Sie sind auf Seite 1von 8

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

 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 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-

 2012 Blackwell Publishing Ltd


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

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

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


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

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

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

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

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

 2012 Blackwell Publishing Ltd


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

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

 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 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).

 2012 Blackwell Publishing Ltd


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

fully amplified using significantly higher annealing tem- 100 120 140 160 180 200 220 240 260
24 000
peratures (up to 65 C, Table 3) further improving PCR
reliability through visible reductions in competing non- 20 000
specific products. These results are not surprising given
16 000

2.D

5.C
1.A

6.B
each universal priming tail has a relatively high Tm value
(approximately 60+ C), and therefore, maximum 12 000
annealing temperature is likely to be limited largely by
8000

4.B
the Tm of the unmodified reverse primer in most cases

3.A
(Tm of the reverse primer should therefore be maximized 4000
when possible). PCR efficiency appeared high using
these universal tails, each amplifying PCR products with 0

high fluorescent intensities and generally requiring dilu-


Fig. 3 Co-amplification of 6 microsatellite loci by multiplex
tion prior to capillary separation.
PCR using multiple primer tails. Peak labels indicate locus num-
Using multiple 5 universal primer tags in a three pri- ber (16) and corresponding universal primer tails (AD). Loci 1,
mer system is an advantage as alternative primers are 3 and 5 are homozygous; loci 2, 4 and 6 are heterozygous.
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 fluorescent PCR primers (as previously outlined in Mis-
primer synthesis costs (e.g. Schable et al. 2002), and each siaggia & Grattapaglia 2006). Here, we provide a suite
of our universal primers is relatively short (1518 bp, of primers that outperform those currently available by
Table 1). (Correction added after publication 24 January improving PCR efficiency and reducing associated labo-
2012: the length range (bp) for our universal primers has ratory labour. Benefits include: (i) each primer can be
been amended.) used to amplify multiple loci without altering locus-
Similar to Missiaggia & Grattapaglia (2006), we found specific PCR conditions; (ii) strong amplifications are
that the universal primers described here can be com- generally achieved owing to incorporation of fluores-
bined with multiple fluorophores to amplify large num- cence in early PCR cycles; (iii) high Tm values enable
bers of size overlapping microsatellites via singleplex high annealing temperatures to be employed thereby
PCR and genotyped by multiplex capillary loading (Strat- improving PCR efficiency through the reduction in
egy II, Table 3). However, unlike Missiaggia & Grattapa- non-specific amplification events; and (iv) universal
glia (2006), large numbers of size overlapping primers described here can be combined with multiple
microsatellites (i.e. 710 loci routinely) were consistently fluorophores to co-amplify large numbers of micro-
co-amplified via multiplex PCR using multiple universal satellite loci effectively via true multiplex PCR (sensu
primers each labelled with unique fluorophores (Strategy Guichoux et al. 2011). These primers have already
III, Table 3). In all cases, individual loci, previously ampli- proved effective in screening microsatellite loci from a
fied singularly and with varying PCR conditions, were diverse range of organisms (see Table 3), including
successfully combined and amplified at a single annealing arthropods, amphibians, birds, fish, mammals, molluscs
temperature (generally 59 C). In each case, optimization and plants.
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 Acknowledgements
59 C annealing temperature). Optimization steps, when
We thank Ary Hoffmann and Michael Kearney (University of
required, typically involved altering the concentrations of
Melbourne) for the use of laboratory facilities; Belinda Apple-
specific primers given some loci amplified stronger than ton and Kate York (University of Melbourne) for assistance
others (independent of tail or fluorescent dye). This is a with employing a fluorescent universal tailed (M13 based)
typical optimization step required for standard fluores- primer system; we thank our collaborators including Josh
cent genotyping using directly labelled primers. Figure 3 Hale (Museum of Victoria), Elizabeth James (Royal Botanic
indicates the quality of genotyping typically acquired Gardens Melbourne), Rhys Coleman, Ary Hoffmann, Michael
Kearney, Andrew Weeks (University of Melbourne) and Kate
using the tailed primers in a multiplex environment.
Bowie (Deakin University) for results from their unpublished
work. We thank the subject editor and referees for very use-
Conclusions ful suggestions that significantly improved this manuscript.
Finally, we thank the Australian Research Council (ARC) for
The costs associated with microsatellite genotyping by infrastructure provided through the Special Research Centre
fluorescence can be significantly reduced using universal 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

References Oetting WS, Lee HK, Flanders DJ, Wiesner GL, Sellers TA, King RA
(1995) Linkage analysis with multiplexed short tandem repeat poly-
de Arruda MP, Goncalves EC, Schneider MPC, da Costa da Silva AL, morphisms using florescence and M13 tailed primers. Genomics, 30,
Morielle-Verute E (2010) An alternative genotyping method using dye- 450458.
labelled universal primer to reduce unspecific amplifications. Molecular Qu W, Shen Z, Zhao D, Yang Y, Zhang C (2009) MFEprimer: multiple
Biology Reports, 37, 20312036. factor evaluation of the specificity of PCR primers. Bioinformatics, 25,
Boutin-Ganache I, Raposo M, Raymond M, Deschepper CF (2001) M13- 276278.
tailed primers improve the readability and usability of microsatellite Roche Applied Science (2006) GS Guide to Amplicon Sequencing. Roche
analyses performed with two different allele-sizing methods. BioTech- Diagnostics. GmbHUSM-00022.B - December 2006.
niques, 31, 2428. Schable NA, Fischer RU, Glenn TC (2002) Tetranucleotide microsatellite
Guichoux E, Lagache L, Wagner S et al. (2011) Current trends in microsat- DNA loci from the dollar sunfish (Lepomis marginatus). Molecular
ellite genotyping. Molecular Ecology Resources, 11, 591611. Ecology Notes, 2, 509511.
Hale JM, Heard GW, Smith KL, Blacket MJ, Austin J, Melville J (2011) Schuelke M (2000) An economic method for the fluorescent labelling of
Microsatellite loci for the endangered growling grass frog (Litoria rani- PCR fragments. Nature Biotechnology, 18, 233234.
formis) with cross amplification in other Australian frog species. Conser- Steffens DL, Sutter SL, Roemer SC (1993) An alternative universal
vation Genetics Resources, 3, 593595. forward primer for improved automated DNA sequencing of M13.
James EA, Brown GK, Citroen R, Rossetto M, Porter C (2011a) Develop- BioTechniques, 15, 580582.
ment of microsatellite loci in Triglochin procera (Juncaginaceae), a poly-
ploid wetland plant. Conservation Genetics Resources, 3, 103105.
Much of this research was conducted in the former Centre for
James EA, Brown GK, Citroen R, Blacket MJ (2011b) Microsatellite marker
development for two species of holly-leafed Grevillea and cross-
Environmental Stress and Adaptation Research (CESAR), which
species amplification in the Aspleniifolia Hookeriana Subgroup investigated how organisms, particularly insects, adapt to envi-
(Proteaceae). Conservation Genetics Resources, DOI 10.1007/s12686-011- ronmental stress. Mark Blacket and Adam Miller are interested
9493-5 in the application of molecular approaches to population genetic
Kibbe WA (2007) OligoCalc: an online oligonucleotide properties calcula- studies of threatened and commercially targeted wildlife groups,
tor. Nucleic Acids Research, 35(Suppl. 2), W43W46, DOI 10.1093/nar/ general phylogeographic and systematic issues and to under-
gkm234 standing climatic adaptation. Charlie Robin and Rob Good apply
Margulies M, Egholm M, Altman WE et al. (2005) Genome sequencing molecular methods to examine insecticide resistance, and the
in microfabricated high-density picolitre reactors. Nature, 437, 376
evolution of genes. Siu Fai Lee is a postdoctoral fellow at Mel-
380.
bourne University. His research focuses on the molecular basis
Miller AD, Umina P, Weeks AR, Hoffmann AA (2011a) Population genet-
ics of the wheat curl mite (Aceria tosichella Keifer) in Australiaimpli-
of climatic adaptation.
cations for the management of wheat pathogens. Bulletin of
Entomological Research, DOI 10.1017/S0007485311000526
Miller AD, Versace VL, Matthews T, Bowie KC (2011b) The development Data Accessibility
of 20 microsatellite loci for the Australian marine mollusk, Donax delto-
ides, through next generation DNA sequencing. Conservation Genetics Websites: NCBI GenBank, http://www.ncbi.nlm.nih.
Resources, DOI 10.1007/s12686-011-9519-z gov/genbank/; OligoCalc, http://www.basic.north
Missiaggia A, Grattapaglia D (2006) Plant microsatellite genotyping with western.edu/biotools/oligocalc.html; MFEprimer-2.0,
4-color fluorescent detection using multiple-tailed primers. Genetics and
Molecular Research, 5, 7278.
http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/.
Neilan BA, Wilton AN, Jacobs D (1997) A universal procedure for primer
labelling of amplicons. Nucleic Acid Research, 25, 29382939.

 2012 Blackwell Publishing Ltd