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Fast and Flexible Single Nucleotide Polymorphism
(SNP) Detection with the LightCycler System
S. Lohmann, L. Lehmann, and K. Tabiti
Roche Molecular Biochemicals, Penzberg, Germany
ntroduction
One of the major objectives of genetics is the association of sequence variations with heritable phenotypes.
Traditional strategies, such as linkage analysis, in
which pedigree analysis track transmission of a disease through a family, have been successfully applied
to in the detection of Mendelian disorders. In recent
years a more powerful approach involving the detection of single nucleotide polymorphisms (SNPs) has
become increasingly popular. By convention, a
nucleotide polymorphism must be present in at least
one percent of the human population to be called an
SNP. SNPs are the most common type of DNA sequence variations and occur once every 100-300 bases.
Researchers looking for associations between a disease and specific sequence differences in a population, use this high degree of variation. As it is easier to
obtain DNA samples from a random set of individuals
in a population, than from every member of a family
over several generations, it is conceivable that researchers may be able to increase the identification of
genes responsible for pathological traits. Due to the
high frequency of SNPs, the chances are high that the
disease is predominantly caused by, or closely associated with, specific SNPs.
SNPs present a potentially vast arena for the detection
of genetic alterations that seem to relate to medically
important differences in disease susceptibility and
drug response. In the near future it may be possible to
identify the genetic basis for complex diseases such as
cancer, cardiovascular disease, mental illness, autoimmune states, and diabetes. The number of polymorphisms known to predispose for a disease is believed
to increase exponentially.
Yet even currently known disease-predisposing polymorphisms may benefit from our expanding knowledge: "old polymorphisms” may be relevant in a novel
context. For example, the E4 genotype of the human
apolipoprotein E gene has been traditionally associated
with lipid disorders. Many recent reports have obser-
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ved a relationship between the Apo E genotype and the
susceptibility to Alzheimer's disease. Even more
recently, scientists have reported that polymorphisms
of the apolipoprotein E gene may influence the outcome after traumatic brain injury and intracerebral
hemorrhage [1]. Thus, one particular haplotype is
informative for several traits.
Besides the above-mentioned common diseases in
humans, the response to medication in terms of both
adverse reactions and beneficial response is closely
regulated by a plenitude of genes. This understanding
is fueling many pharmaceutical companies' interest in
generating SNP libraries, with a view to detecting
genes involved in drug metabolism. One of the most
predominant examples of such an effort is the SNP
Consortium (TSC), a joint nonprofit venture of ten
pharmaceutical companies with the aim of creating
and releasing to the public a library of some 300 000
SNPs [2].
LIGHTCYCLER
I
The possibility of using DNA sequence information to
distinguish those individuals who are likely to benefit
from a new medication from those who could suffer
adverse side-effects, or to determine the optimal dosage, has rekindled the interest in the research area termed pharmacogenetics. The ideas underlying pharmacogenetics are not new: for decades it has been understood that genes affect the interindividual variation in
drug response. The concerted actions in detecting and
analyzing SNPs may now open new avenues for personalized medicine, which should improve therapeutic
outcome, reduce overall healthcare costs and ultimately benefit the individual.
Besides these SNPs involved in drug metabolism, specific SNPs are associated with a higher risk of disease
(i.e. disease associated SNPs). Knowledge of these
SNPs is important for the understanding of processes
in disease development and the role and function of
the genes involved. A future vision is to use this information for improved diagnosis, disease prevention and
causal therapy.
This article gives an overview describing how the
LightCycler Instrument can be applied in genotyping
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SNPs in less than 40 min for 32 research samples and
refers to the most recent publications and LightCycler
Kits in these exciting research areas.
S
NP Detection using the LightCycler System
The LightCycler Instrument enables both the amplification and the real-time, on-line detection of a PCR product, thus allowing accurate quantification.
Furthermore, the system also provides a unique and
innovative approach perfectly suited for the detection
and genotyping of single nucleotide polymorphisms:
the melting curve analysis feature. During the melting
curve analysis the LightCycler Instrument monitors the
temperature-dependent hybridization of the sequencespecific Hybridization Probes to single stranded DNA.
hn
LIGHTCYCLER
A
LC-Red 640
hn
Fluorescein
mutation probe 1
anchor probe 1
LC-Red 705
Fluorescein
mutation probe 2
anchor probe 2
primer
5‘
3‘
codon 112
mutation site
codon 158
mutation site
3‘
5‘
B primer
The genotype of codon 112 is monitored in the F2 detection channel of the
LightCycler Instrument.
Fluorescence -d(F2)/dT
4
Homozygous (CGC)
Heterozygous
Homozygous (TGC)
H2O
3
2
1
0
-0.5
45.0
50.0
55.0
60.0
Temperature (°C)
65.0
70.0
75.0
Fluorescence -d(F3)/dT
The genotype of codon 158 is monitored in the F3 detection channel of the
LightCycler Instrument.
3.2
2.8
2.4
2.0
1.6
1.2
0.8
0.4
0.0
Homozygous (CGC)
Heterozygous
Homozygous (TGC)
H2O
45.0
Ý
50.0
55.0
60.0
Temperature (°C)
65.0
70.0
75.0
Figure 1: Dual color SNP genotyping example using the LightCycler-Apo E Mutation
Detection Kit (Cat. No. 3 004 716). A. Schematic presentation of the PCR fragment, and the ori-
entation of the PCR primers, the anchor probes, and the mutation probes.B. Discrimination of the
different genotypes at codon 112 and 158 of the Apoliprotein E sequence with the melting curve
analysis function. The first negative derivative of the fluorescence versus temperature [-d(F2)/dT]
or [-d(F3/dT] graph shows peaks with different T m. The melting peaks indicate that the fully
homologous sequence (homozygous CGC) has a higher Tm than the sequences that have a mismatch
with the mutation probe (homozygous TGC). Samples containing both sequences (heterozygous
genotypes) display two peaks at exactly the same temperatures as the respective homozygous samples. As a negative control, the template DNA was replaced with PCR-grade water.
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No post-PCR processing is needed and the risk of contamination is minimized, as amplification and genotyping are performed in the same sealed capillary without any further handling steps.
In brief, during the PCR, a DNA fragment of the respective gene is amplified with specific primers from
human genomic DNA. The amplicon is detected by fluorescence using specific pairs of Hybridization Probes.
Hybridization Probes consist of two different oligonucleotides that hybridize to an internal sequence of
the amplified fragment during the annealing phase of
PCR cycles. One probe is labeled at the 5’-end with a
LightCycler–Red fluorophore (LightCycler-Red 640 or
LightCycler-Red 705) and, to avoid extension, is modified at the 3’-end by phosphorylation. The other probe
is labeled at the 3’-end with fluorescein. Only after
hybridization to the template DNA do the two probes
come in close proximity, resulting in fluorescence resonance energy transfer (FRET) between the two fluorophores. During FRET, fluorescein, the donor fluorophore, is excited by the light source of the LightCycler
Instrument, and part of the excitation energy is transferred to LightCycler-Red, the acceptor fluorophore.
The emitted fluorescence of the LightCycler-Red fluorophore is measured.
These Hybridization Probes are also used to determine
the genotype by means of a melting curve analysis
implemented after the amplification cycles are completed and the amplicon is formed. The melting temperature (Tm) of the heteroduplex consisting of
Hybridization Probe and single-stranded target DNA
sequence is dependent on GC content, length, degree
of homology and sequence order [3]. One of the two
Hybridization Probes covers the region of the potential
mutation (mutation probe) and has a lower Tm than the
adjacent probe (anchor probe) (Figure 1). Hybridization
Probe/DNA hybrids containing a mismatch melt at a
lower Tm than perfectly matched probes. Hence, wildtype, mutant and heterozygous genotypes can be
distinguished by different melting temperatures, displayed in the LightCycler software as melting peaks.
The time required to genotype SNPs is reduced to less
than 40 min for 32 research samples, while eliminating
post PCR-processing and minimizing the risk of contamination (Figure 1).
The principle of using Hybridization Probes and melting curving analysis to detect specific mutations and
SNPs has been described in detail elsewhere [4,5]. For
more detailed information please visit our website
http://www.biochem.roche.com/lightcycler
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A Technical Note describing different concepts and
strategies for mutation detection using the LightCycler
Instrument is available on request (TN 14/2000:
General Recommendations for LightCycler Mutation
Analysis Using Dual Color and Melting Temperature).
A
pplication fields
Pharmacogenetics
SNPs and other DNA variations in drug metabolizing
enzymes are related to side effects, incompatibility
and variations in therapeutic efficacy. The human
cytochrome P-450 (CYP) supergene family represents
an important group of enzymes involved in the metabolite activation of procarcinogenesis to produce
potentially carcinogenic metabolites. A rapid and
reliable method has been established for genotyping
the CYP1B1 codon 432-polymorphisms using the
LightCycler Instrument [6].
Variability in drug acetylation is due to geneticallycontrolled expression of the N-acetyltransferase 2
enzyme (NAT2). Several polymorphisms in the NAT2
gene are responsible for a slow acetylator phenotype.
The LightCycler-NAT2 Mutation Detection Kit (Cat.
No. 3 113 914) from Roche Molecular Biochemicals
(RMB) allows the simultaneous detection of the 4
major polymorphisms in the NAT2 gene, using two primer pairs to amplify and cover the different polymorphisms with four different Hybridization Probe pairs.
Dihydropyrimidine dehydrogenase (DPD), thymidylate
synthase (TS) and thymidine phosphorylase (TP) are
involved in fluoropyrimidine metabolism and thus
regulate levels of 5-FU, a drug used in chemotherapy.
Several point mutations in these enzymes are associated with expression levels of these enzymes and
are consequently related to therapeutic success.
Dedicated LightCycler kits for analyzing these polymorphisms are under development with a view to supporting research in this field.
Metabolic disorders
The ability to use the LightCycler System for the rapid
detection of deletion mutations in inherited metabolic
diseases has recently been demonstrated in research
studies for two different parameters [7]. Fabry disease
is an X-linked recessive disorder caused by deficient
activity of a-galactosidase. Using Hybridization Probes
and melting curve analysis, a 2-bp deletion mutation
in genomic DNA of an individual with Fabry disease
has successfully been detected. In addition, the aut-
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hors were able to detect a 9-bp deletion in an individual with carbamyl-phophate synthase I (CPS1) deficiency using the SYBR Green I detection format and
melting curve analysis. CPS1 deficiency is an autosomal recessive disorder caused by deficient activity of
CPS1 in the liver. This study demonstrates, for the first
time, the detection of a 9-bp deletion using the SYBR
Green I detection format. With this latter format the Tm
is influenced primarily by GC content, and to a lower
extent by size.
Two point mutations (Cys282Tyr, His63Asp) in the
human hemochromatosis gene (HFE) are considered
to be responsible for the development of hereditary
hemochromatosis (HH), an autosomal recessive ironloading syndrome. The homozygous Cys282Tyr mutation is present in 80-100% of hemochromatosis cases
[8]. The authors used a multiplex PCR protocol to
amplify the relevant locations. Two sets of
Hybridization Probes, both labeled with LC Red 640,
were used for the genotyping of the SNPs. As shown
by Bernhard et al., it is also possible to use a Dual
Color approach to detect both mutations [9]. A recently described third mutation, S65C, is enriched in HH
individuals with a mild form of the disease and with no
mutations at C282 or H63. Bollhalder et al. published
a LightCycler protocol for the simultaneous detection
of the H63D and S65C mutations of the HFE gene [10].
One primer and probe set can be used to detect both
sites, as the distance between them is just 6 nucleotides (A193T, C187G). One mutation probe was designed to be complementary to the 63D mutant and the
S65 wildtype. Therefore, 65C mutant melts off at the
lowest melting temperature (Tm), followed by the H63
wildtype and the 63D mutant allele showing the highest Tm.
LIGHTCYCLER
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Protease inhibitor 1 (a1-antitrypsin; AT) is the main
serum inhibitor of proteolytic enzymes. In AT deficiency, enzymes such as neurophil elastase can damage
the lung tissues, leading to pulmonary emphysema.
The 3 most important types are: type M (90% of the
population), type S (Pi*S), and type Z (Pi*Z) [11]. A
pair of primers flanking each of the mutations in the
Pi*S and Pi*Z alleles was designed to amplify a 238 bp
and a 253 bp product. Hybridization Probe pairs
homologous to the wildtype sequence were used for
genotyping [12].
Disease-associated applications
The most common causes of inherited thrombophilia
in the Caucasian population are point mutations in the
Factor V gene and the Prothrombin gene (Factor II).
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LIGHTCYCLER
The C677T point mutation in the methylenetetrahydrofolate reductase (MTHFR) gene is also associated with
a higher risk. Over 90% of individuals with a resistance
to activated protein C (APC-resistance), a condition
associated with thrombophilia, are associated with
hetero- or homozygosity for a single point mutation in
the Factor V gene. This G to A substitution in exon 10
(G1691A), also known as Factor V Leiden, leads to an
arginine substituted by glutamine (R506Q) and prevents cleavage by activated protein C that normally
inactivates the coagulation factor. A respective
LightCycler research kit is available from Roche
Molecular Biochemicals: the LightCycler Factor V
Leiden Mutation Detection Kit (Cat. No. 2 212 161). A
methodological comparison between allele-specific
PCR (PCR-SSP) and the LightCycler technology in the
genotyping of the Factor V Leiden mutation in research
samples was recently published [13]. The results generated with both methods showed 100% correlation. For
prothromobin, a G to A substitution (G20210A) in the
3´untranslated region of the human prothrombin gene
leads to a substitution of argine by glutamine. This
polymorphism is associated with an elevated level of
plasma prothrombin. The approach for detecting this
SNP is identical to the Factor V assay. A dedicated
RMB research kit is available for the LightCycler, i.e.
the LightCycler Prothrombin Mutation Detection Kit
(Cat. No 2 236 842). An additional independent risk
factor for thrombophilia is hyperhomocysteinemia. A
point mutation in the MTHFR gene results in reduced
MTHFR activity. Elevated plasma homocysteine levels
are the consequence. Research papers on the genotyping of the relevant point mutation (C677T) in the responsible MTHFR gene with the LightCycler Instrument
have been published [14, 15].
The role of apolipoprotein E (Apo E) in lipid disorders
has already been mentioned. The LightCycler – Apo E
Mutation Detection Kit from RMB (Cat. No. 3 004 716)
can be used for the simultaneous detection of two
point mutations within codons 112 and 158 of the
human apolipoprotein E gene using a dual color
approach (Figure 1). The Hybridization Probe mix provided with the kit includes one LC Red 640 labeled
Hybridization Probe for detecting the nucleotide polymorphism C3932T, while a second Hybridization Probe
labeled with LC Red 705 specifically detects the possible C4070T substitution. A recently published evaluation report shows 100% concordance for the data obtained for 120 DNA samples with allele specific restriction enzyme analysis (ASRA) and genotyping with the
LightCycler-Apo E Mutation Detection Kit, representing a genotype distribution typical for the Caucasian
population [16].
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The respective LightCycler kit from RMB for human
apolipoprotein B (Apo B) is intended as a research tool
to help further understand the relevance of the Apo B
gene in diseases like familial hypercholesterolemia,
atherosclerosis, and ischemic heart disease (Cat. No. 3
004 708). Two relevant point mutations in the Apo B
gene are located in directly adjacent positions: C9774T,
G9775A. Only one Hybridization Probe can be used to
detect these nucleotide polymorphisms since the
C9774T mutation has a smaller impact on Tm than the
G9775A, resulting in different Tms.
A detailed description of the different detection principles used for prothrombin, apolipoprotein B and apolipoprotein E has been published [17].
Monogenic hereditary disease
Cystic fibrosis (CF) is one of the most common autosomal recessive disorders among the Caucasian population, with a frequency of 0.05% and a carrier frequency of 5%. Various disease causing polymorphisms are
associated with CF. The most common mutation is a 3bp deletion that removes a phenylalanine from the protein at codon 508 (F508del). This deletion accounts for
70% of all mutations, and 50% of individuals with CF
are homozygous for this mutation. Amplification and
genotyping with the LightCycler Instrument was achieved in less than 30 min. A total of 105 samples were
genotyped for the deletion F508del. Furthermore, a
single nucleotide polyphorphism leading to the F508C
variant was detected [18].
Polygenic hereditary disease
The majority of disorders such as allergies, diabetes or
cancer are related to a distinct polygenic background.
The individual genetic background may be associated
with a higher risk of cancer development, but complex
events occur during carcinogenesis and tumor progression.
Point mutations in different members of the ras gene
family are involved in a variety of human malignancies.
N- ras mutations are frequently observed in hematological malignancies. A rapid and reliable strategy for
detecting N- ras mutations in acute lymphoblastic leukemia (ALL) has recently been published [19]. The authors designed two sets of primers and Hybridization
Probes to cover ‘hot spot’ regions for mutations within
codons 12, 13, and 61. Amplicons exhibiting an abnormal melting characteristic were directly sequenced.
134 samples from individuals with childhood ALL were
screened for N- ras mutations and 14 mutation-positive cases characterized in less than three days, revealing an incidence for N- ras mutations of 10%, i.e. compatible with previous reports.
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Infectious disease
Helicobacter pylori colonizes the human stomach,
inducing chronic gastritis and playing a major role in
gastric and duodenal ulceration. Clarithromycin is frequently used in macrolide antibiotic therapies.
Resistance to clarithromycin is associated with single
base pair mutations within the peptidyltransferaseencoding region of the 23S rRNA gene. Gibson et al.
use the SYBR Green I detection format in combination
with a single labeled Hybridization Probe to monitor
these polymorphisms using melting curve analysis. The
authors were able to detect the three most relevant
SNPs using this approach [20].
C
onclusions
There are numerous examples for SNP detection in
research applications. In each case a LightCycler
approach can be developed to perform these assays.
Table 1 summarizes the LightCycler Kits currently available from RMB and those applications that have
already been published.
The renewed and extensive interest in genome polymorphism will have a major impact upon population
genetics, drug development, forensic, cancer and
genetic disease research. The optimal genotyping
method should allow the allele determination of SNPs
rapidly and economically. The LightCycler System fulfills these demands and is the perfect tool for reliable
and accurate genotyping of single nucleotide polymorphisms.
Application area
Associated gene
Reference / LightCycler Kit
Pharmacogenetics
Cytochrome P450: CYB1B1
[6]
Cytochrome P450: CYP2D6
N-Acetyltransferase 2 (NAT2)
[21]
Cat.No. 3 113 914
Metabolic disorders
Disease-associated
Monogenic hereditary
disease
Polygenic hereditary
disease
Infectious disease
others
Thiopurine methyltransferase (TPMT*3)
[22]
Carbamyl-phosphate synthase (CPS1)
[7]
Fabry disease
[7]
Hemochromatosis (HFE)
[8, 9, 10, 23]
Protease inhibitor 1 (a1-antitrypsin; AT)
[11, 12]
Angiotensin converting enzyme (ACE)
[24]
Apolipoprotein B (Apo B)
Cat. No. 3 004 708
Apolipoprotein E (Apo E)
Cat. No. 3 004 716
Factor V
Cat. No. 2 212 161
Methylenetetrahydrofolate reductase
(MTHFR)
[14, 15]
Mitochondrial DNA (MELAS3243)
[25]
Plasma plasminogen activator inhibitor-1
(PAI-1)
[26]
Prothrombin (Factor II)
Cat. No. 2 236 842
Cystic fibrosis (CF)
[18]
b-Globin (HbC, HbE, and HbS genotyping)
[27]
BRCA1
[28]
N-ras
[19]
Glycoprotein Ia
[29]
Clarithromycin-resistance associated
gene mutations
[20]
Lamivudine resistance associated gene mutations
[30]
Human platelet antigen-1 (HPA-1)
[31]
LIGHTCYCLER
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Note: LightCycler kits listed are for life science research only.
Ý
Table 1 Summary of research application areas and published literature or respective LightCycler Kit.
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References
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(1999) J. Clin. Microbiol. 37: 3746-3748.
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Product
Cat. No.
Pack Size
LightCycler- NAT2
Mutation Detection Kit
3 113 914
32 reactions
LightCycler-Apo B
Mutation Detection Kit
(codon 3500)
3 004708
32 reactions
LightCycler-Apo E
Mutation Detection Kit
(codon 112 and 158)
3 004716
32 reactions
LightCycler Factor V Leiden 2 212161
Mutation Detection Kit
32 reactions
LightCycler-Prothrombin
(G20210A) Mutation
Detection Kit
2 236842
32 reactions
LightCycler Red 640
NHS ester
2 015 161
1 vial
LightCycler Red 705
Phosphoramidite
2 157 594
1 vial
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