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Detection of the factor V Leiden mutation by direct allele-specific hybridization of PCR amplicons to photoimmobilized locked nucleic acids.

Several inherited abnormalities in the anticoagulant systems currently are recognized to predispose patients to venous thromboembolism (VTE) [4] (1, 2). By far the most frequent of these is resistance to activated protein C (3-5). In 1994, several reports documented that this disorder is almost always the result of a G-to-A transversion at position 1691 in the gene coding for coagulation factor V (6-8). The prevalence of the mutation, commonly referred to as the Leiden mutation, varies widely with ethnic background (9,10). In Caucasian subpopulations, the allele frequency is 1-8.5%, whereas it appears to be absent in African, Chinese, Japanese, and native North American populations. Individuals heterozygous for the mutation have been estimated to have a 3.5-fold (11) to 7-fold (12) higher risk of VTE compared with genetically unaffected individuals. In individuals homozygous for the Leiden mutation, this risk has been estimated to increase 80-fold (12). Nonhereditary factors such as surgery, pregnancy, and the use of oral contraceptives also add to the risk of developing VTE (1,13-15). In the case of oral contraceptives, carriers of the Leiden mutation were reported to have an eightfold increase in the risk of developing VTE compared with noncarriers (15). Thus, there appears to be a need for a factor V assay suitable for routine clinical use.

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 (16-19). 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 (20-24). 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 (20-24). For example, the all oxy-LNA nonamer (5'-GTGATATGC-3') has a melting temperature ([T.sub.m) of 64 and 74 [degrees]C toward its complementary DNA and RNA, respectively, as opposed to 28 [degrees]C (DNA) and 28 [degrees]C (RNA) for the corresponding DNA nonamer (20). Substantial increases in [T.sub.m] 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 ([T.sub.m] = 28 [degrees]C) increases to 44 [degrees]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.

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.

Materials and Methods


DNA and LNA (20) oligomers were synthesized on an automated DNA synthesizer using 2-cyanoethyl-N,N-diisopropyl phosphoramidites. The DNA oligomers were synthesized with a 5'-monomethoxytrityl-NH-thymidine-3'-phosphoramidite prepared according to published procedures (27). After synthesis, the DNA oligomer (still on the control pore glass resin) was transferred to a separate reactor used for peptide synthesis and coupled to the AQ moiety (2-carboxyanthraquinone) via amide formation as follows: the monomethoxytrityl group on the DNA oligomer was first removed by trichloroacetic acid treatment (30 mL/L in dichloromethane), after which the oligomer and resin were neutralized by diisopropylethylamine (50 mL/L in dichloromethane). The 2-carboxyanthraquinone was activated by O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate and allowed to couple to the DNA oligomer for 1 h. Capping was not performed. Deprotection and removal of the conjugate from the control pore glass support was performed according to standard procedures for DNA synthesis.

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 ([C.sup.m]) monomers instead of standard cytosine monomers (because of the ease of synthesis of the [C.sup.m] monomer).

The following DNA and LNA capture probes were used:

WT-LNA8s: AQ-CONH-[([CH.sub.2]).sub.3]tacatgttatgctga[C.sup.m] [C.sup.m]T[C.sup.m]G[C.sup.m][C.sup.m]T-3' (DNA in lower case letters, LNA in upper case letters; "WT" designates that the probe is directed against the wild-type amplicon, "8" designates the size of the probe, and "s" designates that the probe is directed against the sense amplicon strand); WT-LNA9s: AQ-CONH-[([CH.sub.2]).sub.3]-tacatgttatgctgT[C.sup.m][C.sup.m] T[C.sup.m]G[C.sup.m][C.sup.m]T-3'; WT-LNA10s: AQ-CONH-[([CH.sub.2]).sub.3]-tacatgttatgctTT[C.sup.m][C.sup.m] T[C.sup.m]G[C.sup.m][C.sup.m]T-3'; MT-LNA8as: AQ-CONH-[([CH.sub.2]).sub.3]-tacatgttatgctgaAGG[C.sup.m]AAGG-3' ("MT" designates that the probe is directed against the mutant amplicon and "as" designates that the probe is directed against the antisense amplicon strand); MT-LNA9as: AQ-CONH-[([CH.sub.2]).sub.3]-tacatgttatgctgaAGG[C.sup.m]AAGGA-3'; MT-LNA10as: AQ-CONH-[([CH.sub.2]).sub.3]-tacatgttatgctga[C.sup.m]AGG [C.sup.m]AAGGA-3'; WT-DNA8s: AQ-CONH-tacatgttatgctgacctcgcct-3'; MT-DNA8as: AQ-CONH-tacatgttatgctgaaggcaagg-3'.

Oligomers used in [T.sub.m] measurements correspond to the oligomers used for amplicon capture without the AQ and spacer moieties.


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 [micro]L 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 [micro]L of 50 g/L Chelex (Bio-Rad) by vortex-mixing, followed by incubation for 30 min at 56 [degrees]C. After another 10 s of vortex-mixing, the sample was incubated for 10 min at 100 [degrees]C, again vortex-mixed, and centrifuged for 3 min at ~10 000g. The supernatant, containing chromosomal DNA, was used for PCR reactions.


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'-TA000AGGAGACCTAACATGTTC-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/[micro]L.

Plasmid PCR reactions (100 [micro]L) were prepared by mixing 10 [micro]L of plasmid with 40 [micro]L of water, 50 [micro]L of PCR master mix [20 mmol/L Tris-HCI, pH 8.3; 100 mmol/L KCl; 3.0 mmol/L Mg[Cl.sub.2]; 200 [micro]mol/L dATP, dGTP, and dCTP; 600 [micro]mol/L dUTP; 0.8 [micro]mol/L biotinylated forward primer (bio-TTCTGAAAGGTTACTTCA AGGACA-3'; positions 1651-1674); 0.8 [micro]mol/L biotinylated reverse primer (bio-TGCCCAGTGCTTAACAAG AC-3';positions 1782-1801); and 0.5 [micro]L of Taq polymerase (5 U/[micro]L; Boehringer Mannheim)].

Patient sample PCR reactions (100 [micro]L) were prepared by mixing 2 [micro]L of patient DNA with 48 [micro]L of water, 50 [micro]L of PCR master mix, and 0.5 [micro]L of Taq polymerase (5 U/[micro]L; Boehringer Mannheim).

The PCR reactions were performed on a Perkin-Elmer 9600 thermocycler using the following profile: 95 [degrees]C for 5 min; followed by 30 cycles of 30 s at 95 [degrees]C, 30 s at 55 [degrees]C, and 30 s at 72 [degrees]C; followed by 5 min at 72 [degrees]C and a 4 [degrees]C hold temperature.


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 [degrees]C) were diluted in immobilization buffer (200 mmol/L LiCl) to a final concentration of 0.1 [micro]mol/L. To prepare the coated microtiter plates (F8 polysorp unfra; Nalge Nunc International), 100 [micro]L 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 [micro]L 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 [degrees]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.


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 [micro]L) was mixed with 10 [micro]L 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 [micro]L of hybridization buffer (50 mmol/L [Na.sub.2][HPO.sub.4], 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-[micro]L denaturation reaction was transferred to the microtiter plate well, and hybridization between amplicon and the immobilized capture probe was allowed to proceed at 37 [degrees]C for 30 min with gentle shaking. After hybridization, the well was washed five times with 250 [micro]L of washing buffer (10 mmol/L [Na.sub.2][HPO.sub.4], 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 [micro]L 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 [degrees]C for 15 min with shaking. The plate was washed five times with 250 [micro]L of washing buffer, and tetramethylbenzidine substrate (100 [micro]L 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 [micro]L of 500 mmol/L [H.sub.2]S[O.sub.4]. The absorbance was recorded at 450 nm using an ELISA reader (Labsystems Multiscan MS).


The thermostability ([T.sub.m]) 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 [lambda]-2 spectrophotometer equipped with a Peltier thermal block. The hybridization buffer contained 50 mmol/L or 150 mmol/L [Na.sub.2][HPO.sub.4], pH 7.0, 0.1 mmol/L EDTA, and 1.5 [micro]mol/L each of the oligomers. Sigmoid melting curves were obtained with both LNA/DNA and DNA/DNA duplexes.



To test the hypothesis that small LNAs, by virtue of their high affinity, would be effective in sequence-specific capture of PCR amplicons when covalently attached to a polymer surface, we synthesized an octamer, a nonamer, and a decamer LNA oligomer complementary to the wild-type and mutated factor V sequences. To avoid potential steric constraints to hybridization that might arise from the immobilization of the LNAs in the final assay, they were all synthesized with a spacer between the LNA segment and the AQ photoprobe used for attachment to the microtiter plate. Because LNA synthesis is fully compatible with standard DNA synthesis, we chose a spacer consisting of DNA nucleotides. The sequence of this spacer was designed such that it had no obvious regions of complementarity to the factor V amplicons.

The Leiden mutation is a G-to-A transversion at position 1691 in the sense strand of exon 10 of the factor V gene (6-8). Thus, if both wild-type LNA and mutant LNA probes were directed against the sense PCR strand, the wild-type LNA would have a [C.sup.m..sub.LNA]/[A.sub.DNA] mismatch to the mutant amplicon, whereas the mutant LNA would have a [T.sub.LNA]/[G.sub.DNA] 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 [T.sub.LNA]/[G.sub.DNA] mismatch with the wild-type factor V sequence to an [A.sub.LNA]/[C.sub.DNA] mismatch. As is evident from comparison of the [T.sub.m] values for the octamer LNAs in Table 1, this change increases the specificity of the LNA probe somewhat: [DELTA][T.sub.m] = 20 [degrees]C for the [A.sub.LNA]/[C.sub.DNA] mismatch (number 6) as opposed to [DELTA][T.sub.m] = 16 [degrees]C for the [T.sub.LNA]/[G.sub.DNA] mismatch (number 4). Interestingly however, the [A.sub.LNA]/[C.sub.DNA] mismatch (number 6;[DELTA][T.sub.m] = 20 [degrees]C) does not affect the thermostability of the LNA/DNA duplex nearly as much as the [C.sup.m.sub.LNA]/[A.sub.DNA] mismatch (number 3; [DELTA][T.sub.m] = 30 [degrees]C). It is unclear at present whether this difference is attributable to the [C.sup.m] nucleobase in the LNA or whether it is a consequence of the heteroduplex character of the LNA/DNA helix. The [T.sub.m] values in Table 1 further confirm previously reported findings that LNA is a very high-affinity DNA analog ([T.sub.m] increase of ~5 [degrees]C per LNA monomer compared with DNA monomers) (20-24).


The LNAs (10 pmol) were photoimmobilized in polystyrene microtiter plates and hybridized with 1, 5, or 10 [micro]L 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 [T.sub.m] 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).

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 [micro]L 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 [T.sub.m] 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 [T.sub.m] 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.


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 WTLNA8s (Fig. 3A) or MT-LNA8s (Fig. 3B) was hybridized with 1-10 [micro]L 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.

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 [10.sup.2] to [10.sup.6] copies of plasmids per PCR reaction, and 10 [micro]L of the final PCR was used per test. Clearly detectable signals (absorbance >1.0) were obtained with both mutant and wild-type samples containing [10.sup.4] copies of plasmid [corresponding to the number of genomes present in ~1 [micro]L of blood (29)] and with the signal reaching the instrument maximum with samples containing [10.sup.5] copies of plasmid (data not shown). It seems likely that further optimization of the PCR reaction can improve this lower detection limit considerably.



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).

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).


The application of the ELISA format to nucleic acid-based genotyping/mutation detection offers many advantages, including a high degree of automation, high throughput, and the use of procedures that are well established in routine clinical laboratories. To be useful in genotyping/ mutation detection, the assay needs to be both sensitive and specific enough to effectively discriminate between target sequences that differ by as little as one base pair. With DNA probes, these requirements have been difficult to fulfil, primarily because DNA probes have only moderate affinity and, therefore, need to be rather large (and thus moderately sensitive to single-base mismatches) to provide effective capture of amplicons.

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.

We thank Kirsten Lindboe, Tinna Larsen, Annette Dyval, Klaus Bergmann, and Elisabeth Scharling for expert technical assistance and Estrid Hogdall for the factor V plasmid constructs. Exiqon holds significant patent rights to the AQ and LNA technology. H. Orum, T. Koch, and M. H. Jacobsen are minority shareholders in Exiqon, and H. Orum is a member of the Exiqon board.

Received May 11, 1999; accepted July 20, 1999.

[C] 1999 American Association for Clinical Chemistry


(1.) Rao AK, Kaplan R, Sheth S. Inherited thrombophilic states. Semin Thromb Hemost 1998;24:3-12.

(2.) Miletich JP. Thrombophilia as a multigenic disorder. Semin Thromb Hemost 1998;24:13-20.

(3.) Dahlback B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci U S A 1993;90:1004-8.

(4.) Griffin JH, Evatt B, Wideman C, Fernandez JA. Anticoagulant protein C pathway defective in majority of thrombophilic patients. Blood 1993;82:1989-93.

(5.) Svensson PJ, Dahlback B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994;330:517-22.

(6.) Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369:64-7.

(7.) Voorberg J, Roelse J, Koopman R, Buller H, Berends F, ten Cate JT, et al. Association of idiopathic venous thromboembolism with single point mutation at [Arg.sup.506] of factor V. Lancet 1994;343: 1535-6.

(8.) Zoller B, Dahlback B. Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet 1994;343:1536-8.

(9.) Zivelin A, Griffin JH, Xu X, Pabinger I, Samama M, Conard J, et al. A single genetic origin for a common Caucasian risk factor for venous thrombosis. Blood 1997;89:397-402.

(10.) Larsen TB, Lassen JF, Brandslund I, Byriel L, Petersen GB, Norgaard-Petersen B. The Arg506G1n mutation (FV Leiden) among a cohort of 4188 unselected Danish newborns. Thromb Res 1998;89:211-5.

(11.) Rosendaal FR, Koster T, Van denbroucke JP, Reitsma PH. High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance). Blood 1995;85:1504-8.

(12.) Ridker PM, Hennekens CH, Lindpaintner K, Stamper MJ, Eisenberg PR, Miletich JP. Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venous thrombosis in apparently healthy men. N Engl J Med 1995;332: 912-7.

(13.) Dizon-Townson D, Nelson LM, Jang H, Varner MW, Ward K. The incidence of the factor V Leiden mutation in obstetrical population and its relation to deep-vein thrombosis. Am J Obstet Gynecol 1997;176:883-6.

(14.) Bokewara MI, Bremme K, Blomback M. Arg506 to Gin mutation in factor V and risk of thrombosis during pregnancy. Br J Haematol 1996;92:473-8.

(15.) Vandenbroucke JP, Koster T, Briet E, Reitsma PH, Bertina RM, Rosendaal FR. Increased risk of venous thrombosis in oral-contraceptive users who are carriers of factor V Leiden mutation. Lancet 1994;344:1453-7.

(16.) Chevrier D, Popoff MY, Dion MP, Hermant D, Guesdon JL. Rapid detection of Salmonella subspecies by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucle otide on a microplate. FEMS Immunol Med Microbiol 1995;10: 245-52.

(17.) Van der Vliet GME, Hermans CJ, Klatser PR. Simple colorimetric microtiter plate hybridisation assay for detection of amplified Mycobacterium leprae DNA. J Clin Microbiol 1993;32:665-70.

(18.) Fujita S-I, Lasker BA, Lott TJ, Reiss E, Morrison CJ. Microtiter plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood. J Clin Microbiol 1995;33:962-7.

(19.) Pina M, Occhialini A, Monteiro L, Doermann H-P, Megraud F. Detection of point mutations associated with resistance of Helicobacter pylori to clarithromycin by hybridisation in liquid phase. J Clin Microbiol 1998;36:3285-90.

(20.) Koskin AA, Singh SK, Nielsen P, Rajwanshi VK, Kumar R, Meldgaard M, et al. LNA (locked nucleic acids): synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 1998;54:3607-30.

(21.) Singh SA, Nielsen P, Koshkin AA, Wengel J. LNA (locked nucleic acids): synthesis and high affinity nucleic acid recognition. Chem Commun 1998;455-6.

(22.) Singh SK, Kumar R, Wengel J. Synthesis of 2'-amino-LNA: a novel conformation ally restricted high-affinity oligonucleotide analogue with a handle. J Org Chem 1998;63:10035-9.

(23.) Kumar R, Singh SK, Koshkin AA, Rajwanshi VK, Meldgaard M, Wengel J. The first analogues of LNA (locked nucleic acids): phosphorothioate-LNA and 2'-thio-LNA. Bioorg Med Chem Lett 1998;8:2219-22.

(24.) Singh SK, Kumar R, Wengel J. Synthesis of novel bicyclo[2.2.1] ribonucleosides: 2'-amino- and 2'-thio-LNA monomeric nucleosides. J Org Chem 1998;63:6078-9.

(25.) Jacobsen MH, Koch T, inventors. Method of photochemical immobilisation of ligands using quinones. World patent WO 96/31557, 1996.

(26.) Jensen SP, Rasmussen SE, Jacobsen MH. Photochemical coupling of peptides to polystyrene microwell plates. In: Epton R, ed. Innovation and perspectives in solid phase synthesis and combi natorial libraries. Birmingham, UK: Mayflower Scientific, 1996: 419-22.

(27.) Bannwarth W. Solid-phase synthesis of oligodeoxynucleotides containing phosphoramidite internucleotide linkages and their specific chemical. Helv Chim Acta 1998;71:5973-88.

(28.) Allawi HT, SantaLucia J Jr. NMR solution structure of a DNA dodecamer containing single G-T mismatches. Nucleic Acids Res 1998;26:4925-34.

(29.) Kawasaki ES. Sample preparation from blood, cells, and other fluids. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. New York: Academic Press, 1990:146-52.


[1] PNA Diagnostics A/S, Ronnegade 2, DK-2100 Copenhagen, Denmark.

[2] Exiqon A/S, Bygstubben 9, DK-2950 Vedbaek, Denmark.

[3] Department of Clinical Biochemistry, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark.

* Author for correspondence. Fax 45-44-44-03-73; e-mail oerum@

[4] Nonstandard abbreviations: VTE, venous thromboembolism; LNA, locked nucleic acid; [T.sub.m], melting temperature; AQ, anthraquinone; RFLP, restriction fragment length polymorphism; and [C.sup.m], methylcytosine.
Table 1. Thermostability of the LNA and DNA octamers against
complementary and single base-mismatched DNA targets.

No. Probe Sequence

3 WT-LNA8s 5'-C'C'TC'GC'C'T-3'
4 MT-LNA8s 5'-C'C'TTGC'C'T-3'

5 MT-DNA8as 5'-AGGCA_AGG-3'
6 MT-LNA8as [5'-AGGC.sup.m]AGG-3'

 WT (a) target MT target
No. 5'-AGGCGAGG-3' (b) 5'-AGGCAAGG-3'

1 44 [degrees]C <15 [degrees]C
2 26 [degrees]C 36 [degrees]C
3 80 [degrees]C 50 [degrees]C
4 55 [degrees]C 71 [degrees]C
 5'-CCT_CGCCT-3' 5'-CCT_TGCCT-3'
5 <15 [degrees]C 36 [degrees]C
6 57 [degrees]C 77 [degrees]C

No. [DELTA][T.sub.m.sup.c]

1 >29[degrees]C
2 10 [degrees]C
3 30 [degrees]C
4 16 [degrees]C

5 >21 [degrees]C
6 20 [degrees]C

(a) WT, wild type; MT, mutant.

(b) Underlined bases are those that are complementary or mismatched.

(c) [T.sub.m] values were determined in 150 mmol/L
[Na.sub.2][HP0.sub.4], pH 7.0, 0.1 mmol/L EDTA.
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Article Details
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Title Annotation:Molecular Diagnostics and Genetics
Author:Orum, Henrik; Jakobsen, Mogens H.; Koch, Troels; Vuust, Jens; Borre, Martin B.
Publication:Clinical Chemistry
Article Type:Clinical report
Date:Nov 1, 1999
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