An Innovative System for Improving DNA Isolation from Diverse Plant Species

Transcription

1 An Innovative System for Improving DNA Isolation from Diverse Plant Species Presented by: Jackson Moeller, Ph.D. Research Specialist, University of Wisconsin Madison and Chris Moreland Global Product Manager, Proprietary Information. Not for further distribution.

2 Webinar Outline Introduction Challenges for isolation DNA from plant samples Maxwell 16 Plant DNA Kit Maxwell 16 Instrument Design and Function The Evaluation Performance Results Conclusions Proprietary Information. Not for further distribution. 2

3 Why Plant Extractions Are Challenging Plant nucleic acid concentration can vary due to the: Source of the starting sample - leaf, node, roots, seed Species Age of sample Condition of the leaf material i.e., dry, tough fibrous material Traditional methods are time consuming- mortar and pestle/ctab phenol/chloroform isolation Downstream amplification issues due to extracted DNA quality (tied to chemistry) Compounds (i.e. polyphenol, polysaccharides) that have been implicated in low yield and downstream inhibition Proprietary Information. Not for further distribution. 3

4 Maxwell 16 LEV Plant DNA Kit Optimized for Plant Genomics Research Research Type Agro Biotech Basic Plant and Ag Research Applications and Areas of Interest Disease resistance, yield, genetic variance for breeding Transgenic seed development, integration of novel genes to create new traits and seed quality Biodiversity, disease resistance, yield improvements, basic and applied research, population genetics, plant stress responses, plant physiology, biofuels Maxwell 16 Plant DNA Kit Proprietary Information. Not for further distribution. 4

5 Maxwell 16 LEV Plant DNA Kit High-quality, amplifiable DNA from corn, soybean and Arabidopsis plant tissue samples Designed for downstream applications like qpcr and next-generation sequencing 48 isolation cartridges and processing reagents Automated extraction from up to 16 samples in the Maxwell 16 Instrument Maxwell 16 Instrument Efficiency and Performance: Automated Extraction of High Quality DNA from Difficult Plant Samples Proprietary Information. Not for further distribution. 5

6 Concentration ( ng/µl) Efficient Purification from a Wide Variety of Plant Sample Types 10 Higher Concentrations (Yield) Promega Competitor Starting Input Samples Soybean/tomato: 20-25mg tissue Wheat: 1 kernel, 25mg Arabidopsis: 20mg tissue Extracted and eluted purified DNA in 100µl Soybean Wheat Arabidopsis Tomato Proprietary Information. Not for further distribution. 6

7 Maxwell Instruments are Automated Magnetic Particle Mover Maxwell 16 Instrument Purification Process Mixing Add Sample LEV Plunger Parallel processing of 1-16 samples Capture Reagent Cartridge Binding Washing & Elution Elution Tube (30-100µl) Proprietary Information. Not for further distribution. 7 7

8 Maxwell 16 Plant DNA Kit Leaf Extraction Protocol 1. Place up to 20mg leaf tissue into bottom of each extraction tube or well. Place beads (as recommended by manufacturer) into each tube or well. Add 300µl tissue lysis buffer, 10 µl RNase A, and 30 µl of Proteinase K (20 mg/ml) to each tube or well. 2. Run the bead beating device using time and speed recommended by the manufacturer. 3. Centrifuge briefly to remove any solid particles from the lysate 4. Add 300 µl of nuclease free water to well #1 of each Maxwell cartridge. Transfer each plant lysate from extraction tube or plate to well #1 of the Maxwell cartridge. 5. Process samples on the Maxwell 16 Instrument using the Maxwell 16 DNA Plant Protocol (~40 minute run time) Proprietary Information. Not for further distribution. 8

9 Maxwell 16 Plant DNA Kit is Compatible with Different Mechanical Leaf Tissue Disruption Methods Grind sample in liquid N 2, add TLA buffer + RNase A Bead-beat samples in TLA buffer + RNase A Bead-beat samples in TLA buffer + RNase A 1. Add 300 µl of water to LEV Blood DNA Cartridge 2. Run with LEV Blood v method 3. Elute into 50 µl of elution buffer Proprietary Information. Not for further distribution. 9

10 Evaluation: University of Wisconsin Department of Botany The Background Dimensions of Biodiversity Grant Analyses of trait variation of native plants population DNA extraction from plant species Employing next gen technology for single nucleotide polymorphisms (SNPs) genotyping Running isolations two or three times Isolation Problem No single DNA isolation method results in high concentration and amplifiability for all species We are gearing up to extract DNA from a diverse set of plant species - with multiple populations and individuals / pop for each. Don Waller Proprietary Information. Not for further distribution. 10

11 Bringing Together a Solution UW s Requirements Consistent highly concentrated and amplifiable DNA across all species within the study Minimal training required Reduced sample processing time Compatibility of purified gdna with downstream applications Proprietary Information. Not for further distribution. 11

12 Experimental Design to Compare Three gdna Purification Methods CTAB - Concentrated and amplifiable DNA - Minimal training - Reduced sample processing time - Downstream assay compatibility 1. DNA isolation using 25 different plant species 2. Three different isolation techniques a. CTAB (Doyle and Doyle, 1987) b. Qiagen DNeasy c. Maxwell 16 Plant DNA kit 3. Compare yield, purity, and amplifiability Proprietary Information. Not for further distribution. 12

13 Purifications Were Performed on 25 Species of Native Plants Genus Species Common Ageratina altissima White snakeroot Alliaria petiolata Garlic mustard Amphicarpea bracteata Hog-peanut Arisaema triphyllum Jack in the pulpit Carex albursina White bear sage Caulophyllum thalictroides Papoose root Clintonia borealis Blue bead lily Circaea lutetiana Enchanters nightshade Leaf tissue samples from four individuals of each of 25 plant species growing in Wisconsin forests Proprietary Information. Not for further distribution. 13

14 Work Flow and Molecular Analysis Plant Samples Homogenization Purification Quality Quantify Amplify Maxwell Homogenize 16 DNA kit, CTAB DNeasy, 100 µl elution Gel Fluorescent detection Purify electrophoresis Quantify qpcr The Analysis Gel electrophoresis using 1% agarose Quantification by fluorescent dye Amplifiability analyzed by qpcr (subset) Sequencing Amplify Proprietary Information. Not for further distribution. 14

15 Results Summary Maxwell 16 DNA kit Outperforms the Competition Outcomes Maxwell 16 Plant DNA Kit produced: Two-fold higher yield More consistent purity More time for other experiments due to simple automation Higher yield from very difficult species Upshot Maxwell 16 Plant DNA kit was shown as a promising solution for plant DNA isolation by: Producing consistently high DNA yields across all species within the study Required minimal amounts of training Reduced the amount of time for sample processing Maxwell is clearly outperforming the DNeasy and CTAB in quantity and quality It's a great product. - Jackson R Moeller, Research Associate UW Madison Proprietary Information. Not for further distribution. 15

16 Performance Results Paramagnetic Cellulose DNA Isolation Improves DNA Yield and Quality Among Diverse Plant Taxa Moeller, J., N. Moehn, D.M. Waller, and T.J. Givnish Paramagnetic cellulose DNA isolation improves DNA yield and quality among diverse plant taxa. Applications in Plant Sciences 2: Proprietary Information. Not for further distribution.

17 Relative Quantity and Quality of DNA Proprietary Information. Not for further distribution. 17

18 DNA concentration (ng/ul) Maxwell 16 DNA Kit Produced Higher Average Concentrations for All Species * (p < , 5E-7) PMC DNeasy CTAB Proprietary Information. Not for further distribution. 18

19 Maxwell 16 DNA Kit Provides an Advantage In Absolute Yield Proprietary Information. Not for further distribution. 19

20 Maxwell 16 DNA Kit Provides Advantage In Absolute Yield Proprietary Information. Not for further distribution. 20

21 Maxwell 16 DNA Kit Produced More Amplifiable DNA Proprietary Information. Not for further distribution. 21

22 Study Conclusions Maxwell 16 Plant DNA Kit provides an advantage over competitive techniques such as CTAB and silica-based spin columns because Less variability Yields amplifiable DNA from challenging samples Greater extraction efficiency Smaller amounts of starting material Less laborious and time-consuming Proprietary Information. Not for further distribution. 22

23 Bringing Together a Solution UW Requirement Consistent highly concentrated and amplifiable DNA across all species within the study Minimal training required Reduced sample processing time Compatibility with downstream applications Resolution Highly concentrated DNA with less variability compared to traditional methods Easy to use, no organic extractions, minimal steps with very little hands-on time Process up to 16 samples in less than an hour Designed for downstream applications like qpcr and next-gen sequencing Proprietary Information. Not for further distribution. 23

24 Maxwell 16 DNA Kit is a Superb Solution for Plant DNA Isolation Maxwell 16 Plant DNA kit is a promising solution for plant DNA isolation by: Producing consistently high DNA yields across all species within the study Requiring minimal training to get up and running Reducing the amount of time for sample processing Producing clean DNA that is highly amplifiable Maxwell 16 Plant DNA Kit (and Instrument) provides an automated, easy-to-use, and consistent nucleic acid extraction system for challenging plant samples Proprietary Information. Not for further distribution. 24

25 Technical Services Scientists Ready to Help By phone: Available 6am-5pm MST M-F By Online promega.com Available 6am-5pm MST M-F Global Chat with Branch office tech serv scientists, too, after hours (language dependent) By techserv@promega.com Guaranteed answers within 24hr Most responses within 2hrs Proprietary Information. Not for further distribution. 25

26 Questions? UW Madison contacts: Dr. Jackson Moeller Professor Thomas Givnish Thank You! Proprietary Information. Not for further distribution. 26