Molecular Ecology Resources (2012) 12, 456463 doi: 10.1111/j.1755-0998.2011.03104.

Universal primers for fluorescent labelling of PCR

fragmentsan efficient and cost-effective approach to
genotyping by fluorescence
M. J. BLACKET,* C. ROBIN,* R. T. GOOD,* S. F. LEE* and A. D. MILLER
*Department of Genetics, Bio21 Institute, The University of Melbourne, Parkville, Vic. 3010, Australia, Department of Zoology,
The University of Melbourne, Parkville, Vic. 3010, Australia, Department of Primary Industries, Knoxfield, Vic. 3180, Australia,
Cesar, 293 Royal Parade, Parkville, Vic. 3052, Australia

Abstract
Directly labelling locus-specific primers for microsatellite analysis is expensive and a common limitation to small-budget
molecular ecology projects. More cost-effective end-labelling of PCR products can be achieved through a three primer PCR
approach, involving a fluorescently labelled universal primer in combination with modified locus-specific primers with 5
universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of avail-
able universal primers suitable for co-amplifying large numbers of size overlapping loci and without requiring locus-spe-
cific PCR conditions to be modified. In this study, we report a suite of four high-performance universal primers that can be
employed in a three primer PCR approach for efficient and cost-effective fluorescent end-labelling of PCR fragments.
Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ C) that enhance pri-
mer versatility and enable higher annealing temperatures to be employed compared with commonly used universal prim-
ers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co-amplify
multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to
microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer
performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single
and multiplex PCR across a wide range of taxa.

Keywords: fluorescent labelling, genotyping, microsatellites, multiplex PCR, tailed primers, universal primers
Received 23 June 2011; revised 11 November 2011; accepted 18 November 2011

monly achieved by directly labelling one locus-specific

Introduction
Genotyping by fluorescence has revolutionized molecu-
lar marker-based analyses over the past decade (Guic-
houx et al. 2011), allowing more rapid data collection
compared to earlier methods such as those based on
radioactive isotopes and silver staining. The field of pop-
ulation genetic research in particular has benefited
greatly from this advancement, and fluorescence is now
the method of choice for projects utilizing microsatellite
and amplified fragment length polymorphism (AFLP)
markers. Fluorescent genotyping typically involves using
fluorescently labelled locus-specific primers that ensure
polymerase chain reaction (PCR) amplicons are end-
labelled with fluorophores prior to analysis. This is com-
Correspondence: Mark J. Blacket, Fax: +61 (03) 9800 3521;
E-mail: mark.blacket@dpi.vic.gov.au
primer with a fluorescent tag, which is then incorporated
during PCR. However, synthesizing fluorescent primers
is expensive, and for molecular ecological studies that
typically use 815 microsatellite markers, this is an
expensive outlay and a common limitation for small-
budget projects.
Costs can be significantly reduced by using fluores-
cently labelled universal primers in a three primer sys-
tem (Fig. 1). This process involves using a fluorescently
labelled universal primer and a modified locus-specific
primer with a 5 universal primer sequence tail. During
PCR, the tailed locus-specific primer is depleted owing to
a low starting concentration and the fluorescently
labelled universal primer is subsequently incorporated
(Fig. 1). Theoretically, this approach can be used for both
single and multiplex PCRs provided target loci do not
overlap in size and can be differentiated by capillary

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 U N I V E R S A L P R I M E R S F O R F L U O R E S C E N T G E N O T Y P I N G 457

(I) Singleplex PCR (II) Singleplex PCR (III) Multiplex PCR

(Single universal primer/Single fluorophore) (Single universal primer/Multiple fluorophores) (Multiple universal primers/Multiple fluorophores)
Ta
Universal tail Universal tail FAM il A
1
Locus-specific 1 FAM Tail A
(Tail A) (Tail A) or One universal
MasterMix PET forward primer
Ta
il B
2 forward primers 2 VIC Tail B Multiple universal
FAM Ta
(Tail A) with a il C with multiple forward primers (Tails
NED 3 3 NED Tail C
Locus-specific Locus-specific Single universal forward Locus-specific Locus-specific choice of possible Ta
il D
Universal tails Locus-specific A, B, C, D) each with
forward primer reverse primer primer (Tail A) with fluorophore forward primer reverse primer VIC fluorophores 4 (A, B, C, D) 4 reverse primers PET Tail D a different fluorophore

FAM VIC

1 2
CACACACACACACACA GAGAGAGAGAGAGAGA
FAM FAM
GTGTGTGTGTGTGTGT CTCTCTCTCTCTCTCT
1 2
Initial PCR CACACACACACACACA CACACACACACACACA

amplification GTGTGTGTGTGTGTGT GTGTGTGTGTGTGTGT

NED PET

3 4
CAACAACAACAACAACAA AAGAAGAAGAAGAAGAAG

GTTGTTGTTGTTGTTGTT TTCTTCTTCTTCTTCTTC
3 4

1 2
FA CACACACACACACACA VIC GAGAGAGAGAGAGAGA
M

CACACACACACACACA GAGAGAGAGAGAGAGA

CACACACACACACACA CACACACACACACACA
GTGTGTGTGTGTGTGT CTCTCTCTCTCTCTCT
FA FA
M 1
M 2
Early PCR
CACACACACACACACA CACACACACACACACA
amplifications
GTGTGTGTGTGTGTGT GTGTGTGTGTGTGTGT
3 4
NE CAACAACAACAACAACAA PE AAGAAGAAGAAGAAGAAG
D T

CAACAACAACAACAACAA AAGAAGAAGAAGAAGAAG

GTTGTTGTTGTTGTTGTT TTCTTCTTCTTCTTCTTC
3 4

FA VIC
M

CACACACACACACACA GAGAGAGAGAGAGAGA

FA FA
M M
GTGTGTGTGTGTGTGT CTCTCTCTCTCTCTCT
1 2
Subsequent PCR CACACACACACACACA CACACACACACACACA

amplifications NE PE
GTGTGTGTGTGTGTGT D T
GTGTGTGTGTGTGTGT

CAACAACAACAACAACAA AAGAAGAAGAAGAAGAAG

GTTGTTGTTGTTGTTGTT TTCTTCTTCTTCTTCTTC
3 4

Fig. 1 Amplification of PCR fragments with fluorescently labelled high annealing temperature universal primers and locus-specific
tailed forward primers. Amplification is indicated by dashed lines. Labelled universal primers begin to be incorporated into PCR frag-
ments in early PCR cycles, tailed forward primers are exhausted in early cycles and subsequent PCR cycles incorporate fluorophores into
PCR fragments. Strategies I, II and III are alternative amplification procedures, outlined further in the text and Table 2.

separation. A number of existing three primer protocols a feasible alternative to standard fluorescent genotyping
have been developed (e.g. Steffens et al. 1993; Oetting in many situations (i.e. analysis of large numbers
et al. 1995; Neilan et al. 1997; Schuelke 2000; Boutin- samples and loci).
Ganache et al. 2001; Missiaggia & Grattapaglia 2006, de Existing protocols have another major limitation
Arruda et al. 2010) and widely adopted, which together caused by the common use of the M13 primer as a uni-
have more than one thousand citations to date. versal tag. The primer requires low annealing tempera-
While the three primer system has been a cost-effec- tures for optimal performance and generally requires
tive alternative for some studies, the wider adoption of locus-specific PCR conditions to be modified whereby
the technique is currently limited by the lack of reliable annealing temperatures in final PCR cycles are reduced
universal primers described in the literature. Current to 53 C for M13 to be incorporated in PCR fragments
protocols remain largely dependent on the use of single (e.g. Schuelke 2000; de Arruda et al. 2010). As a conse-
universal tails (most commonly M13) and single fluo- quence, this can reduce the efficiency of fragment label-
rescent dyes, consequently limiting the number of size ling (resulting in lower fluorescent signals) and also
overlapping microsatellite markers that can be com- promote the amplification of non-specific genomic prod-
bined for analysis or co-amplified by multiplex PCR. ucts. Alternative universal tails have been trialled with
Missiaggia & Grattapaglia (2006) reported a set of alter- limited success, while some studies have even advocated
native universal primers that can be combined with interrupting PCR cycling to add the universal primer for
multiple fluorophores to enable multiple microsatellite the final cycles to avoid non-specific products (e.g. de
loci to be genotyped by multiplex loading, i.e., each Arruda et al. 2010).
locus amplified separately and subsequently combined In this study, we identify a suite of universal primers
for simultaneous capillary separation. However, to date, and demonstrate their utility in amplifying multiple
no protocol using multiple universal primers for microsatellite loci using both single and multiplex PCR
co-amplification of multiple loci via multiplex PCR has methodologies. Unlike primers previously developed for
been formally described. As a result, current three pri- tailed primer genotyping, primers described here maxi-
mer protocols for fluorescent genotyping do not provide mize PCR efficiency and reliability by enabling locus-spe-

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458 M . J . B L A C K E T E T A L .

cific PCR conditions to be maintained. We demonstrate
how the primers can be combined with multiple fluores-
cent dyes to co-amplify large numbers of loci using
a modified three primer PCR protocol. The methods
we outline maximize locus amplification efficiency and
reliability, and significantly reduce the cost of fluorescent
microsatellite genotyping.
noting the level of identity for the best fifty matches
(Table 1). Additionally, we used the MFE primer 2.0 pro-
gram (Qu et al. 2009) to test all four primers together
for potential non-specific PCR amplification products
in three complete genomes (Human, Drosophila melano-
gaster, Caenorhabditis elegans).

Real-time PCR (RT-PCR) comparison of primers

Materials and methods
Novel universal primers
Four novel universal primers were identified and tested
in the current study (Table 1). Primers Tail A and
Tail B are commonly used as DNA adapters in 454
DNA sequencing protocols (Margulies et al. 2005),
while primers Tail C and Tail D were designed by
the authors. All primers described here are character-
ized by high GC contents and Tm values. Primer viabil-
ity analyses were conducted using the Oligonucleotide
Properties Calculator (Kibbe 2007) to test for potential
issues associated with cross-primer and self-comple-
mentarity.
It is particularly important that universal primers
share limited or no identity with the target genome. Mis-
siaggia & Grattapaglia (2006) counteracted this problem
by using human microsatellite primers as universal prim-
ers for the amplification of plant microsatellites. To test
for potential universal primer matches, we initially per-
formed identity comparisons using similarity searches
(BLASTN) for each of the four universal primers against
sequence databases (i.e. nucleotide collection (nr nt) on
GenBank). However this proved difficult owing to a large
number of sequences that have been submitted without
the removal of 454 adapter sequences. As an alternative,
we chose to compare the universal primers against the
complete Human and Drosophila melanogaster genomes,
To assess the performance of tailed vs. non-tailed prim-
ers, we performed quantitative real-time PCR (RT-PCR)
on the LightCycler 480 system (Roche Applied Sciences,
Castle Hill, Australia). Each 10 lL reaction contained
5 lL Qiagen multiplex mix, 0.2 lM each of forward and
reverse primers, and 0.25 lL of the LightCycler 480
High Resolution Melting Master kit (Roche). Cycling con-
ditions were 95 C for 15 min followed by 40 cycles of
94 C for 30 s, 59 C for 1.5 min and 72 C for 1 min.
Fluorescence signal was captured after each 72 C elon-
gation step. Duplicate reactions were conducted using
either: (i) non-tailed forward primers or (ii) tailed for-
ward primers (i.e. universal primer tail sequence 5 to
locus-specific forward primer, see below). Three genomic
DNA templates were tested, and three technical replica-
tions were performed per DNA sample per primer
combination.
Alternative PCR strategies and primer performance
Primer utility was tested using a variety of PCR strategies
(Tables 2 and 3, Fig. 1), including: Strategy Iloci ampli-
fied via singleplex PCR using a single universal primer
and fluorophore (historically, the most commonly used
three primer system); Strategy IIloci amplified via sin-
gleplex PCR using a single universal primer and multiple
fluorophores; Strategy IIImultiple loci amplified by
multiplex PCR using multiple universal primers each

Table 1 Universal primers utilized and or designed in this study

Blastn results Blastn

Universal Multiplex Length GC content Primer (% match to results
Primer Tail sequence (53) PCR fluorophore (bp) (%) Tm Reference primer) (e-value)

Tail A GCCTCCCTCGCGCCA FAM 15 80 63 C Roche Applied 80100* 14217*

(Primer A) Science. 2006 6087 3.5844
Tail B GCCTTGCCAGCCCGC VIC 15 80 57 C Roche Applied 6384* 3.5858*
(Primer B) Science. 2006 5379 0.44427
Tail C CAGGACCAGGCTACCGTG NED 18 67 59 C This study 6783* 14217*
5678 1.8427
Tail D CGGAGAGCCGAGAGGTG PET 17 71 59 C This study 71100* 0.89858*
5982 1.3320

Blast searches were conducted against the *Human (Build 37), and Drosophila melanogaster (Build 9) genomes. (Correction added after
publication 24 January 2012: Table 1 has been corrected.)

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 U N I V E R S A L P R I M E R S F O R F L U O R E S C E N T G E N O T Y P I N G 459

Table 2 Alternative PCR strategies for employing labelled universal primers with tailed forward primers

PCR
Strategy Procedure Advantages Disadvantages Example

I A single universal primer
used as a tail on each
locus-specific forward
primer and a single universal
primer with a single type of
fluorophore (e.g.
PrimerA-FAM)
Only requires the purchase of
a single labelled primer for
screening many loci.
Each similar sized locus
requires singleplex PCR.
Similar-sized alleles from
different loci cannot be
screened together in the
same lane (i.e. increased
allele screening costs).
Steffens et al. (1993),
Oetting et al. (1995),
Neilan et al. (1997),
Schuelke (2000),
Present study.

II A single universal primer
used as a tail on each
locus-specific forward
primer and a single universal
primer with multiple
fluorophores (e.g. Primer
A-FAM, A-VIC, A-PET,
A-NED)
Many loci with similar-sized
alleles can be pooled for
allele screening in a single
lane.
Simple to change locus
fluorophores if required (i.e.
no need to purchase a new
locus-specific tailed primer).
Each similar-sized locus Hale et al. (2011),
requires singleplex PCR, James et al. (2011a,b),
with a different fluorophore. Present study.
Slightly higher cost of
purchasing multiple labelled
universal primers.

III Multiple universal primers
used as tails on each
locus-specific forward
primer and multiple
universal primers each with
different fluorophores (e.g.
Primer A-FAM, B-VIC,
C-NED, D-PET)
Multiple loci with
similar-sized alleles can be
co-amplified in a single PCR
reaction and analyzed
simultaneously.
Most cost-effective method for
genotyping by fluorescence
when dealing with relatively
moderate to large numbers
of samples and loci
Slightly higher cost of
purchasing multiple labelled
universal primers.
Marker combination requires
some optimization prior to
genotyping
Missiaggia &
Grattapaglia (2006)
(pooling after
singleplex PCR, i.e.
pool-plexing, not
through multiplex
PCR), Miller et al.
(2011a,b), Present
study.

Table 3 Studies employing novel high annealing temperature labelled universal primers (from Table 1) with tailed locus-specific
primers

Number of PCR PCR Universal

Organism microsatellite loci PCR Ta conditions strategy* primer Reference

Flies: Drosophila melanogaster 8 5860 C Singleplex I Tail A Robin & Good (in prep)
Stick Insects: Sipyloidea sp. 6 5055 C Singleplex II Tail A Blacket & Kearney (in prep)
Grasshoppers: Warramaba sp. 7 5065 C Singleplex II Tail A Blacket & Kearney (in prep)
Frogs: Litoria sp. & Cyclorana sp. 9 50 C Singleplex II Tail A Hale et al. (2011)
Plants: Triglochin sp. 11 55 C Singleplex II Tail A James et al. (2011a)
Plants: Grevillea sp. 14 55 C Singleplex II Tail A James et al. (2011b)
Aceria mites: Aceria tosichella 9 59 C Multiplex III Tail A, B, C, D Miller et al. (2011a)
Marsupial: Perameles gunnii 7 59 C Multiplex III Tail A, B, C, D Weeks et al. (in review)
Marsupial: Burramys parvus 8 59 C Multiplex III Tail A, B, C Weeks et al. (in review)
Fish: Galaxias fuscus 12 59 C Multiplex III Tail A, B, C, D Miller et al. (in review)
Molluscs: Donax deltoides 11 59 C Multiplex III Tail A, B, C, D Miller et al. (2011b)
Birds: Neophema chrysogaster 15 59 C Multiplex III Tail A, B, C, D Coleman et al. (in prep)

*Strategies are outlined in Figure 1, Table 2 and text.

Universal primers (see Table 1 and text).

tagged with a unique fluorophore (Table 1, see below).

Strategy ISingleplex using a single fluorophore
An overview of each PCR strategy, research applications
and relative advantages and disadvantages are discussed The simplest approach requiring only a single labelled
below and in Table 2. universal primer to be purchased. Any locus can be

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460 M . J . B L A C K E T E T A L .
amplified by singleplex PCR providing one locus-spe-
cific primer is modified with a corresponding universal
primer sequence tail. PCR products can be combined
post-PCR for multiplex capillary loading; however, simi-
lar markers with overlapping allelic ranges cannot be
analyzed simultaneously. This approach is particularly
suited for smaller projects (i.e. relatively small numbers
of samples and few loci) and may be used on mono-
laser screening systems, such as the Licor (Table 2,
Fig. 1).
Strategy IISingleplex using multiple fluorophores
This strategy alleviates some of the limitations
described for Strategy I by allowing multiple loci with
similar-sized alleles to be amplified and pooled follow-
ing PCR for multiplex capillary loading (typically on a
multi-colour screening system, such as ABI capillary
analyzers). Individual loci with similar allelic ranges
are differentiated using different fluorescent dyes (e.g.
FAM, VIC, NED, PET on the same universal primer
sequence), thus allowing larger numbers of loci to be
analyzed simultaneously (as outlined in Missiaggia &
Grattapaglia 2006). This strategy provides significant
cost benefits but can be quite labour intensive when
dealing with large numbers of samples and loci, given
individual loci require separate PCR amplification (i.e.
singleplex PCRs using a universal primer with differ-
ent fluorophores) prior to analysis. This approach is
therefore best suited to projects involving relatively
small to moderate numbers of samples and loci
(Table 2, Fig. 1), where cost savings from using a sim-
ple protocol outweigh expenses associated with opti-
mizing multiplex PCR conditions (Strategy III).
Strategy IIIMultiplex with multiple fluorophores
This strategy involves using multiple universal prim-
ers each labelled with a unique fluorescent tag (e.g.
FAM, VIC, NED, PET) to co-amplify multiple loci,
including size overlapping markers. This is the most
cost-effective method for genotyping large numbers of
samples and loci and provides the same level of mul-
tiplexing attained by microsatellite fluorescent detec-
tion assays using directly labelled primers. Optimizing
PCR conditions for multiplexing multiple loci can be a
challenging task given that all primers are required to
perform effectively under identical PCR conditions.
However, the optimization procedure can be simpli-
fied using commercially available multiplex PCR kits
that promote co-amplification of multiple loci. This
application of universal primers is generally suited for
larger projects (relatively high numbers of samples
and loci) where the reduced costs associated with mul-
tiplex PCR outweigh the additional optimization
required relative to adopting Strategy II (i.e. amplify-
ing all loci individually) (Table 2, Fig. 1).
Species markers
A series of previously developed microsatellite marker
sets representing a wide range of taxa were employed to
test primer performance under PCR Strategies I, II and III
(Table 3). Additional details concerning specific library
development have either been previously reported or
remain yet to be published (Table 3). Locus-specific
primers were modified by resynthesizing a single primer
from each primer pair with the addition of a universal
sequence tail to its 5 end (i.e. 5-Universal tail sequence +
Locus-specific primer sequence-3).
Allele screening
Following primer modification, locus-specific PCR condi-
tions were re-optimized via gradient PCR and visualiza-
tion on 2% agarose gels. Singleplex PCRs were
performed in 10 uL reactions consisting of 1x PCR buffer,
1 mM dNTPs, 0.2 lM fluorescently labelled universal pri-
mer, 0.15 lM forward tailed primer, 0.5 lM reverse primer
(singleplex primers were in a 0.4:0.3:1 ratio, to deplete the
forward tailed primer during early PCR cycles, Fig. 1)
and 0.2 units of taq polymerase (New England Biolabs).
Multiplex PCR trials employed the Qiagen multiplex kit
(Qiagen) following the manufacturers recommendations.
PCRs were performed in a 10 lL reaction consisting of
5 lL Qiagen multiplex mix, 0.1 lM forward tailed primer,
0.2 lM reverse primer and 0.1 lM fluorescently tagged
universal primer corresponding to each tailed primer.
Finally, optimized conditions (Table 3) were used to
amplify microsatellite fragments using Eppendorf Mas-
tercycler Gradient S PCR cycler (Fig. 1), and fragment
analyses were performed on an Applied Biosystems
ABI3730 DNA analyzer using a LIZ-500 size standard.
Allele sizes were subsequently assessed and scored using
GENEMAPPER version 4.0 (Applied Biosystems). Obviously,
future studies could further reduce costs by adding the
appropriate universal tail to forward primers when they
are first purchased, prior to any optimization.
Results and Discussion
Primer viability tests indicated that each universal primer
had low cross-primer and self-complementarity, high-
lighting its potential utility for singular (Strategy I & II)
and multiple (Strategy III) primer applications. The prim-
ers also generally exhibited low similarity to the Human
and D. melanogaster genomes (Table 1). MFE primer tests
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 U N I V E R S A L P R I M E R S F O R F L U O R E S C E N T G E N O T Y P I N G 461

indicated that potential interactions between universal Table 4 Comparison of crossing points between tailed and non-
primers, when used in combination and at annealing tem- tailed primers using quantitative RTPCR
peratures less than 53 C, may lead to the amplification
Genomic Cp difference
of small numbers of loci (36, 2 and 2 loci in the
DNA (CpLocus + Tail)
Human, D. melanogaster and C. elegans genomes, respec- sample Crossing point CpLocus)
tively). Although we have only tested a limited number
of genomes here, it is likely that non-target potential Locus 1 Locus 1 +
amplicons could be avoided by increasing the stringency Tail A
of the PCR conditions. DNA 1 22.60 22.83 0.23
Primer efficiency was validated through RT-PCR DNA 2 22.48 22.71 0.22
DNA 3 22.88 23.11 0.23
testing. These tests indicated that primers modified
Locus 2 Locus 2 +
with universal tails amplified target loci as effectively Tail B
as equivalent locus-specific non-tailed primers. The DNA 1 19.47 19.79 0.32
PCR amplification profiles of these primer combinations DNA 2 19.46 19.66 0.20
are shown in Fig. 2. PCR was robust, and amplification DNA 3 19.92 20.00 0.09
profiles were comparable across all primer pairs (Fig. 2, Locus 3 Locus 3 +
Table 4). Slightly higher crossing point values (approxi- Tail C
DNA 1 21.46 21.75 0.29
mately 0.2 cycles) were observed when using the tailed
DNA 2 21.25 21.48 0.23
primers (Table 4). Regardless of primer combination, all DNA 3 21.36 21.62 0.26
PCR reactions showed a typical sigmoidal curve and Locus 4 Locus 4 +
went to completion well before cycle 40 (Fig. 2). Tail D
Compared with the standard M13 primer (Tm approx- DNA 1 20.56 20.68 0.12
imately 53 C), all universal tails described in this study DNA 2 20.09 20.51 0.42
proved superior through allowing locus-specific PCR DNA 3 20.48 20.54 0.06
conditions to be maintained. Loci could also be success-

2.9 2.9

2.4 Locus 1 + Tail A 2.4

Locus 2 + Tail B

Locus 2
1.9 1.9
Locus 1

1.4 1.4

0.9 0.9
Flourescence level

0.4 0.4
14 16 18 20 22 24 26 28 30 32 34 36 38 40 14 16 18 20 22 24 26 28 30 32 34 36 38 40

2.9 2.9

Locus 4 + Tail D
2.4 2.4
Locus 3 + Tail C

Locus 4
1.9 1.9
Locus 3

1.4 1.4

0.9 0.9

0.4 0.4
14 16 18 20 22 24 26 28 30 32 34 36 38 40 14 16 18 20 22 24 26 28 30 32 34 36 38 40

PCR cycle number

Fig. 2 PCR amplification profiles between tailed and non-tail primers on three genomic DNA samples. Each curve represents an aver-
age of three technical replications (see Table 4).

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462 M . J . B L A C K E T E T A L .
fully amplified using significantly higher annealing tem-
peratures (up to 65 C, Table 3) further improving PCR
reliability through visible reductions in competing non-
specific products. These results are not surprising given
each universal priming tail has a relatively high Tm value
(approximately 60+ C), and therefore, maximum
annealing temperature is likely to be limited largely by
the Tm of the unmodified reverse primer in most cases
(Tm of the reverse primer should therefore be maximized
when possible). PCR efficiency appeared high using
these universal tails, each amplifying PCR products with
high fluorescent intensities and generally requiring dilu-
tion prior to capillary separation.
Using multiple 5 universal primer tags in a three pri-
mer system is an advantage as alternative primers are
available when there are negative interactions with
locus-specific DNA sequences, e.g., through the forma-
tion of hairpins (e.g. Schable et al. 2002). It is also an
advantage to use short universal primers to help reduce
primer synthesis costs (e.g. Schable et al. 2002), and each
of our universal primers is relatively short (1518 bp,
Table 1). (Correction added after publication 24 January
2012: the length range (bp) for our universal primers has
been amended.)
Similar to Missiaggia & Grattapaglia (2006), we found
that the universal primers described here can be com-
bined with multiple fluorophores to amplify large num-
bers of size overlapping microsatellites via singleplex
PCR and genotyped by multiplex capillary loading (Strat-
egy II, Table 3). However, unlike Missiaggia & Grattapa-
glia (2006), large numbers of size overlapping
microsatellites (i.e. 710 loci routinely) were consistently
co-amplified via multiplex PCR using multiple universal
primers each labelled with unique fluorophores (Strategy
III, Table 3). In all cases, individual loci, previously ampli-
fied singularly and with varying PCR conditions, were
successfully combined and amplified at a single annealing
temperature (generally 59 C). In each case, optimization
steps were minimal, with markers successfully trans-
ferred to the three primer approach in a single step (pri-
mer modification and universal PCR protocol using a
59 C annealing temperature). Optimization steps, when
required, typically involved altering the concentrations of
specific primers given some loci amplified stronger than
others (independent of tail or fluorescent dye). This is a
typical optimization step required for standard fluores-
cent genotyping using directly labelled primers. Figure 3
indicates the quality of genotyping typically acquired
using the tailed primers in a multiplex environment.
Conclusions
The costs associated with microsatellite genotyping by
fluorescence can be significantly reduced using universal
100 120 140 160 180 200 220 240 260
24 000
20 000
16 000
2.D
5.C
1.A
6.B
12 000
8000
4.B
3.A
4000
0
Fig. 3 Co-amplification of 6 microsatellite loci by multiplex
PCR using multiple primer tails. Peak labels indicate locus num-
ber (16) and corresponding universal primer tails (AD). Loci 1,
3 and 5 are homozygous; loci 2, 4 and 6 are heterozygous.
fluorescent PCR primers (as previously outlined in Mis-
siaggia & Grattapaglia 2006). Here, we provide a suite
of primers that outperform those currently available by
improving PCR efficiency and reducing associated labo-
ratory labour. Benefits include: (i) each primer can be
used to amplify multiple loci without altering locus-
specific PCR conditions; (ii) strong amplifications are
generally achieved owing to incorporation of fluores-
cence in early PCR cycles; (iii) high Tm values enable
high annealing temperatures to be employed thereby
improving PCR efficiency through the reduction in
non-specific amplification events; and (iv) universal
primers described here can be combined with multiple
fluorophores to co-amplify large numbers of micro-
satellite loci effectively via true multiplex PCR (sensu
Guichoux et al. 2011). These primers have already
proved effective in screening microsatellite loci from a
diverse range of organisms (see Table 3), including
arthropods, amphibians, birds, fish, mammals, molluscs
and plants.
Acknowledgements
We thank Ary Hoffmann and Michael Kearney (University of
Melbourne) for the use of laboratory facilities; Belinda Apple-
ton and Kate York (University of Melbourne) for assistance
with employing a fluorescent universal tailed (M13 based)
primer system; we thank our collaborators including Josh
Hale (Museum of Victoria), Elizabeth James (Royal Botanic
Gardens Melbourne), Rhys Coleman, Ary Hoffmann, Michael
Kearney, Andrew Weeks (University of Melbourne) and Kate
Bowie (Deakin University) for results from their unpublished
work. We thank the subject editor and referees for very use-
ful suggestions that significantly improved this manuscript.
Finally, we thank the Australian Research Council (ARC) for
infrastructure provided through the Special Research Centre
and Discovery Project Schemes.
 2012 Blackwell Publishing Ltd
 U N I V E R S A L P R I M E R S F O R F L U O R E S C E N T G E N O T Y P I N G 463
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Much of this research was conducted in the former Centre for
Environmental Stress and Adaptation Research (CESAR), which
investigated how organisms, particularly insects, adapt to envi-
ronmental stress. Mark Blacket and Adam Miller are interested
in the application of molecular approaches to population genetic
studies of threatened and commercially targeted wildlife groups,
general phylogeographic and systematic issues and to under-
standing climatic adaptation. Charlie Robin and Rob Good apply
molecular methods to examine insecticide resistance, and the
evolution of genes. Siu Fai Lee is a postdoctoral fellow at Mel-
bourne University. His research focuses on the molecular basis
of climatic adaptation.
Data Accessibility
Websites: NCBI GenBank, http://www.ncbi.nlm.nih.
gov/genbank/; OligoCalc, http://www.basic.north
western.edu/biotools/oligocalc.html; MFEprimer-2.0,
http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/.