service for genome editing



TAL effector technologyHow TALs function

• Bacterial pathogen proteins redirect transcription of host plants upon infection

• TAL proteins use discrete domains to recognize A, T, G, and C nucleotides in dsDNA

Engineered system

• Modular assembly of domains allows for creation of sequence specific DNA binding proteins

Why is this technology so compelling?

• Simple code for creating engineered TAL proteins: no bias except for a 5’ T

• More predictable than Zn fingers

• One-to-one correspondence between the identity of two critical amino acids in each repeat and each DNA base in the target sequence



53

QC1) Submit preverification2) Intermediate assembly sequence3) TAL terminus sequence4) TAL size

•Access the GeneArt® web portal from the Life Technologies website

•Download and complete the order form

•Email the completed form to [email protected]•All inquiries will be answered within 24 hours•Production starts within 24 hours of ordering•3 weeks from order to deliver

GeneArt® Precision TALs—ordering

http://www.invitrogen.com/site/us/en/home/Global/forms/precision-tal-request-form.html

mailto:[email protected]



Gene expression analysis

RNA-Seq for whole transcriptome sequencing

Targeted mRNA expression using TaqMan® Assays

NCode™ miRNA qRT-PCR Kits

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/agricultural-biotechnology/plant-gene-expression-analysis.html


Plant DNA isolation solutionsTotal RNA

LysisSolution

TaqMan® Sample-to-

SNP™ Kit

Plant DNAzol® Reagent

ChargeSwitch® gDNA Plant Kit

PureLink®

Genomic Plant Kit

MagMAX™

DNA Multi-Sample Kit

Key features Bulk buffers for crude extraction

Extraction+ TaqMan® Assay combined

Most efficient for large amounts of tissue

Most suitable for GMO testing

Low abundance DNA samples

Low abundance DNA samples

Downstream applications

PCR, real-time PCR

Real-time PCR

All All All All

Protocol time <15 min 5 min <60 min <15 min <40 min <40 min

Starting materials

≥0.1 g 2–3 mm punch

≥0.1 g 50–100 mg 100 mg 5–10 mg

Yield Varies Varies Varies Up to 7 µg Up to 14.6 µg Up to 14.6 µg

Isolation technologies

Bulk lysis buffer

Lysis solution

Organic extraction

ChargeSwitch® + magnetic beads

Silica spin column

Magnetic beads

Automatable No Yes No Yes No Yes

High-throughput

Yes Yes Yes Yes No Yes



Plant RNA isolation solutions

Plant RNA Reagent MagMax™-96 RNA Isolation Kit

PureLink® RNA Mini Kit

mirVana™ miRNAIsolation Kit

Key features Great for difficult samples (conifer tissue and seeds)

Rapid and fully automated

Quick and easy to use

Efficient recovery of miRNA and small RNA

Protocol time 60 min <45 min <20 min 30 min

Starting materials Up to 1 g Up to 10 mg <50 mg 0.5–200 mg

Isolation technologies

Organic extraction Magnetic beads Silica column Organic extraction and silica column

High-throughput compatible

No Yes No No



Full-spectrum genetic analysis and beyond Solutions in plant agriculture biotechnology

Genome sequencing

Transcriptome sequencing

Genotyping by sequencing

SNP discovery

QTL mapping

Marker-assisted selection

Targeted sequencing

Sequence confirmation

SNP confirmation

Microsatellite/SSR analysis


QTL analysis


SNP confirmation

HRM genotyping

GMO testing

Sample QC

Rare allele detection

Targeted gene expression

miRNA analysis


Gene synthesis

Genome editing

(TAL effectors)

Plant DNA cloning

Plant transformation

Mutagenesis

Discover EngineerConfirm and screen

Plant RNA & DNA isolation and purification


One globe, one team, one goalQuestions or comments? Looking for custom solutions?

---- We are always here to support you

• Contact us at [email protected]

• Visit us at www.lifetechnologies.com/plants

For Research Use Only. Not for use in diagnostic purposes.© 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners.

mailto:[email protected]

http://www.lifetechnologies.com/plants

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Genetic Analysis Solutions for Plant Sciences

Education