Download Use of a single primer to fluorescently label selective amplified

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Use of a single primer to fluorescently
label selective amplified fragment length
polymorphism reactions
Ledare Habera, Naomi Smith, Ryan Donahoo, and Kurt Lamour
University of Tennessee, Knoxville, TN, USA
BioTechniques 37:902-904 (December 2004)
902 BioTechniques
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et al. through the selective amplification
step with the addition of a labeling step
(6). Specifically, unlabeled selective
reactions are diluted 500× and used as
template for an additional nonselective
reaction using a fluorescently labeled
preselective primer. We have tested
this strategy on a variety of organisms
including Phytophthora capsici,
horseweed (Conyza Canadensis),
dogwood (Cornus florida), and soybean
cyst nematodes (SCN) (Heterodera
glycines) comparing AFLP profiles
generated using fluorescently labeled
selective primers versus AFLP profiles
labeled using nonselective primers. In
each case, the profiles generated were
identical (Figure 1).
In brief, high molecular weight DNA
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Amplified
fragment
length
polymorphism (AFLP) markers are
used for a variety of genetic applications including population genetic
studies (1,2), mapping (3), and gene
discovery (4). Genomic DNA or cDNA
is digested with restriction enzymes
in the presence of synthetic adaptors
in a “restriction/ligation” reaction that
produces a population of anonymous
DNA fragments with known ends.
Selective primers complementary to
the adaptors with additional 3′ nucleotides are then used to amplify specific
subsets of the modified fragments under
stringent PCR conditions. Typically, the
resulting AFLP profiles are separated
using polyacrylamide, and the markers
are scored for presence or absence (5).
One of the advantages of AFLP
is that a large number of markers can
be generated based on differential
combinations of selective primers
(5). The use of fluorescently labeled
selective primers in conjunction with a
capillary sequencing device provides a
robust platform for resolving fragment
profiles, but a limiting factor for this
type of resolution is the cost of fluorescently labeled selective primers. Cost
includes the relatively short lifespan
of fluorescently labeled primers. We
report here a strategy for labeling
selective AFLP products using a single
fluorescently labeled AFLP primer.
This strategy requires no new technical
skills on the part of the investigator and
provides greater consistency as batchto-batch variation in primer synthesis is
lessened.
Our AFLP protocol is very similar to
the original description of AFLP by Vos
was extracted from approximately
10 mg of freeze-dried material using
DNeasy® mini-kits (Qiagen, Valencia,
CA, USA). Freeze-dried starting
material consisted of Phytophthora
mycelium grown in antibiotic-amended
V8 broth, SCN eggs, and leaf discs of
horseweed and dogwood leaves. DNA
was quantified on an agarose gel with
known standards. EcoRI was obtained
from Invitrogen (Carlsbad, CA, USA),
while MseI and T4 DNA ligase were
obtained from Takara (Madison, WI,
USA). EcoRI and MseI adapters and
primers for ligation and amplification
reactions were obtained from Integrated
DNA Technologies (Coralville, IA,
USA). For sequences for the adapters
and primers, see Vos et al. (6). The
fluorescently labeled primers were
obtained from Proligo (Boulder, CO,
USA) and are comprised of the EcoRI
core sequence with and without any
selective nucleotides and either the
WellRED D4-PA label (Proligo) or the
WellRED D3-PA label at the 5′ end.
Restriction and ligation reactions
were carried out simultaneously in
11-μL volumes containing 150 ng of
genomic DNA in 1× T4 ligase buffer,
0.5 M NaCl, 0.045 M bovine serum
albumin (BSA), 0.5 μM EcoRI adapter,
5.0 μM MseI adapter, 5 U EcoRI, 1
U MseI, and 1 U T4 DNA Ligase.
Reactions were placed on a Dyad®
PTC-220 thermal cycler (MJ Research,
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Figure 1. Phytophthora capsici amplified fragment length polymorphism (AFLP) electropherograms generated using selective primers E+AC/M+CC. (A) The fragments were labeled using a labeled E+AC in a standard reaction. (B) The fragments were labeled using a labeled E+00 in an additional nonselective labeling reaction. Fragments were resolved and visualized using a CEQ 8000 Genetic
Analysis System. nt, nucleotides; a.u., arbitrary units.
Vol. 37, No. 6 (2004)
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Waltham, MA, USA) for 37°C for 2 h
followed by 72°C for 15 min or allowed
to incubate overnight at room temperature. Following the restriction-ligation
reactions, the template was diluted by
the addition of 189 μL 10 mM Tris.
Preamplification
reactions
were carried out in 20-μL volumes
containing 5.0 μL dilute restriction
ligation products, 1× PCR buffer, 0.2
mM dNTPs, 0.275 μM EcoRI and MseI
primers, and 1 U Taq DNA polymerase
(Fisher Scientific, Pittsburgh, PA,
USA). Preamplification reactions were
performed on a Dyad PTC-220 thermal
cycler using the following cycling
parameters: initial incubation at 72°C
for 2 min, followed by 20 cycles of
94°C for 30 s, 56°C for 30 s, and 72°C
for 2 min, with a final extension at
72°C for 2 min, and a final incubation
at 60°C for 30 min. Upon completion
of the preamplification, the reaction
products were diluted by adding 135
μL 10 mM Tris.
Selective amplification reactions
were carried out with both labeled and
unlabeled selective primer pairs in 20-μL
volumes containing 5.0 μL dilute preamplification products, 1× PCR buffer, 0.2
mM dNTPs, 0.275 μM EcoRI + 2 primer
(E+2), and MseI + 2 primer (M+2) for
Phytophthora and SCN, and EcoRI
+ 3 primer (E+3) and MseI + 3 primer
(M+3) for horseweed and dogwood, and
1 U Taq DNA polymerase. Selective
amplification reactions were performed
using the following cycling parameters:
initial incubation at 94°C for 2 min,
followed by one cycle of 94°C for 20
s, 66°C for 30 s, and 72°C for 2 min;
cycles 2–11: 94°C for 20 s, decrease
1°C every cycle for 30 s, and 72°C for 2
min; cycles 12–31: 94°C for 20 s, 56°C
for 30 s, and 72°C for 2 min, followed
by a final incubation at 60°C for 30 min.
Upon completion of the program, the
unlabeled selectively amplified products
were diluted 1:500 in 10 mM Tris.
For the selective reactions
performed with unlabeled selective
primers, a second round of selective
amplification was performed using the
1:500 dilute products as template. We
tested 1:10, 1:100, 1:500, 1:750, and
1:1000 dilutions, and 1:500 provided
the optimal signal. This extra labeling
reaction is accomplished by substituting the E+2 or the E+3 with an E+00
904 BioTechniques
EcoRI primer containing either the
WellRED D4-PA or the WellRED D3PA label.
Fluorescent products from both
the traditional and second round
selective amplification were analyzed
on a CEQ™ 8000 Genetic Analysis
System (Beckman Coulter, Fullerton,
CA, USA) using the manufacturer’s
protocols. Figure 1 illustrates typical
results from selective AFLP reactions
labeled using this single primer
strategy versus AFLP reactions using
fluorescently labeled selective primers.
We have tested this strategy on over
100 genomic DNA samples comparing
both labeling methods and have found
no significant differences between the
AFLP profiles generated. Co-loading of
AFLP fragments generated using two
different WellRED dyes (multiplexing)
gave identical results to loading each
product separately. The key to this
procedure is the 500-fold dilution of
the unlabeled selective PCR products.
Considering the cost of individual dyelabeled primers and the limited lifespan
of fluorescent dyes, this strategy can
significantly reduce the cost of generating AFLP markers from multiple
selective primer combinations.
am. Plant Dis. 87:841-845.
3.Van der Lee, T., I. De Witte, A. Drenth, C.
Alfonso, and F. Govers. 1997. AFLP linkage
map of the oomycete Phytophthora infestans.
Fungal Genet. Biol. 21:278-291.
4.Durrant, W.E., O. Rowland, P. Piedras,
K.E. Hammond-Kosack, and J.D.G. Jones.
2000. cDNA-AFLP reveals a striking overlap in race-specific resistance and wound
response gene expression profiles. Plant Cell
12:963-977.
5.Blears, M.J., S.A. De Grandis, H. Lee,
and J.T. Trevors. 1998. Amplified fragment
length polymorphism (AFLP): a review of the
procedure and its applications. J. Ind. Micro.
Biotech. 21:99-114.
6.Vos, P., R. Hogers, M. Bleeker, M. Reijans,
T. van der Lee, M. Hornes, A. Frijters, J.
Pot, et al. 1995. AFLP: a new technique for
DNA fingerprinting. Nucleic Acids Res.
23:4407-4414.
Received 19 July 2004; accepted
9 August 2004.
Address correspondence to Kurt Lamour,
The University of Tennessee, Institute of
Agriculture, Knoxville, TN 37996-4560,
USA. e-mail: [email protected]
ACKNOWLEDGMENTS
We are grateful to Matt Smith,
Sharyce Banks, Catherine Zama, Beth
Wilson, and Melinda Tierney for technical assistance. This work was supported in part by funds from the Tennessee Soybean Promotion Board.
COMPETING INTERESTS
STATEMENT
The authors declare no conflicts of
interest.
REFERENCES
1.Lamour, K.H. and M.K. Hausbeck. 2001.
The dynamics of mefenoxam insensitivity in
a recombining population of Phytophthora
capsici characterized with amplified fragment
length polymorphism markers. Phytopathology 91:553-557.
2.Lamour, K.H. and M.K. Hausbeck. 2003.
Effect of crop rotation on the survival of Phytophthora capsici and sensitivity to mefenoxVol. 37, No. 6 (2004)