Special Report

For reprint orders, please contact: reprints@futuremedicine.com

Development of a new genotyping assay for

detection of the BDNF Val66Met polymorphism
using melting-curve analysis
Brain-derived neurotrophic factor (BDNF) plays a critical role in the growth, differentiation and survival
of neurons in the CNS. Recent research has suggested that BDNF may be implicated in the etiology of
mood disorders and schizophrenia, as well as in the therapeutic action of some drugs, such as antidepressants
and antipsychotics. This study aimed to develop a simple, fast and accurate new method for detecting the
Val66Met polymorphism of the BDNF gene in schizophrenia patients using melting-curve analysis and a
DNA-specific dye, SYBR® Green I. A group of 30 schizophrenia patients were analyzed to detect the BDNF
Val66Met polymorphism (rs6265) using the new genotyping method based on the analysis of fluorescence
melting curves of PCR products that were labeled with SYBR Green I. The genotype results were confirmed
for all 30 samples using the specific BDNF TaqMan® allele discrimination assay. This new method allows
the analysis of both alleles in the same reaction tube using SYBR Green I, with no need for additional
steps. The addition of a GC clamp makes this method universally applicable, since the melting temperature
of one allele can be adjusted as necessary to give the distinctive separation of melting curves. Therefore,
this new method is simple, fast and accurate for determining the presence of the BDNF Val66Met
polymorphism. It may also be useful for the analysis of other SNPs in pharmacogenetic studies.

KEYWORDS: BDNF n melting curve n schizophrenia patients n SYBR María Antonia

n Val66Met polymorphism
Brain-derived neurotrophic factor (BDNF) is a
member of the superfamily of the neurotroph-
ins and plays a critical role in promoting and
modify­ing the growth, differentiation and sur-
vival of neurons in the CNS. Being the most
abundant of the neurotrophins in the brain,
BDNF is important for guiding the neurons
of the CNS during their development and for
maintaining their survival in adulthood [1] .
Neurodevelopmental studies suggest that a com-
bination of genetic and environmental factors is
required for the manifestation of some psychi-
atric disorders. For instance, altered synthesis
and/or release of neurotrophins during defined
developmental periods could affect how neu-
ronal networks are formed or maintained, and
thus, could possibly be an underlying cause of
the development of disease [2] . Moreover, both
antidepressive and antipsychotic drug treat-
ments regulate the levels of these trophic fac-
tors in the brain. Thus, the regulation of neu-
rotrophic factors could be a crucial factor in
psychiatric drug treatments.
The BDNF gene, encoded on human chromo-
some 11p13, consists of upstream untranslated
exons that are alternatively spliced and a com-
mon downstream exon IX containing the coding
region and the 3´ untranslated region. Of par-
ticular interest is a G>A nucleotide substitution
Sánchez‑Romero,
Pedro Dorado†,
in the BDNF coding region at position 196 Estrella Guarino &
(dbSNP rs6265) that encodes a Val66Met Adrián Llerena
amino-acid substitution. †
Author for correspondence:
The influence of a BDNF gene polymorphism Clinical Research Centre
on response to neuroleptic drugs [3–9] and lith- (CICAB), Extremadura
ium [10–12] has been demonstrated. The Val66Met University Hospital, Servicio
polymorphism is also implicated in individual dif- Extremeño de Salud (SES),
ferences in brain structure of the prefrontal cortex 06080 Badajoz, Spain
and hippocampus, hippocampus function such Tel.: +34 924 218 040;
as memory performance, experience-dependent Fax: +34 924 218 040;
plasticity in the motor cortex and age-related pdorado@unex.es
reasoning skills [13–15] . This polymorphism has
been demonstrated to be implicated in modifying
the risk of onset of Parkinson’s disease, childhood
onset mood disorder, substance abuse and neuro-
cognitive dysfunction in systemic lupus erythema­
tosus, and in the etiology of bipolar disorder,
schizophrenia and geriatric depression [16–24] .
A wide variety of SNP genotyping methods have
been developed, from a standard geno­typing assay
involving PCR amplification followed by restric-
tion enzyme digestion to novel genotyping meth-
ods, including the oligonucleotide ligation assay
(OLA) [25] , genetic bit analysis [26] , mass spectros-
copy [27] , ‘chip’ technology [28] , TaqMan® [29] and
dynamic allele-specific hybridization (DASH) [30] .
Nevertheless, no single technology has emerged
that is clearly superior owing to limitations such
as cost, complexity and accuracy.

10.2217/PGS.09.44 © 2009 Future Medicine Ltd Pharmacogenomics (2009) 10(6), 989–995 ISSN 1462-2416 989
Special Report Sánchez-Romero, Dorado, Guarino & Llerena
The real-time PCR techniques can be used
for either nonspecific or specific detection using
unspecific DNA-binding dyes (SYBR® Green I)
or target-specific probes (TaqMan assays),
respectively [31,32] . In addition, real-time PCR
equipment allows the analysis of the melting
curves of the products at the end of the reac-
tion as well as the checking of PCR product
purity. The double stranded DNA-specific dye,
SYBR Green I, has been used to analyze the
melting curves of PCR products. These PCR
product melting curves are characterized by a
rapid loss of fluorescence as the temperature is
raised through the sample’s melting temperature
(Tm) [33] .
This study aimed to develop a simple,
fast and accurate new method to detect the
Val66Met polymorphism of the BDNF gene by
melting-curve analysis.
Materials & methods
„„ Samples & instrumentation
The DNA samples were collected from 30 white
European Spanish schizophrenia patients
(Diagnostic and Statistical Manual of Mental
Disorders, 4th Edition [DSM-IV] criteria) and
only individuals without any relevant organic
disease that is treated with aripiprazole were
included in the study for the determination of
the BDNF Val66Met polymorphism. A routine
clinical examination was performed and a medi-
cal history was taken prior to the study. The
subjects were informed about the aims of the
study and each gave their consent to participate.
This study was performed in accordance with
the Helsinki Declaration and was approved
by the Ethical Committee of the Extremadura
University Hospital (Badajoz, Spain).
For PCR amplification and fluorescence
melting-curve analysis, an ABI 7300 instrument
(Applied Biosystems [CA, USA]) was used. The
PCR was performed in standard 96-well plates,
sealed with optical adhesive covers.
For genotyping, 5 ml blood samples were col-
lected in ethylenediaminetetraacetic acid (EDTA)
tubes and DNA was extracted using the QIAamp®
DNA blood kit (Qiagen [Hilden, Germany]).
Table 1. Primer sequences, PCR product length, GC content and predicted product melting temperature.
Variant Forward primer Common reverse primer
(5´>3´) (5´>3´)
Val66 TGGCTGACACTTTCGAACtCG CCGAACTTTCTGGTCCTaAT
Met66 CGCGGCCGGCCTGGCTGACACTTTCGAACcCA CCGAACTTTCTGGTCCTaAT
The polymorphic base is in bold, the 3´ mismatch is in lowercase and the GC clamp is underlined.
Tm: Melting temperature.
990 Pharmacogenomics (2009) 10(6)
The SNP rs6265 of the BDNF gene (GenBank
accession: AF411339; at position 95422) was
analyzed. A total of 30 samples of genomic DNA
were amplified in each reaction using TaqMan
master mix (Applied Biosystems) or Power
SYBR Green I master mix (Applied Biosystems),
according to the genotyping method.
„„ BDNF genotyping using TaqMan®
Amplification conditions consisted of a 10 min
pre-incubation period at 95°C to activate the
Taq DNA polymerase, followed by 40 cycles of
denaturation at 95°C for 15 s and then primer
annealing and extension carried out for 1 min
at 60°C.
Genomic DNA was amplified and analyzed
using the appropriate TaqMan assay, which
includes specific primers and probes for Val66Met
(ID: C_11592758_10) and the Universal PCR
Master Mix, No AmpErase UNG (Applied
Biosystems), which contains AmpliTaq Gold
DNA polymerase, dNTP, buffers and passive
internal reference based on ROX (reference dye).
„„ BDNF genotyping using SYBR®
Green I & melting-curve analysis
Amplification conditions and samples of
genomic DNA were the same as for TaqMan.
Power SYBR Green I master mixture contains
SYBR Green I dye, AmpliTaq Gold DNA poly-
merase, a dNTPs mixture, optimized buffers
and passive internal reference based on ROX
dye. No fluorescent probes were used for this
method and specific primers were designed for
this SNP (Table 1) .
The fluorescence melting curve was analyzed
immediately following amplification. After
amplification, the fluorescence intensity of the
PCR product was measured from 60–92°C
at a temperature gradient of 0.2°C/min. The
ABI 7300 automatically calculates the negative
derivative of the change in fluorescence. When
plotted on a graph, this yields a peak at the Tm
of the PCR product.
Products were designed to be in the 50–100 bp
range, as recommended for optimal SYBR
Green I fluorescence melting-curve analysis.
Product GC Predicted
length content Tm
(bp) (%) (ºC)
59 49 74
70 57 79
future science group
 New genotyping assay for detection of the BDNF Val66Met polymorphism
Dissociation curve
0.32
0.28
0.24
0.20
0.16
Derivative
0.12
0.08
Val/Met
0.04
NTC
0.00
-0.04
60 65 70
Temperature (°C)
Figure 1. Melting-curve analysis of the BDNF Val66Met genotype.
NTC: Not template control.
For these products, a single base change does
not affect the Tm sufficiently to distinguish the
SNPs. To increase the differences in the Tm
between different alleles, a random GC segment
was added to the 5´ end of one of the specific
forward primers. The GC clamp was added to
the primer of the product with the highest initial
Tm to achieve a difference of 4°C between the
two alleles [34] .
For analysis of allelic variants, two forward
primers were designed with the 3´  base of
each primer matching only one of the biallelic
SNP bases to be evaluated. Incorporation of
a primer mismatch at the third base from the
3´ end of the primer has been demonstrated to
enhance the specificity of the PCR by further
destabilizing the extension of the double mis-
matched primer. In addition, to distinguish
the allelic primers during amplification, dif-
ferent mismatches in the third 3´ base of both
forward primers were employed. A common
reverse primer was designed downstream of the
future science group
Special Report
Val/Val
Met/Met
75 80 85 90 95
polymorphic site [35] . Both alleles were analyzed
in the same reaction and in real time, with no
need for additional steps.
Primer design was based on the pub-
lished sequence (sequence accession num-
ber AF411339) using the Primer3 program
(Whitehead Institute for BioMedical Research
[MA, USA]). Primers were synthesized by
Stabvida (Lisbon, Portugal). Forward and
reverse primers were used at 100 nM (Table 1) .
Predicted product Tm was calculated by the
oligonucleotide properties calculador computer
program (OligoCalc, Northwestern University,
IL, USA) [36] .
To determine the optimal annealing tem-
perature of the SYBR Green  I assay, a tem-
perature gradient was employed, the specificity
was evaluated through gel and melting-curve
analysis of the PCR product and the repro-
ducibility of the assay was determined by
standard deviations between replicates (data
not shown).
www.futuremedicine.com 991
Special Report Sánchez-Romero, Dorado, Guarino & Llerena

Results confirmed using specific BDNF rs6265 TaqMan

Figure 1 shows the melting-curve peaks of four
samples (Val66 homozygote, Met66 homo­
zygote, heterozygote and not template control).
This method clearly distinguished homozygous
from heterozygous individuals, where hetero­
zygous melting curves were composites of the
two homozygous melting curves. The Val66
homozygote demonstrated a peak at 74°C, the
Met66 homozygote peaked at 79°C and the
heterozygote included both peaks.
The method used for predicting Tm was a sim-
ple salt-adjusted formula obtained by the oligonu-
cleotide properties calculator [36] . The predicted
and observed Tms were similar. The predicted
Tm of the 59-bp BDNF Val66 product was 74°C.
Adding an 11-bp GC clamp to the BDNF Met66
allele increased the calculated Tm to 79°C.
The fluorescence melting curves of each of
the 30 samples of genomic DNA were ana-
lyzed (Figur e  2) . These genotype results were
minor groove binder allele discrimination
probes (Figure 3) .
In this population, 53.3, 33.3 and 13.3% of
subjects were Val/Val, Met/Met and Val/Met,
respectively. The frequencies of Val66 and
Met66 were 0.6 and 0.4, respectively.
Discussion
The method reported here is a simple, fast and
accurate real-time PCR-based genotyping assay
to detect the Val66Met polymorphism. The use
of SYBR Green I is relatively inexpensive, which
may make the present approach more cost-
effective in comparison with other fluorescence-
based PCR techniques for SNP detection. This
method is similar in price to others (e.g., the
TaqMan assay) but the cost is less for smaller
sample sizes since the reagents can be bought
in smaller quantities and the SYBR Green I
dye is cheaper than others. Thus, this method

Dissociation curve

0.32

0.28

0.24

0.20

0.16
Derivative

0.12

0.08

0.04

0.00

-0.04
60 65 70 75 80 85 90 95

Temperature (°C)

Figure 2. Melting curve analysis of the BDNF Val66Met genotype in 30 samples of

genomic DNA.

992 Pharmacogenomics (2009) 10(6) future science group

 New genotyping assay for detection of the BDNF Val66Met polymorphism
2.00
1.60
Allele Y (BDNF A [Met])
1.20
0.80
0.40
0.00
-0.40
0.00 0.20 0.40
Allele X
Figure 3. Cluster plot of 30 samples of genomic DNA and two not template controls that
were genotyped using a TaqMan® SNP genotyping assay.
X, Y and both represent a Val66 homozygote, Met66 homozygote and heterozygote, respectively.
NTC: Not template control.
may permit the analysis of both BDNF alleles,
Val66 and Met66, in the same reaction with
no need for additional steps. The allele-specific
amplification with the additional 3´ GC clamp
enables discrimination between the two alleles,
but the difference in hybridization temperature
of a primer with two mismatches as opposed
to one is probably insufficient to differentially
amplify two alleles. In this assay, specificity is
provided by the in­e fficient extension by the
Taq DNA polymerase from a primer with an
unmatched 3´  end. The allele-specific prim-
ers can be designed either in their forward or
reverse direction; however, the primer orienta-
tion should be the same (forward or reverse) if
both reactions are performed in the same tube.
The use of allele-specific primers containing
an additional mismatch in the third 3´  base
of both forward primers eliminates the need
for extensive optimization of PCR conditions,
which reduces the time and effort required dur-
ing assay setup and increases robustness during
sample analysis  [35,37] . Furthermore, the addi-
tional GC clamp makes this method universally
applicable since the Tm of one allele can be
adjusted as necessary to give distinct separation
of melting curves. The GC clamp works well
for samples of a variety of GC content and Tms.
The addition of the GC-rich sequence to the
future science group
Special Report
Allelic discrimination
0.60 0.80 1.00 1.20 1.40 1.60 1.80
Allele X (BDNF G [Val])
Allele Y Both NTC
5´ end of the primer does not interfere with its
annealing to the original sequence [34,38–40] and
ensures a readily measurable difference in the
Tm of two alleles amplified in the same reaction
tube. Primers are standard DNA oligonucle-
otides, so there is no need to use additional
fluorescent probes other than the generic SYBR
Green I fluorescent dye. Moreover, all reactions
and measurements take place in a single closed
tube, removing the need to manipulate the
PCR product and reducing the risk of post-PCR
contamination. Thus, the use of both allele-
specific PCR and GC clamp/differential melt-
ing analysis generates a powerful SNP genotyp-
ing method that is also reliable, inexpensive and
simple to perform.
In conclusion, the newly developed method
of PCR with melting-curve analysis is simple,
fast and accurate for the determination of the
BDNF Val66Met polymorphism and would
be useful if applied to SNP analysis in other
pharmacogenetic studies.
Future perspective
The GC clamp method is extensively applica-
ble since the addition of a GC clamp allows to
adjust the Tm of one allele to distinguish dif-
ferent melting curves. Theoretically, using this
methodology, it would be possible to analyze
www.futuremedicine.com 993
Special Report Sánchez-Romero, Dorado, Guarino & Llerena

Executive summary
ƒƒ This method permits the analysis of both the BDNF alleles Val66 and Met66 in the same reaction with no need for additional steps.
ƒƒ The influence of the BDNF gene polymorphisms on response to neuroleptic drugs and lithium has been demonstrated.
ƒƒ The developed method could have a general application in pharmacogenetic studies since it could be adapted to evaluate rapidly and
routinely different SNPs on different genes.
ƒƒ The addition of a GC clamp makes this method universally applicable since the melting temperature of one allele can be adjusted as
necessary to give distinctive separation of melting curves.
ƒƒ All reactions and measurements take place in a single closed tube, which removes the need to manipulate the PCR products and reduces
the risk of post-PCR contamination.

multiple SNPs in the same reaction tube and
primers can be designed with an adequate Tm
difference. This application would be useful in
pharmacogenomic studies.
Acknowledgements
The collaboration of Eva Peñas-LLedó MD PhD and
Alfredo de la Rubia MD PhD is gratefully acknowledged.
Financial & competing interests disclosure
This study was supported in Spain by the Ministries of
Education and Science (SAF2006/13589) and Health,
Instituto de Salud Carlos III-FIS and European Union
(FEDER): CIBERSAM, PI06/1681 and CP/06/0030
(Pedro Dorado). The study was coordinated in the net-
work Red Iberoamericana de Farmacogenética y
Farmacogenómica (CYTED206RT0290). The authors
have no other relevant affiliations or financial involve-
ment with any organization or entity with a financial
interest in or financial conflict with the subject matter or
materials discussed in the manuscript apart from
those disclosed.
No writing assistance was utilized in the production of
this manuscript.

6 Flanagin BA, Cook EH Jr, de Wit H: An 12 Dmitrzak-Weglarz M, Rybakowski JK,

Bibliography
Papers of special note have been highlighted as:
n of interest
1 Thoenen H: Neurotrophins and neuronal
plasticity. Science 270, 593–598 (1995).
2 Durany N, Michel T, Zochling R et al.:
Brain-derived neurotrophic factor and
neurotrophin 3 in schizophrenic psychoses.
Schizophr. Res. 52, 79–86 (2001).
3 Krebs MO, Guillin O, Bourdell MC et al.:
Brain derived neurotrophic factor (BDNF )
gene variants association with age at onset
and therapeutic response in schizophrenia.
Mol. Psychiatry 5, 558–562 (2000).
n Suggests a contribution of BDNF gene
variants to a treatment-sensitive form of
schizophrenia and thus, BDNF appears to
be associated with developmental features
of schizophrenia.
4 Hong CJ, Yu YW, Lin CH, Tsai SJ: An
association study of a brain-derived
neurotrophic factor Val66Met polymorphism
and clozapine response of schizophrenic
patients. Neurosci. Lett. 349, 206–208
(2003).
5 Tsai SJ, Cheng CY, Yu YW, Chen TJ,
Hong CJ: Association study of a brain-
derived neurotrophic-factor genetic
polymorphism and major depressive
disorders, symptomatology, and
antidepressant response. Am. J. Med.
Genet. B Neuropsychiatr. Genet. 123,
19–22 (2003).
association study of the brain-derived
neurotrophic factor Val66Met polymorphism
and amphetamine response. Am. J. Med. Genet.
B Neuropsychiatr. Genet. 141, 576–583 (2006).
n Suggests that BDNF is related to response to
low doses of amphetamine.
7 Choi MJ, Kang RH, Lim SW, Oh KS, Lee MS:
Brain-derived neurotrophic factor gene
polymorphism (Val66Met) and citalopram
response in major depressive disorder. Brain
Res. 1118, 176–182 (2006).
8 Kato M, Serretti A: Review and meta-analysis
of antidepressant pharmacogenetic findings in
major depressive disorder. Mol. Psychiatry
4 (2008) (Epub ahead of print).
9 Monteleone P, Maj M: Genetic susceptibility to
eating disorders: associated polymorphisms and
pharmacogenetic suggestions.
Pharmacogenomics 9, 1487–1520 (2008).
10 Rybakowski JK, Suwalska A, Skibinska M
et al.: Prophylactic lithium response and
polymorphism of the brain-derived
neurotrophic factor gene. Pharmacopsychiatry
38, 166–170 (2005).
n First demonstration of the association
between the Val66Met polymorphism and
response to lithium prophylaxis.
11 Masui T, Hashimoto R, Kusumi I et al.:
Lithium response and Val66Met
polymorphism of the brain-derived
neurotrophic factor gene in Japanese patients
with bipolar disorder. Psychiatr. Genet. 16,
49–50 (2006).
Suwalska A et al.: Association studies of the
BDNF and the NTRK2 gene polymorphisms
with prophylactic lithium response in bipolar
patients. Pharmacogenomics 9, 1595–1603
(2008).
13 Bueller JA, Aftab M, Sen S,
Gomez‑Hassan D, Burmeister M, Zubieta
JK: BDNF Val66Met allele is associated with
reduced hippocampal volume in healthy
subjects. Biol. Psychiatry 59, 812–815 (2006).
n The presence of the BDNF Met66 allele was
found to be associated with reduced
hippocampal formation in
healthy volunteers.
14 Kleim JA, Chan S, Pringle E et al.: BDNF
Val66Met polymorphism is associated with
modified experience-dependent plasticity in
human motor cortex. Nat. Neurosci. 9,
735–737 (2006).
15 Nemoto K, Ohnishi T, Mori T et al.: The
Val66Met polymorphism of the brain-
derived neurotrophic factor gene affects
age-related brain morphology. Neurosci. Lett.
397, 25–29 (2006).
16 Duman RS: Role of neurotrophic factors in
the etiology and treatment of mood
disorders. Neuromolecular Med. 5, 11–25
(2004).
17 Schumacher J, Jamra RA, Becker T et al.:
Evidence for a relationship between genetic
variants at the brain-derived neurotrophic
factor (BDNF) locus and major depression.
Biol. Psychiatry 58, 307–314 (2005).

994 Pharmacogenomics (2009) 10(6) future science group

 New genotyping assay for detection of the BDNF Val66Met polymorphism
18 Zhang H, Ozbay F, Lappalainen J et al.: Brain
derived neurotrophic factor (BDNF) gene
variants and Alzheimer’s disease, affective
disorders, posttraumatic stress disorder,
schizophrenia, and substance dependence.
Am. J. Med. Genet. B Neuropsychiatr. Genet.
141B, 387–393 (2006).
19 Gratacos M, Gonzalez JR, Mercader JM,
de Cid R, Urretavizcaya M, Estivill X:
Brain-derived neurotrophic factor Val66Met
and psychiatric disorders: meta-analysis of
case-control studies confirm association to
substance-related disorders, eating disorders,
and schizophrenia. Biol. Psychiatry 61,
911–922 (2007).
n Meta-analysis study that confirms the
association of Val66Met with substance-
related disorders, eating disorders
and schizophrenia.
20 Neves-Pereira M, Mundo E, Muglia P,
King N, Macciardi F, Kennedy JL: The
brain-derived neurotrophic factor gene confers
susceptibility to bipolar disorder: evidence
from a family-based association study. Am. J.
Hum. Genet. 71, 651–655 (2002).
21 Karamohamed S, Latourelle JC, Racette BA
et al.: BDNF genetic variants are associated
with onset age of familial Parkinson disease:
GenePD Study. Neurology 65, 1823–1825
(2005).
22 Lohoff FW, Sander T, Ferraro TN, Dahl JP,
Gallinat J, Berrettini WH: Confirmation of
association between the Val66Met
polymorphism in the brain-derived
neurotrophic factor (BDNF) gene and
bipolar I disorder. Am. J. Med. Genet. B
Neuropsychiatr. Genet. 139, 51–53 (2005).
23 Neves-Pereira M, Cheung JK, Pasdar A et al.:
BDNF gene is a risk factor for schizophrenia
in a Scottish population. Mol. Psychiatry 10,
208–212 (2005).
24 Oroszi G, Lapteva L, Davis E et al.: The
Met66 allele of the functional Val66Met
polymorphism in the brain-derived
neurotrophic factor gene confers protection
against neurocognitive dysfunction in
systemic lupus erythematosus. Ann. Rheum.
Dis. 65, 1330–1335 (2006).
future science group
25 Landegren U, Kaiser R, Caskey CT, Hood L:
DNA diagnostics – molecular techniques and
automation. Science 242, 229–237 (1988).
26 Nikiforov TT, Rendle RB, Goelet P et al.:
Genetic Bit Analysis: a solid phase method
for typing single nucleotide polymorphisms.
Nucleic Acids Res. 22, 4167–4175 (1994).
27 Griffin S, Wyllie SG, Markham J:
Determination of octanol-water partition
coefficient for terpenoids using reversed-
phase high-performance liquid
chromatography. J. Chromatogr. A 864,
221–228 (1999).
28 Wang E, Lacelle C, Xu S, Zhao X, Hou M:
Designer microarrays: from soup to nuts.
J. Gerontol. A Biol. Sci. Med. Sci. 57,
B400–B405 (2002).
29 Livak KJ, Flood SJ, Marmaro J, Giusti W,
Deetz K: Oligonucleotides with fluorescent
dyes at opposite ends provide a quenched
probe system useful for detecting PCR
product and nucleic acid hybridization. PCR
Methods Appl. 4, 357–362 (1995).
30 Howell WM, Jobs M, Gyllensten U,
Brookes AJ: Dynamic allele-specific
hybridization. A new method for scoring
single nucleotide polymorphisms. Nat.
Biotechnol. 17, 87–88 (1999).
31 Higuchi R, Fockler C, Dollinger G,
Watson R: Kinetic PCR analysis: real-time
monitoring of DNA amplification reactions.
Biotechnology (NY) 11, 1026–1030 (1993).
32 Gupta M, Yates CR, Meibohm B: SYBR®
Green-based real-time PCR allelic
discrimination assay for b2-adrenergic
receptor polymorphisms. Anal. Biochem.
344, 292–294 (2005).
33 Ririe KM, Rasmussen RP, Wittwer CT:
Product differentiation by analysis of DNA
melting curves during the polymerase chain
reaction. Anal. Biochem. 245, 154–160
(1997).
n Suggests that the ana­lysis of melting curves
can extend the dynamic range of initial
template quantification when amplification
is monitored with double-stranded
DNA‑specific dyes.
www.futuremedicine.com
Special Report
34 Sheffield VC, Cox DR, Lerman LS,
Myers RM: Attachment of a 40-base-pair
G + C-rich sequence (GC-clamp) to
genomic DNA fragments by the
polymerase chain reaction results in
improved detection of single-base changes.
Proc. Natl Acad. Sci. USA 86, 232–236
(1989).
35 Okimoto R, Dodgson JB: Improved PCR
amplification of multiple specific alleles
(PAMSA) using internally mismatched
primers. Biotechniques 21, 20–22, 24, 26
(1996).
36 Kibbe WA: OligoCalc: an online
oligonucleotide properties calculator.
Nucleic Acids Res. 35, W43–W46
w(2007).
n OligoCalc is web-accessible, with three
common methods for calculating
oligonucleotide-melting temperatures.
37 Akey JM, Sosnoski D, Parra E et al.:
Melting-curve analysis of SNPs (McSNP):
a gel-free and inexpensive approach for SNP
genotyping. Biotechniques 30, 358–362,
364, 366–367 (2001).
38 Papp AC, Pinsonneault JK, Cooke G,
Sadee W: Single nucleotide polymorphism
genotyping using allele-specific PCR and
fluorescence melting curves. Biotechniques
34, 1068–1072 (2003).
n A PCR method for the identification of
SNPs by incorporating a GC-rich sequence
into one of the two allele-specific primers
to increase the melting temperature.
39 Boniotto M, Hazbon MH, Jordan WJ
et al.: Novel hairpin-shaped primer assay to
study the association of the -44 single-
nucleotide polymorphism of the DEFB1
gene with early-onset periodontal disease.
Clin. Diagn. Lab. Immunol. 11, 766–769
(2004).
40 Germer S, Holland MJ, Higuchi R:
High-throughput SNP allele-frequency
determination in pooled DNA samples by
kinetic PCR. Genome Res. 10, 258–266
(2000).
995