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Articles |
Department of Clinical Chemistry, Georg-August-University, Robert-Koch-Strasse 40, 37075 Goettingen, Germany.
a Author for correspondence. Fax 49-551-39-8551; e-mail nahsen{at}gwdg.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We investigated the ability of a genotyping assay based on hybridization of labeled oligonucleotides to detect and discriminate known and as yet unknown mutations in the factor V and apolipoprotein B-100 genes. Expected melting points were calculated using a nearest-neighbor model for nucleic acid duplex stability and compared with experimental findings derived from LightCycler melting curves. A method for genotyping the apolipoprotein B-100 G10699A and C10698T mutations is presented.
Results: All mismatches tested for in the probed sequence could be detected with a single probe. The measured melting points were in good agreement with their values predicted using the nearest-neighbor model (r = 0.96; y = 0.98x + 1.18; Sy|x = 0.96; n = 24).
Conclusions: This procedure not only allows the identification of the mutation of interest but also enables the discrimination from other potential mutations in the vicinity of the former. The nearest-neighbor model is valid for hybridization probe assays on the LightCycler and should be of general value in setting up such assays. We have shown for two clinically relevant genotyping examples that the LightCycler mutation detection system has superior discriminatory performance compared with conventional RFLP or ASA PCR-based methods for molecular diagnostic purposes. With this method, in every hybridization probe assay, all mutations under a properly designed probe should be detectable, but they will not necessarily be discriminated from each other in all cases.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Another approach is the use of oligonucleotide probes for mutation detection. Probes added to PCR reaction will specifically hybridize to their complementary strand depending on the experimental conditions. Mutations under the probe decrease the stability of the duplex and lead to a decreased melting temperature (Tm) (3). This principle of mutation detection together with a rapid PCR cycling can be realized by use of the LightCyclerTM (Roche Diagnostics), as has already been described for factor V (4), methylenetetrahydrofolate reductase (5), prothrombin G20210A (6), and HFE (7) genotyping. When two fluorescent dye-labeled probes in the assay hybridize on a PCR amplicon at adjacent sites, then fluorescence resonance energy transfer occurs, producing a specific fluorescence emission, which is then detected. The emission is disrupted if the temperature in the device is incrementally increased above the specific melting point of the probe/ single-stranded DNA duplex. The appearance of an emission is indicative of specific product accumulation and confirms successful PCR amplification. Mutation detection then occurs in the same closed tube without any analytical postamplification steps. The stability of nucleic acids can be predicted by a nearest-neighbor model (8)(9), and even the influence of single-base mismatches on the stability can be taken into account (10)(11)(12)(13)(14).
Little is known about the applicability of nearest-neighbor duplex stability calculations for the specific situation of the hybridization probe assay on the LightCycler. We therefore investigated the sensitivity and specificity of this method toward other mutations near the classical mutation sites of factor V (G1691A) and apolipoprotein B-100 (G10699A). In familial defective apolipoprotein B-100, a G10699A mutation produces an arginine-to-glutamine exchange at position 3500, which leads to impaired binding to the LDL receptor protein and, consequently, increased LDL-cholesterol with risk for cardiovascular disease. Another mutation in the same codon (C10698T) causes an arginine-to-tryptophan exchange at position 3500 in the protein. This mutation has less effect on the phenotype and is not significantly associated with coronary heart disease (15). However, the restriction enzyme recognition site used for genotyping is also destroyed. Using data calculated with the nearest-neighbor model, we expected that both mutations would show sufficient difference in their melting behavior to be clearly distinguished. We have compared these data with observed results to assess the validity of predicted melting points for molecular diagnostic purposes and to find whether this could be useful for probe design.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ApoB 3500 GENOTYPING
A 305-bp fragment of the apolipoprotein B-100 gene
(GenBank accession no. M14162) was amplified using the primers ApoB
3500 for 5'-CCT CAC CTC TTA CTT TTC C-3' and ApoB 3500 rev 5'-CTT TGC
TTG TAT GTT CTC C-3' (Fig. 1
).
The mutation site is covered by a wild-type complementary detection
probe: ApoB 3500 R 5'-AGA GCA CAC GGT CTT CAG T-3'-FLU labeled with
fluorescein as indicated (Fig. 1
). The adjacent anchor probe ApoB 3500
anchor 5'-LC-Red640-TGC AGG GCA CTT CCA AAA TTG ATG A-3'-PHO (Fig. 1
)
is 5' labeled with the LC-Red640 dye (Roche Biochemica) and 3'
phosphorylated by using a 3' phosphate controlled-pore glass.
The phosphorylation was to prevent probe elongation by the Taq
polymerase. If these probes lie adjacent to each other on a DNA strand,
fluorescence resonance energy transfer occurs and specific emission is
detected by the LightCycler. PCR amplification and detection occur in
the same closed tube in ~40 min. Oligonucleotides were synthesized by
standard phosphoramidite chemistry.
LC-Red640-N-hydroxysuccimide ester was linked with the
respective oligonucleotide via an amino linker and purified by HPLC.
PCR reactions were carried out in a final volume of 10 µL in the
LightCycler glass capillaries. The reaction mixture consisted of 1 µL
of genomic DNA solution, 0.5 µmol/L amplification primers ApoB 3500
for and ApoB 3500 rev, 0.1 µmol/L ApoB 3500 R probe, 0.3 µmol/L
ApoB 3500 anchor, 0.5 U of Taq DNA polymerase (Boehringer Mannheim), 1
µL of 10x PCR buffer (Boehringer Mannheim), 0.2 mmol/L each dNTP
(Boehringer Mannheim), 2.5 mmol/L MgCl2, 500 mg/L
bovine serum albumin (New England BioLabs), and 50 mL/L dimethyl
sulfoxide (Sigma). PCR-grade water was added to the final volume
of 10 µL. The cycling program consisted of a 30-s initial
denaturation at 95 °C and 55 cycles of 95 °C for 0 s,
50 °C for 5 s, and 72 °C for 5 s, with maximum ramp
rate. The program for analytical melting was 95 °C for 30 s,
40 °C for 30 s, increasing to 70 °C at a 0.1 °C/s ramp
rate. The PCR conditions and cycler program are essentially the same
that we have used before (6), thereby fully integrating the
apolipoprotein B-3500 genotyping into our single master mixture and
single cycler program approach for diagnostic genotyping.
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The typical G10699A mutation was found in a patient with a
LDL-cholesterol concentration of 2900 mg/L. Its presence was
confirmed after cloning and sequencing as described above. The C10698T
mutation was constructed using site-directed mutagenesis with a
megaprimer approach essentially as described (16). Briefly,
the amplicon from the apolipoprotein B-100 PCR was cloned into a TOPO
TA vector. It was then amplified with a proofreading enzyme mixture
(ExpandTM PCR system; Boehringer Mannheim) to
minimize the generation of nontemplate adenosine overhangs using a
mutagenic primer that introduces the C10698T mutation into the
sequence. The product of this PCR was purified (PCR purification kit;
Qiagen) and used as megaprimer in a second PCR with the same vector
containing the wild-type sequence as template. The resulting amplicon
was cloned into a TOPO TA vector, and successful mutagenesis was
confirmed by sequencing as described above. In the same way, other
mutations were created for investigation of their melting behavior. The
observed melting points (Tm determined
by LightCycler Software Package Ver. 3.1 Data Analysis Module) together
with the predicted melting points for the ApoB 3500R or alternatively
the ApoB 3500W probe (5'-AGA GCA CAT GGT CTT CAG T-3'-FLU) in this
assay are summarized in Table 1
.
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nearest-neighbor stability calculations
The stability of double-stranded DNA is related to the number of
hydrogen bonds formed. GC-rich sequences pair with greater stability
than AT-rich sequences. Sequence ordering is also a component of
stability. A newly unified data set describing the nearest-neighbor
interactions is available and was used for this study (9).
Furthermore, the destabilizing impact of mismatches in a given sequence
context is now well investigated (10)(11)(12)(13)(14). These data enable
prediction of the Tm of
oligonucleotide sequences with or without single base pair mismatches
with an error of <2 °C (8)(10)(11)(12)(13)(14). Results
of probe hybridization experiments (Tm
determined by LightCycler Software Package Ver. 3.1 Data Analysis
Module) together with the predicted melting points for given
probe/mismatch settings are shown in Table 1
. Formulas used for
Tm calculation are given in the
Appendix. The PCR product concentration will be different in
every PCR, depending for example, on template quality and amplification
primer efficiency. However, from a practical standpoint we found the
best fit of melting point predictions when we set CT equal to the
detection probe concentration used in the assay. It seems that probe
concentrations in the range of 0.1–0.3 µmol/L, which are commonly
used in these assays, reflect the specific situation of hybridization
probe assays on the LightCycler. Our experimental conditions were 0.1
µmol/L probe concentration. The sodium equivalent of our PCR buffer
was 350 mmol/L.
We performed stability calculations with a spreadsheet application running under Microsoft Excel® 4.0 and later (E. Schütz and N. von Ahsen, submitted for publication). The program is available for downloading at http://server1.medikc.med.uni-goettingen.de/meltcalc.htm. Results are equivalent to predictions from the Hyther program, which is available at http://jsl1.chem.wayne.edu.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A and Table 1
).
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Examples of genotyping results for the apolipoprotein B-3500 mutation
are given in Fig. 2B
, and the Tm
values are summarized in Table 1
. Values represent the mean from
determinations on at least five different days. The mean CV for
all melting points was 0.7% ± 0.26%.
All mutations under a wild-type-compatible probe were detected (100%
discrimination), and observed shifts in the
Tm values were 1.8–10.9 °C for
single-base pair mismatches. There was a certain specificity inasmuch
that the two known mutations of apolipoprotein B-100 (G10699A, C10698T)
were well discriminated by the detection assay as well as the factor V
mutations (G1691A and A1692C). The values predicted from the
nearest-neighbor model suggest that mutations are possible that
destabilize the double-stranded DNA to the same extent and, therefore,
should not be discriminated from each other. As an example, we
constructed the mutations G10700T and T10704A by site-directed
mutagenesis because from the prediction model we expected these
mutations to have almost the same melting points as the investigated
G10699A mutation. We found that when using the wild-type-compatible
probe, the observed melting points of the two mutations (54.7 and
53.4 °C) did not differ sufficiently from that of the typical
G10699A mutation (54.0 °C) to allow reliable discrimination.
Only by using a specific mutation-compatible probe can these mutations
be detected again with an almost 100% discriminating efficacy. Another
important question is whether any mutation might remain undiscovered
under a 19mer probe. We therefore investigated the A10695C mutation,
which has of all possible mutations under the probe the lowest
calculated melting point difference from the wild type. This mutation
did indeed have the lowest melting point shift (1.8 °C) compared
with the wild type, but it is reliably detected by the LightCycler
hybridization probe assay. There was a good correlation between the
observed and predicted melting points for all investigated mutations
under the wild-type- (3500R) or a mutation-compatible (3500W) probe
(Pearson r = 0.96; Fig. 3
). The mean difference between the observed and predicted
Tms was 0.1 ± 1.34 °C.
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The mean observed melting shift caused by mutations under the
wild-type-compatible probe was 6.3 °C, whereas it was 9.3 °C for
the mutation-compatible probe that contained an additional mismatch.
From our experimental data (mean SD of
Tms in our assay, 0.35 °C), we
expect that mismatches causing a 
Tm
>1.0 °C can be reliably discriminated from the wild type. We
performed melting point calculations for all possible 3 x
106 mismatches under 60 000 randomly chosen
19mer oligonucleotides and found that only 0.055% cause a melting
shift smaller than 1.25 °C (Fig. 4
). For this purpose, the sequence of a starting probe was
randomly altered by exchanging nucleotides by a computer program. The
randomization algorithm was programmed to produce a gaussian-shaped
distribution with respect to probe GC content and a preponderance of
probes with 50–60% GC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The stability of single-base mismatches next to Watson-Crick pairs is
now well characterized and also depends on the interaction with the
neighboring bases. This is impressively illustrated by the more than
sixfold range in the observed melting point differences. When a
wild-type-compatible probe is used, the apolipoprotein B-3500 G10699A
mutation causes a G-T mismatch in the trimer
CGG/GTC, which is more stable than the C-A
mismatch in the trimer ACG/TAC caused by the
C10698T mutation (10)(11). This is reflected by
the higher Tm of the G10699A mutation
compared with the C10698T mutation. The ApoB 3500W detection probe
complementary to the C10698T genotype produces the highest
Tm if this particular mutation is
present. For all other mutations, a lower melting point than that
observed with the wild-type probe results. This is because all other
genotypes lead to two mismatches under the mutation-specific probe. It
must be mentioned that the G10699A mutation under the ApoB 3500W probe
produces two immediately adjacent mismatches. For this situation, only
a few nearest-neighbor data are available, including that of the
present resulting ATGG/TGTC mismatch
(10). The melting point prediction from nearest-neighbor
parameters was not compromised by two mismatches under a 19mer if these
were not immediately adjacent. The result from this and other studies
show that all existing polymorphisms of factor V (G1691A,
A1692C, and G1689A) (23) and apolipoprotein B-100
(G10699A and C10698T) are discriminated using a single wild-type probe.
We have extended these results and found that some possible mutations
at the apolipoprotein B-100 locus (e.g., T10704A and
G10700T) are not appreciably discriminated from the G10699A mutation.
Therefore, only a confirmation assay with a mutation-specific probe
will give almost 100% sensitivity and specificity for this mutation
detection. All samples with unexpected melting behavior must be further
investigated before reliable genotyping results are obtained. Our
experimental findings are in good correlation with calculated values.
From thermodynamic predictions, we expect that every mutation under a
19mer probe should be detected in this assay. We have tried to extend
these findings to every possible sequence under a 19mer, but the
calculation of all possible mismatches in 419
oligonucleotides is not easily achieved. However, we calculated the
minimal Tm shift caused by any
possible mutation occurring in >60 000 randomly chosen sequences
(Fig. 4
) and found a distinct pattern. In 19mers with a very high GC
content (>80%), a relatively stable mismatch such as
GAC/CGG will probably not be detected. Several
solutions are possible in this situation: (a) Siting the
detection probe on the antisense strand changes the mismatch to
CTG/GCC, which is an unstable mismatch and is
easily detected. (b) Introduction of additional mismatches
into the detection probe has also been shown to increase the melting
point difference (25). (c) Alternatively, the
detection probe may be shortened by a few nucleotides to reduce the
Tm and to increase the
Tm shift caused by the mismatch. The
practical endpoint for the LightCycler genotyping is that the
mismatched probe should still have a
Tm >45 °C because the instrument
is not equipped with active cooling. In all of these cases, prediction
of the melting behavior is clearly advantageous, and such model-based
probe design can greatly improve the discriminatory performance of
these genotyping assays. The only limitations of the model are caused
by missing thermodynamic data for dangling ends and double and terminal
(i.e., penultimate) mismatches because these situations have their own
thermodynamics that must be considered. Currently, only the Hyther
program (see Materials and Methods) can account for these
mismatches, but it uses as yet unpublished nearest-neighbor
parameters for the calculation of these situations. Accordingly, we
omitted these cases from our analysis, such as the G10699T mutation,
which produces a double mismatch when probed with the 3500W probe
(Table 1
). As a consequence, our results apply only to the central 17
bases covered by the probe because the outermost bases will appear as
dangling ends when mismatched. We could not find any evidence that the
labeling of probes per se or the different types of labeling that were
used substantially changes the Tm
(data not shown).
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Appendix |
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H), entropy (S), and free energy G° of an
oligonucleotide duplex are calculated as the sum of the entire
Watson-Crick nearest neighbors plus helix initiation and symmetry
terms:
 | (1) |
For S and G°, the same formula applies. The H, S,
and G° values for Watson-Crick nearest neighbors for matched and
mismatched base pairs as well as those for initiation and symmetry
correction were taken from published data (9)(10)(11)(12)(13)(14).
Apart from entropy and enthalpy of the nucleotides in the sequence, the
concentration of monovalent ions and oligonucleotides must be taken
into account because the data provided by SantaLucia and Allawi
(8), Allawi and SantaLucia (10)(11)(12)(13), and Peyret et
al. (14) are derived at 1 mol/L NaCl. Because
enthalpy is independent of salt concentration, only entropy values for
a given [Na+] were calculated according to the
formula:
 | (2) |
where N is the total number of phosphates in the duplex divided by 2, which equals the length minus 1 (9).
Na+ equivalents of the PCR assay buffer were
calcu- lated based on the 140-fold higher stabilizing effect of
Mg2+ on duplexes compared with monovalent ions
(26):
 | (3) |
 | (4) |
where R is the gas constant (1.987 cal/K · mol), for non-self-complementary sequences and CT is the concentration of oligonucleotides divided by 4. The dimethyl sulfoxide-induced Tm reduction was 0.6 °C for each percent of change in the dimethyl sulfoxide concentration (27).
In conclusion, we have demonstrated the applicability and benefits of a thermodynamic nearest-neighbor nucleic acid melting prediction model for diagnostic genotyping. A method for apolipoprotein B-3500 genotyping is presented and evaluated against different known and potential mutations at this locus. Observed Tms were in good agreement with calculated values. We have shown for two clinically relevant genotyping examples that the LightCycler mutation detection system has superior discriminatory performance compared with conventional RFLP or ASA PCR-based methods for molecular diagnostic purposes. With this method, in every hybridization probe assay, all mutations under a properly designed probe should be detected, but they will not necessarily be discriminated from each other in all cases.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, single mismatches; 
, two-point
mismatches.