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Articles |
1
PNA Diagnostics A/S, Rønnegade 2, DK-2100 Copenhagen, Denmark.
2
Exiqon A/S, Bygstubben 9, DK-2950 Vedbæk, Denmark.
3
Department of Clinical Biochemistry, Statens Serum
Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark.
a Author for correspondence. Fax 45-44-44-03-73; e-mail oerum{at}euroconnect.dk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: LNA octamer probes complementary to the factor V wild-type or mutated sequence were covalently attached to individual wells of a microtiter plate. Biotinylated factor V amplicons were added, and hybridization to the immobilized LNA probes was scored colorimetrically using a horseradish peroxidase-anti-biotin Fab conjugate and tetramethylbenzidine substrate.
Results: In a prospective study of 53 patients, the assay reproducibly scored both factor V homozygotes and heterozygotes with excellent sensitivity and specificity. All results were in complete agreement with the results obtained with the conventional PCR-restriction fragment length polymorphism technique.
Conclusions: The simplicity of the assay and its procedural relatedness to the widely used ELISA format should make it useful for routine factor V testing in the clinical laboratory.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immunodiagnostic format, ELISA, usually carried out in 96-well microtiter plates, is widely used in clinical laboratories for the detection of antigens and antibodies. For this reason, much effort has been spent to expand its use to the emerging field of DNA diagnostics, in particular to the detection of infectious diseases (16)(17)(18)(19). Currently, there are two tests available that use DNA capture probes immobilized in microtiter plates in combination with PCR to diagnose the presence of an infectious agent by an ELISA-like assay: a test for Chlamydia trachomatis (Amplicor Chlamydia; Roche) and a test for Mycobacterium tuberculosis (Amplicor Mycobacterium; Roche). The development of similar tests suitable for routine diagnosis of genetic diseases, which often require single-base specificity, however, has been a serious challenge, primarily because of difficulties in designing DNA probes and assay conditions that are sufficiently discriminatory.
Locked nucleic acids (LNAs; Fig. 1
) are a novel class of bicyclic DNA analogs in which the 2' and
4' positions in the furanose ring are joined via an O-methylene
(oxy-LNA), S-methylene (thio-LNA), or amino-methylene (amino-LNA)
moiety (20)(21)(22)(23)(24). Common to all of these LNA variants is an
affinity toward complementary nucleic acids, which is by far the
highest reported for a DNA analog (20)(21)(22)(23)(24). For example, the
all oxy-LNA nonamer (5'-GTGATATGC-3') has a melting temperature
(Tm) of 64 and 74 °C toward its
complementary DNA and RNA, respectively, as opposed to 28 °C (DNA)
and 28 °C (RNA) for the corresponding DNA nonamer (20).
Substantial increases in Tm are also
obtained when LNA monomers are used in combination with standard DNA or
RNA monomers. For example, the affinity of the above DNA nonamer
(5'-GTGATATGC-3') for its complementary DNA
(Tm = 28 °C) increases to 44 °C
when the three T residues are replaced by oxy-LNA-T monomers
(20)(21). Given the very high affinity for
complementary nucleic acids, we reasoned that small LNA oligomers would
perform as well in sequence-specific capture of PCR amplicons as
substantially larger probes based on DNA, RNA, or other
moderate-affinity analogs. Because the specificity of a probe is
inversely related to its size, we anticipated that such small probes
would be able to provide point mutation specificity when used in an
ELISA-like detection assay.
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In this report, we demonstrate that LNA probes as small as octamers are able to capture PCR amplicons very efficiently and provide exquisite discrimination of amplicons differing by one base pair when covalently attached to a solid phase. To attach the LNAs covalently to microtiter plate wells, we used a photochemical approach, which exploits the highly photoreactive properties of anthraquinone (AQ) (25). This photoprobe has been used successfully to covalently attach several different macromolecules to a range of different polymers (25)(26) and also provided an effective and convenient means of immobilizing LNA in a hybridization competent manner.
Genomic DNA from a total of 53 different patients, previously characterized for their factor V status by the PCR-restriction fragment length polymorphism (RFLP) technique described by Bertina et al. (6), was subjected to the new microtiter plate ELISA-like test. In our test system, 6 of the 53 patients were heterozygous and 1 was homozygous for the Leiden mutation. These results were 100% concordant with the results obtained with the reference method.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The AQ-LNA oligomers were synthesized directly on the DNA synthesizer. This was achieved by using an AQ-C3-linker-phosphoramidite, N-3[(2-cyanoethyl-N,N-diisopropyl phosphoramidite)propyl]-2-carbamoyl anthraquinone. All LNAs were synthesized using methylcytosine (Cm) monomers instead of standard cytosine monomers (because of the ease of synthesis of the Cm monomer).
The following DNA and LNA capture probes were used:
Oligomers used in Tm measurements correspond to the oligomers used for amplicon capture without the AQ and spacer moieties.
clinical samples
Patient blood samples were received as blood with no additions or
occasionally with EDTA added to a final concentration of ~5 nmol/L.
An aliquot of 50 µL of blood (or the liquid above the blood clot) was
added to 1 mL of distilled water and incubated at room temperature for
30 min with occasional slight agitation. Lysed cells and DNA were
pelleted by centrifugation for 3 min at ~10 000g. The
pellet was resuspended in 200 µL of 50 g/L Chelex (Bio-Rad) by
vortex-mixing, followed by incubation for 30 min at 56 °C. After
another 10 s of vortex-mixing, the sample was incubated for 10 min
at 100 °C, again vortex-mixed, and centrifuged for 3 min at
~10 000g. The supernatant, containing chromosomal DNA,
was used for PCR reactions.
factor v amplification
Plasmids containing a fragment of the wild-type or the
Leiden-mutated form of the factor V gene were made by PCR amplification
of wild-type DNA or DNA from a patient with the Leiden mutation,
using the primers SSI1 [5'-GGAACAACACCATGATCAGAGCA-3' (positions
1605–1627)] and SSI2 [5'-TAGCCAGGAGACCTAACATGTTC-3' (positions
1870–1892)]. Amplicons were cloned into pBluescript II SK+ digested
with EcoRV and furnished with a T overhang. Wild-type and
mutant plasmids used for PCR were dissolved in water to a concentration
of ~20 ng/µL.
Plasmid PCR reactions (100 µL) were prepared by mixing 10 µL of plasmid with 40 µL of water, 50 µL of PCR master mix [20 mmol/L Tris-HCl, pH 8.3; 100 mmol/L KCl; 3.0 mmol/L MgCl2; 200 µmol/L dATP, dGTP, and dCTP; 600 µmol/L dUTP; 0.8 µmol/L biotinylated forward primer (bio-TTCTGAAAGGTTACTTCAAGGACA-3'; positions 1651–1674); 0.8 µmol/L biotinylated reverse primer (bio-TGCCCAGTGCTTAACAAGAC-3';positions 1782–1801); and 0.5 µL of Taq polymerase (5 U/µL; Boehringer Mannheim)].
Patient sample PCR reactions (100 µL) were prepared by mixing 2 µL of patient DNA with 48 µL of water, 50 µL of PCR master mix, and 0.5 µL of Taq polymerase (5 U/µL; Boehringer Mannheim).
The PCR reactions were performed on a Perkin-Elmer 9600 thermocycler using the following profile: 95 °C for 5 min; followed by 30 cycles of 30 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C; followed by 5 min at 72 °C and a 4 °C hold temperature.
preparation of oligomer-coated, 96-well microtiter plates
The AQ moiety has been shown to be a highly effective photoprobe
for the covalent attachment of diverse macromolecules to polymer
surfaces (25)(26). Concentrated stocks of AQ-LNA
and AQ-DNA probes in water (stored in dark tubes at -20 °C) were
diluted in immobilization buffer (200 mmol/L LiCl) to a final
concentration of 0.1 µmol/L. To prepare the coated microtiter plates
(F8 polysorp unfra; Nalge Nunc International), 100 µL of either
wild-type or mutant AQ probe was applied per well and subjected to 20
min of 350 nm light from a ULS-20-2 illuminator (UV-Lights Systems).
This illuminator was equipped with 28 Philips Cleo Compact 25W-S light
bulbs (14 located above and 14 located below the glass plate
sample holder). After irradiation, the plate was washed three times in
200 µL of wash buffer (400 mmol/L NaOH, 20 mL/L Tween 20) and
three times with water in an automated plate washer.
Typically, the coated plates were used immediately after preparation. For longer storage, the plates were blotted dry on paper towels and stored at 4 °C in a sealed plastic bag together with a 5-g package of silica gel desiccant (Sigma). Stored plates were used within 2 weeks, in which period no decrease in performance was observed.
detection of the leiden mutation in amplicons
Analysis for the Leiden mutation by the PCR-RFLP technique was
carried out essentially as described by Bertina et al. (6),
using the MnlI restriction endonuclease for the digestion of
PCR amplicons.
Analysis for the Leiden mutation using the microtiter plate assay was carried out as follows. PCR reaction mixture (10 µL) was mixed with 10 µL of denaturation buffer (125 mmol/L NaOH, 8 mmol/L EDTA, 0.2 g/L thymol blue) and incubated at room temperature for 5 min. In the meantime, 100 µL of hybridization buffer (50 mmol/L Na2HPO4, pH 7.0, 1 g/L bovine serum albumin and 2 mL/L Tween 20) was added to the microtiter plate well containing the photoimmobilized AQ-LNAs or control DNA capture probes. The 20-µL denaturation reaction was transferred to the microtiter plate well, and hybridization between amplicon and the immobilized capture probe was allowed to proceed at 37 °C for 30 min with gentle shaking. After hybridization, the well was washed five times with 250 µL of washing buffer (10 mmol/L Na2HPO4, pH 7.3, 150 mmol/L NaCl, 1.25 mL/L Tween 20, and 1 mmol/L EDTA) in an automated plate washer (Labsystems). Conjugate solution [100 µL of horseradish peroxidase-anti-biotin Fab fragment (Boehringer Mannheim) diluted 1:1000 in 500 mmol/L Tris, pH 7.0] were added to the well and incubation was continued at 37 °C for 15 min with shaking. The plate was washed five times with 250 µL of washing buffer, and tetramethylbenzidine substrate (100 µL of Boehringer Mannheim Blue POD substrate) was added. The microtiter plate was incubated in the dark for 10 min at room temperature, and the reaction was stopped by the addition of 100 µL of 500 mmol/L H2SO4. The absorbance was recorded at 450 nm using an ELISA reader (Labsystems Multiscan MS).
Tm MEASUREMENTS
The thermostability (Tm) of
the LNA/DNA and the corresponding DNA/DNA duplexes (without the AQ and
spacer moieties; Table 1
) was determined spectrophotometrically at 260 nm with a
Perkin-Elmer 
-2 spectrophotometer equipped with a Peltier thermal
block. The hybridization buffer contained 50 mmol/L or 150 mmol/L
Na2HPO4, pH 7.0, 0.1 mmol/L
EDTA, and 1.5 µmol/L each of the oligomers. Sigmoid melting curves
were obtained with both LNA/DNA and DNA/DNA duplexes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Leiden mutation is a G-to-A transversion at position 1691 in the
sense strand of exon 10 of the factor V gene (6)(7)(8). Thus,
if both wild-type LNA and mutant LNA probes were directed against the
sense PCR strand, the wild-type LNA would have a
CmLNA/ADNA
mismatch to the mutant amplicon, whereas the mutant LNA would have a
TLNA/GDNA mismatch to the
wild-type amplicon. The latter mismatch is known to be among the least
destabilizing mismatches in DNA duplexes (28), and this also
appears to be the case with LNA/DNA duplexes (20). To avoid
this mismatch, we designed the mutant LNA against the factor V
antisense strand, thus converting the
TLNA/GDNA mismatch with the
wild-type factor V sequence to an
ALNA/CDNA mismatch.
As is evident from comparison of the
Tm values for the octamer LNAs in
Table 1
, this change increases the specificity of the LNA probe
somewhat: 
Tm = 20 °C for the
ALNA/CDNA mismatch (number
6) as opposed to Tm = 16 °C for
the TLNA/GDNA mismatch
(number 4). Interestingly however, the
ALNA/CDNA mismatch (number
6; Tm = 20 °C) does not affect
the thermostability of the LNA/DNA duplex nearly as much as the
CmLNA/ADNA
mismatch (number 3; Tm = 30 °C).
It is unclear at present whether this difference is attributable to the
Cm nucleobase in the LNA or whether it is a
consequence of the heteroduplex character of the LNA/DNA helix. The
Tm values in Table 1
further confirm
previously reported findings that LNA is a very high-affinity DNA
analog (Tm increase of ~5 °C per
LNA monomer compared with DNA monomers) (20)(21)(22)(23)(24).
sequence-specific capture of pcr amplicons
The LNAs (10 pmol) were photoimmobilized in polystyrene microtiter
plates and hybridized with 1, 5, or 10 µL of wild-type or mutant
factor V amplicons in buffers of different ionic strength. Fig. 2
shows the results obtained with the octamer LNAs
directed against the sense strand of the wild-type amplicon (WT-LNA8s)
and against the antisense strand of the mutant amplicon (MT-LNA8as). As
expected from the high Tm values of
these probes, both LNAs are highly efficient in capturing their
complementary amplicons, giving clearly detectable signals under all
conditions tested. Under similar conditions, the corresponding DNA
probes did not produce a detectable signal (data not shown).
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In the 50 mmol/L phosphate buffer, both WT-LNA8s (Fig. 2A
) and
MT-LNA8as (Fig. 2B
) exhibited an excellent ability to discriminate
between their complementary and single-base mismatched amplicons. Here,
a saturated signal was obtained with 5 µL of the complementary
amplicon, whereas the signal with the mismatched amplicon did not
increase above background. When the ionic strength of the hybridization
buffer was increased to 100 mmol/L and further to 150 mmol/L, a modest
but detectable increase in the capture of mismatched amplicon was
observed. This is consistent with the increase in the
Tm for the mismatched target that
results from increasing the ionic strength of the hybridization buffer
(data not shown).
The nonamer and decamer LNAs were also highly efficient in capturing their complementary amplicons, but consistent with their increased Tm relative to the octamer LNAs, they displayed less specificity than the octamer LNAs when tested under identical hybridization conditions (data not shown). Because attaining maximum specificity in the assay was a key objective, we used the octamer LNAs and the 50 mmol/L phosphate hybridization buffer for all further experiments.
analytical sensitivity
The concentration of LNA capture probes required for sensitive and
specific amplicon capture was determined by analyzing the performance
of plates that had been prepared with 5–50 pmol of the AQ-LNAs. When
WT-LNA8s (Fig. 3
A) or MT-LNA8s (Fig. 3B
) was hybridized with 1–10 µL of
wild-type or mutant amplicons, good signals were obtained with the
complementary amplicons in all cases. With the WT-LNA8s probe, the
background signals from the mutant amplicon remained very low over the
entire LNA concentration range tested (absorbance <0.2). In contrast,
the background signals from the MT-LNA8as hybridized with wild-type
amplicon increased with increasing LNA concentration and reached an
absorbance of 0.43 at 50 pmol of LNA probe. This increase was also
observed in the control reactions without added amplicon, indicating
that it was not attributable to a lack of specificity of the MT-LNA8as
but rather some unspecific adsorption of the conjugate to the coated
plates. In any case, plates coated with up to 10 pmol of MT-LNA8as and
WT-LNA8s gave an excellent signal-to noise-ratio and were used as the
standard in subsequent experiments.
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The analytical sensitivity of the assay was determined by performing the test on diluted samples of the wild-type and mutant plasmids. The concentration range tested was from 102 to 106 copies of plasmids per PCR reaction, and 10 µL of the final PCR was used per test. Clearly detectable signals (absorbance >1.0) were obtained with both mutant and wild-type samples containing 101 copies of plasmid [corresponding to the number of genomes present in ~1 µL of blood (29)] and with the signal reaching the instrument maximum with samples containing 105 copies of plasmid (data not shown). It seems likely that further optimization of the PCR reaction can improve this lower detection limit considerably.
application of the factor v microtiter plate assay to clinical
samples
DNA was extracted from blood samples from 53 patients. The
chromosomal DNA was amplified by PCR and analyzed by digestion with the
restriction endonuclease, MnlI, essentially as described by
Bertina et al. (6) or analyzed by the factor V microtiter
plate assay as described in Materials and Methods. Each PCR
was tested in duplicate against both WT-LNA8s and MT-LNA8as. Fig. 4
shows the result from the first microtiter plate test of the 53
patient samples. As is evident, patients carrying the Leiden mutation
(patients 4, 6, 13, 16, 17, 35, 46, and 49) were easily differentiated
from unaffected patients. Moreover, the assay also allowed clear
differentiation between factor V Leiden heterozygotes (patients 6, 13,
16, 17, 35, 46, and 49) and homozygotes (patient 4).
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To evaluate the reproducibility of the assay, the entire test was repeated with new PCR amplification and detection. The signals from the positive samples in the second test were somewhat lower than those in the first test (~14% of positive signals between 1.0 and 1.5 in the second test as opposed to only 3% in the first test). The results, however, again allowed clear and unambiguous assignment of both factor V Leiden heterozygotes and homozygotes as well as unaffected individuals, and the conclusions were similar to those drawn on the basis of the first test.
All 53 patients samples were subjected to the LNA microtiter plate assay without prior knowledge of the results obtained by the PCR-RFLP reference method. Subsequent comparison of the results showed complete agreement between the two methods (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here we described that a novel class of high affinity DNA analogs, termed LNAs, are able to capture amplicons with high efficiency when covalently attached to a solid surface. We further demonstrated that LNAs as small as octamers can be used and that such small LNAs are able to effectively discriminate between PCR amplicons that differ by only a single base.
The covalent attachment of the LNA probes to the microtiter plates was performed photochemically using an AQ photoprobe located at the 5' end of the oligomer. This approach was simple and rapid, and plates with LNA probes remained fully functional when tested over a 2-week period (data not shown). A particularly interesting aspect of the photocoating technology is that it is applicable to a wide selection of polymers and is ideally suited for miniaturization. Hence, it should be relatively straightforward to transfer the assay to other detection platforms such as strips and arrays.
Using two LNA octamers, we developed a 1-h ELISA-like assay for genotyping patients for the presence of the Leiden mutation. In a prospective study of 53 patients, the assay reproducibly scored both factor V Leiden heterozygotes and homozygotes as well as unaffected individuals. Moreover, the results were in 100% concordance with the PCR-RFLP reference method.
The continued success of DNA-based diagnostics will largely depend on the development of fully automated, high-throughput systems that integrate the entire procedure of sample preparation, amplification, and detection. Of importance to this process is the development of simple and robust technologies for each of the steps. The demonstration that small LNAs are able to effectively capture PCR amplicons with exquisite specificity and reproducibility in a simple solid-phase hybridization assay places LNA as an interesting tool in the detection step. Is seems likely that future research will uncover more advantages of using LNA in nucleic acid diagnostics.
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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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The following articles in journals at HighWire Press have cited this article:
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  J. D. Levin, D. Fiala, M. F. Samala, J. D. Kahn, and R. J. Peterson Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers Nucleic Acids Res., November 6, 2006; 34(20): e142 - e142. [Abstract] [Full Text] [PDF]
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 J. Rupp, W. Solbach, and J. Gieffers Single-Nucleotide-Polymorphism-Specific PCR for Quantification and Discrimination of Chlamydia pneumoniae Genotypes by Use of a "Locked" Nucleic Acid. Appl. Envir. Microbiol., May 1, 2006; 72(5): 3785 - 3787. [Abstract] [Full Text] [PDF]
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Y. You, B. G. Moreira, M. A. Behlke, and R. Owczarzy Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res., January 1, 2006; 34(8): e60 - e60. [Abstract] [Full Text] [PDF]
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M. P. Johnson, L. M. Haupt, and L. R. Griffiths Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR Nucleic Acids Res., March 26, 2004; 32(6): e55 - e55. [Abstract] [Full Text] [PDF]
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D. A. Di Giusto and G. C. King Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays Nucleic Acids Res., February 18, 2004; 32(3): e32 - e32. [Abstract] [Full Text] [PDF]
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D. A. Braasch, Y. Liu, and D. R. Corey Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design Nucleic Acids Res., December 1, 2002; 30(23): 5160 - 5167. [Abstract] [Full Text] [PDF]
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N. Jacobsen, J. Bentzen, M. Meldgaard, M. H. Jakobsen, M. Fenger, S. Kauppinen, and J. Skouv LNA-enhanced detection of single nucleotide polymorphisms in the apolipoprotein E Nucleic Acids Res., October 1, 2002; 30(19): e100 - e100. [Abstract] [Full Text] [PDF]
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A. Simeonov and T. T. Nikiforov Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection Nucleic Acids Res., September 1, 2002; 30(17): e91 - e91. [Abstract] [Full Text] [PDF]
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N. Zammatteo, L. Lockman, F. Brasseur, E. De Plaen, C. Lurquin, P.-E. Lobert, S. Hamels, T. Boon, and J. Remacle DNA Microarray to Monitor the Expression of MAGE-A Genes Clin. Chem., January 1, 2002; 48(1): 25 - 34. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or mutant (
) amplicon,
or no amplicon (
) in 50–150 mmol/L phosphate buffer, pH 7.0, 0.1
mmol/L EDTA. (B), same conditions as in panel
A, with MT-LNA8as as the capture probe. Each PCR was tested
in duplicate, and results are plotted as the mean values (SD <14%).
