Direct DNA Amplification from Plants
Leta Steffen, Samantha Lewis, Doug Wieczorek, Sandra Nguyen, Trista Schagat, and Doug Storts
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI, 53711
4. Direct amplification from oil palm leaf
Plant Direct Amp System
Direct amplification without purification is now a workflow option for a wider
selection of plants, enabling high throughput genetic mapping, genotyping,
transgenic screening, and sexing.
#2
Tomato (lat52)
#1
Lysates shown in Fig.3 were
stored at 4°C for 11 months
and re-amplified with plantspecific primers in triplicate
20µL reactions with GoTaq®
qPCR Master Mix. (Tomato
lysates were tested
undiluted; Soy and corn
lysates were diluted 1/5
before amplification; NTC
reactions are shown in
green.)
#3
Oil Palm (shell)
DNA was extracted from fresh 5mm oil palm leaf punches in triplicate on
two separate occasions. Lysates were diluted 1/10, and 2µL of diluted
lysate amplified in duplicate 20µL endpoint PCR reactions using primers to
the shell gene5 with GoTaq® Green MasterMix (above left), or 20µL dyebased PCR reactions using universal plant primers and GoTaq® qPCR
Master Mix (above right, NTC reactions are shown in black).
5. Direct amplification from ground seeds
Soy (lectin)
Genetic marker-assisted breeding of plants is limited by the need to purify
nucleic acids from plant tissues. Non-commercial protocols for preparing
crude extracts suffer from limited utility across diverse plant species,
limited utility in high-throughput applications, and the use of high
temperature acids and bases.
We have developed a reagent for preparing crude DNA extracts from a
variety of plant species that can be directly amplified in endpoint PCR,
dye-based real time PCR, and probe-based real time PCR. Lysis can be
performed in 5 minutes, uses no toxic or reactive chemicals, and is
compatible with high-throughput automation. Using single 6-8mm leaf
punches from tomato, soy, corn, and cucumber leaves, crude lysates that
were prepared with the Promega Plant Direct Amp System showed earlier
and more consistent amplification than lysates prepared using Hot Sodium
Hydroxide and Tris (HotSHOT) methods. Amplifiable DNA can also be
extracted from seeds, and from difficult plant species (e.g. oil palm) using
the Plant Direct Amp System. Lysates are compatible with standard and
fast cycling using GoTaq® qPCR Master Mix and GoTaq® Probe qPCR
Master Mix, and can be decanted and stored at 4°C for at least a year, or
further purified as needed.
7. Leaf lysates are amplifiable for > 1 year
The same samples were
amplified again after 14
months storage at 4°C in
triplicate 20µL reactions with
GoTaq® G2 Hot Start Green
MasterMix and analyzed on a
2% agarose/TBE Gel with
6µL BenchTop PCR Markers.
Corn (hmg)
1. Abstract
2. Fast and easy extraction of amplifiable
DNA from plants
#1
Prepare
sample
Lyse
sample
Amplify
Grind seeds in tubes or plates
Tomato (lat52)
Add lysis reagent; heat 5 min at 70-85°C*
Add lysis reagent; heat 5 min at 70-85°C*
DNA was extracted from ground pumpkin, tomato, and watermelon seeds
(n=4 each, various strains), and diluted 1/10. Samples were amplified in
triplicate 20µL PCR reactions with plant-specific primers (A-H) using
GoTaq® qPCR MasterMix. Mean Cq values are plotted for each sample.
Use in
5-20µL
reactions
Endpoint PCR
Probe-based real-time PCR
Dye-based real-time PCR
6. Superior amplification performance, even
with HotSHOT lysates
DNA was extracted from leaf punches using the HotSHOT method and 1µL
was amplified in triplicate 5µL reactions using Promega GoTaq® Probe
qPCR Master Mix, TaqMan® GTExpress Master Mix, or TaqMan®
Universal II Master Mix.
3. Amplifiable DNA from a variety of plant
leaves
Tomato (lat522)
Tomato (lat52)
Promega
GoTaq Probe
Lysates shown in Fig.4 were further purified using the Maxwell® 16 FFS
Nucleic Acid Extraction System, Custom (X9431) or the Maxwell® RSC
Cell DNA Purification Kit (AS1370) (data not shown) and eluted in 50µL.
Purified DNA was analyzed undiluted, and diluted 1/10, in 20µL reactions
with universal plant primers in GoTaq® qPCR Master Mix. (NTC reactions
are shown in black.) The ΔCq between dilutions is 3.28; 3.33 is expected
for samples free of qPCR inhibition.
TaqMan
GTExpress
HotSHOT
Soy (lectin)
9. Conclusions
Quick and easy protocol for direct amplification
• Lyse leaf punches or ground seeds in just 5 minutes
• Reagent is non-toxic and room temperature stable
Lysates can be stored at 4°C or further purified
• Direct amplification method allows for rapid primary screening of
plant samples
• Lysate is stable, and compatible with traditional purification
chemistries
TaqMan
Universal II
DNA was extracted from fresh 6-8mm leaf punches in triplicate from
tomato, soy, or corn leaves. DNA from crude lysates was amplified (n=3)
using 1µL in a 5µL reaction with plant-specific primers/probe and GoTaq®
Probe qPCR Master Mix. For comparison, equivalent samples were
prepared using the HotSHOT1 method (10 minutes, 95°C extraction in
alkaline extraction buffer, followed by acid neutralization) and similarly
amplified. NTC reactions are shown in black. Lysis with the Plant Direct
Amp System is faster, demonstrates more robust performance with
difficult plants such as tomato, and yields DNA that amplifies earlier than
HotSHOT-prepared samples.
Soy (lectin3)
8. Lysates can be further purified for
sensitive downstream applications
Centrifuge, transfer lysate, dilute 1/10
*depending on heat block and plate format
Plant Direct Amp System
#3
Array leaf punches in tubes or plates
Transfer lysate
Transfer
lysate
#2
References:
1Truett
GE et al. Preparation of PCR quality mouse genomic DNA with Hot Sodium
Hydroxide and Tris (HotSHOT). BioTechniques 29, 52-54 (2000).
The Plant Direct Amp System offers maximum flexibility for a
range of throughputs
• Compatible with leaf and seed from a wide variety of plants
• Automation and high-throughput friendly
• Compatible with endpoint, dye-, and probe-based PCR
• Lysates can be used in 5µL – 20µL PCR reactions
Corn (hmg4)
2Yang
L et al. Validation of a tomato-specific gene, LAT52, used as an endogenous
reference gene in qualitative and real-time quantitative PCR detection of transgenic
tomatoes. J Agric Food Chem 53, 183-190 (2005).
3Moriuchi
R et al. Applicability of quantitative PCR to soy processed foods containing
Roundup Ready Soy. Food Control 18, 191-195 (2007).
4Chiueh
L et al. Establishment of quantitative method for five events of genetically modified
maize (Bt11, Event176, GA21, MON810 and T25) by real-time qPCR. J Food Drug Anal
12(4), 316-323 (2004).
Acknowledgements: Ground seeds, primer sequences, and
cycling conditions used in Fig. 5 were generously provided by CSP
Labs, Inc.
5Singh
R et al. The oil palm SHELL gene controls oil yield and encodes a homologue of
SEEDSTICK. Nature 500, 340-344 (2013).
www.promega.com
Corresponding author: [email protected]