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Soeren Junge Nielsen

Jens F. Rehfeld
Jens Peter Goetze
Department of Clinical Biochemistry
University of Copenhagen
* Address correspondence to the author at:
Department of Clinical Biochemistry
9 Blegdamsvej, DK-2100
Copenhagen, Denmark.
Fax 4535454640
e-mail soerenjunge@gmail.com
DOI: 10.1373/clinchem.2007.096172
Hexaprimer Amplification
Refractory Mutation System
PCR for Simultaneous Single-
Tube Genotyping of 2 Close
To the Editor:
Tetraprimer amplification refrac-
tory mutation system PCR (T-
ARMS-PCR) is a simple and inex-
pensive genotyping method for
differentiating both alleles of a
nucleotide polymorphisms and
small insertions/deletions) with a
single-tube PCR (1). In T-ARMS-
PCR, a pair of common (outer)
primers produces a nonallele-
specific control amplicon and in
combination with 2 allele-specific
(inner) primers (designed to anneal
in the opposite orientation) pro-
duces 2 allele-specific amplicons.
These allele-specific amplicons have
different sizes because the poly-
morphism/mutation is asymmetri-
cally located with respect to the
common primers. Thus, the ampli-
cons can be separated by standard
gel electrophoresis. T-ARMS-PCR
has alsobeendesignedina multiplex
fashion to genotype more than one
polymorphism/mutation by a sin-
gle-tube PCR (2).
We describe a modified multi-
plex T-ARMS-PCR, the hexaprimer
is for when 2 polymorphisms are
close in the sequence. H-ARMS-
PCR uses only 6 primers and pro-
vides direct information about hap-
lotype structure.
The CTLA4 gene (cytotoxic T-
lymphocyteassociated protein 4;
also known as CD152) is a negative
regulator of T-cell function (3).
The CTLA4 polymorphisms 318
CT (rs5742909) and 49 AG
(rs231775) are associated with
susceptibility to autoimmune dis-
eases and cancer (3, 4). To geno-
type these 2 polymorphisms,
which are only 365 bp apart in the
5 region of the CTLA4 gene, we
designed an H-ARMS-PCR that
combines a single pair of com-
mon primers and 2 pairs of allele-
specific primers in the same tube
(Fig. 1A).
The PCR reaction (25 L)
contained 200 mol/L de-
oxynucleoside triphosphates, 100
ng of genomic DNA, 1.25 U of
HotStarTaq DNAPolymerase with
its buffer (Qiagen), and the follow-
ing primers at the indicated con-
centrations (deliberate mis-
matches from the reference
sequence, GenBank no. M74363,
are in boldface italics): 318fo, 5-
TG-3 (0.5 mol/L); 49ro, 5-TA
3 (0.8 mol/L); 318fi(c), 5-CTC
(2.5 mol/L); 318ri(t), 5-AC
(0.5 mol/L); 49fi(a), 5-GCACA
3 (0.1 mol/L); and 49ri(g),
GTCCTAGC-3 (0.5 mol/L).
Cycling conditions were 12
min at 95 C, followed by 5 cycles
of 94 Cfor 30 s, 62 Cfor 30 s, and
72 Cfor 45 s; 30 cycles of 94 Cfor
30 s, 57 Cfor 30 s, and 72 Cfor 45
s; and a final cycle at 72 C for 10
We genotyped both the 318
CT and 49 AG CTLA4 poly-
morphisms with this newly de-
signed H-ARMS-PCR(Fig. 1A) for
samples from 80 individuals that
had previously been characterized
with 3 established methods [re-
strictionfragment lengthpolymor-
phism (RFLP) PCR, direct DNA
sequencing, and T-ARMS-PCR]
(4) and observed no discrepancies.
Moreover, H-ARMS-PCR provides
both an additional internal control
and direct information about the
haplotype structure by generating an
amplicon that is specific for 1 of the
4 haplotypes that 2 polymorphisms
can theoretically have. In this case,
the haplotype-specific amplicon is
observed when the alleles 318C
and 49Gare on the same chromo-
some (Fig. 1A).
We further tested this ap-
proach by implementing
H-ARMS-PCR in the genotyping
of 2 polymorphisms that are 142
bp apart in the 3 untranslated re-
gion (UTR) of the CYP19A1 gene
(cytochrome P450, family 19, sub-
family A, polypeptide 1): rs10046
(CT) and rs4646 (GT).
CYP19A1 encodes aromatase, the
enzyme responsible for the final
step of estrogen biosynthesis.
CYP19A1 polymorphisms have
been associated with estrogen con-
centrations in woman and with
susceptibility to breast and pros-
tate cancers (5).
We used the PCR to analyze
these 2 CYP19A1 polymorphisms
in the 3 UTR (Fig. 1B; see legend
for PCR conditions) in 100 in-
dividuals already characterized
by RFLP PCR and/or direct DNA
sequencing (P.P. and R.N., un-
published data) and observed no
discrepancies. A haplotype-specific
amplicon is present when alleles
rs10046C and rs4646T are on the
same chromosome. This H-ARMS-
PCRis currentlybeingusedinamul-
Clinical Chemistry 54:1 (2008) 227
ticenter Italian clinical trial (GIM5)
to test the possible association of
CYP19A1 polymorphisms with the
efficacy of adjuvant therapy with an
aromatase inhibitor (letrozole) after
tamoxifen treatment in postmeno-
pausal patients with early breast
In addition, we genotyped a
series of 40 individuals for both
CTLA4 and CYP19A1 polymor-
phisms by H-ARMS-PCR analysis
with different thermal cyclers
(iCycler by Bio-Rad, Px2 by
Hybaid, and PTC-100 by MJ Re-
search) and in different laborato-
ries, and we obtained fully concor-
dant results, demonstrating the
robustness and reproducibility of
this approach. All human samples
were collected after informed con-
sent was obtained according to in-
stitutional procedure.
We have shown that H-ARMS-
PCR is an effective and robust tech-
nique for genotyping 2 close poly-
morphisms (in a range of about
100400 bp) with only 6 primers.
With appropriate long-range Taq
DNA polymerase, H-ARMS-PCR
may work for genotyping even more
distantly separated polymorphisms,
likely withina distance of about 5kb.
In addition, by providing direct in-
formation about one defined haplo-
type, H-ARMS-PCR, may be useful
for the identification of potentially
ambiguous haplotypes in individu-
als who are double heterozygotes.
Grant/fundingSupport: This work
is supported in part by grants from
Ministero della Salute (Italy), Com-
pagnia di San Paolo (Torino, Italy),
and CARIGE (Genova, Italy). P.P. is
Fig. 1. Polymorphism genotyping by H-ARMS-PCR.
(A), H-ARMS-PCR for CTLA4 318 CT (rs5742909) and 49 AG (rs231775) polymorphisms: representative results for 6
individuals. PCR products were separated by electrophoresis on a 17.5-g/L agarose gel. Lane MW, molecular size markers; lane
1, 318 C/C and 49 A/A; lane 2, 318 C/C and 49 G/G; lane 3, 318 C/T and 49 A/G; lane 4, 318 C/C and 49 A/G;
lane 5, 318 C/T and 49 A/A; lane 6, 318 T/T and 49 A/A. Amplicon sizes are as follows: C (control amplicon), 701 bp;
318C allele, 597 bp, 318T allele, 149 bp; 49A allele, 230 bp; 49G allele, 517 bp; Hap CG [haplotype 318C- and
49G-specific amplicon (lanes 2, 3, 4)], 420 bp.
(B), H-ARMS-PCR for CYP19A1 rs10046 (CT) and rs4646 (GT) polymorphisms: representative results for 6 individuals. Lane
MW, molecular size markers; lane 1, rs10046 T/T and rs4646 G/G; lane 2, rs10046 C/C and rs4646 G/G; lane 3, rs10046 C/C
and rs4646 G/T; lane 4, rs10046 C/C and rs4646 T/T; lane 5, rs10046 C/T and rs4646 G/G; lane 6, rs10046 C/T and rs4646 G/T.
PCR reactions (see text) were with the following primers (deliberate mismatches from the GenBank sequence, no. M30804, are
in boldface italics): CYP19-Fo, 5-CAGAAGATACACGACTTGTCCTTGCACCCAG-3 (0.25 mol/L); CYP19-Ro, 5-CCGACTATT
GTGCTACTG-3 (2.0 mol/L); rs4646-Ri(T), 5-GTTCTCTGGTGTGAACAGGAGCAGATGTCA-3 (2.0 mol/L). Cycling conditions
were 12 min at 95 C, followed by 20 cycles of 94 C for 30 s, 67 C for 30 s, and 72 C for 30 s; 15 cycles of 94 C for 30 s,
63 C for 30 s, and 72 C for 30 s; and a final cycle at 72 C for 10 min. PCR products were separated by electrophoresis on
25-g/L agarose gels (Agarose MS; Roche Diagnostics). Amplicon sizes are as follows: C (control amplicon), 342 bp; rs10046C
allele, 255 bp; rs10046T allele, 149 bp; rs4646T allele, 289 bp; rs4646G allele, 112 bp; Hap CT [haplotype rs10046C- and
rs4646T-specific amplicon (lanes 3, 4, 6)], 202 bp.
Because of the tight linkage of these polymorphisms, only 3 (combined in 6 genotypes) of the 4 possible haplotypes have been
observed for the 2 genes. The SNP nomenclature is according to NCBI at: http://www.ncbi.nlm.nih.gov/SNP/index.html.
228 Clinical Chemistry 54:1 (2008)
supported by Oncotech, Naples,
Financial Disclosures: None de-
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Patrizia Piccioli
Martina Serra
Simona Pedemonte
Giuseppe Balbi
Fabrizio Loiacono
Sonia Lastraioli
Lucia Gargiulo
Anna Morabito
Daniela Zuccaro
Lucia Del Mastro
Maria Pia Pistillo
Marco Venturini
Maria De Angioletti
Rosario Notaro
Laboratory of Human Genetics
Medical Oncology C
Laboratory of Translational Research A
Medical Oncology A
IST, Istituto Nazionale per la
Ricerca sul Cancro
Genoa, Italy
Department of Oncology,
Biology and Genetics
University of Genoa
Genoa, Italy
Medical Oncology
Ospedale Sacro Cuore Don Calabria
Verona, Italy
Istituto di Genetica e Biofisica
Adriano Buzzati Traverso (IGB-CNR)
Naples, Italy
Laboratory of Genetics and Gene
Core Research Laboratory Istituto
Toscano Tumori (CRL-ITT)
Florence, Italy
* Address correspondence to this author at:
Laboratory of Genetics and Gene Transfer
Core Research Laboratory Istituto
Toscano Tumori (ITT-CRL)
Viale Pieraccini 6 (Cubo)
50139 Florence, Italy
Fax 39-055-427-1280
e-mail rosario.notaro@ittumori.it

These authors contributed equally

to this work.
DOI: 10.1373/clinchem.2007.095703
Clinical Chemistry 54:1 (2008) 229