Single-stranded conformation polymorphism analysis of Vitamin D receptor gene by capillary electrophoresis with laser-induced fluorescence detection

Analytica Chimica Acta 445 (2001) 197–204

Single-stranded conformation polymorphism analysis of Vitamin D receptor gene by capillary electrophoresis with laser-induced fluorescence detection Hidetoshi Arakawa a,∗ , Hisako Igarashi a , Hiroyuki Kashiwazaki a , Masako Maeda a , Akifumi Tokita b , Yuichirou Yamashiro b a

School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan Department of Pediatrics, Juntendo University School of Medicine, Tokyo 113-8421, Japan

b

Received 3 May 2001; received in revised form 12 July 2001; accepted 12 July 2001

Abstract A highly sensitive single-stranded conformation polymorphism method for analysis of Vitamin D receptor (VDR) gene polymorphism was developed employing laser-induced fluorescence capillary electrophoresis (LIF-CE). LIF-CE was conducted utilizing a linear polyacrylamide solution as entangled polymer and acridine orange as a fluorescent dye of single-stranded DNA. Effect of acridine orange, size of PCR product and running temperature were investigated by LIF-CE in order to analyze the two polymorphisms of the allelic variation of the Bsm I and Taq I sites in intron 8 and exon 9, respectively, of the VDR gene. The developed method was simple, rapid (<16 min for Taq I type, <20 min for Bsm I type) and highly sensitive for VDR gene polymorphism. VDR gene polymorphism in 32 subjects was determined by the proposed method. The results were consistent with those obtained by restriction fragment-length polymorphism (RFLP) analysis using gel electrophoresis. The proposed method can be employed among the various VDR gene polymorphism analyses related to osteoporosis. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Single-stranded conformation polymorphism; Vitamin D receptor gene; Laser-induced fluorescence capillary electrophoresis

1. Introduction Osteoporosis is a multifactorial disease involving a reduction in bone mineral density (BMD) and bone strength, which leads to increased risk of fracture. With increasing life expectancy, osteoporosis is becoming a major public health concern resulting in significant morbidity and mortality in elderly persons. A decrease of one standard deviation in BMD within the normal range increases the risk of osteoporotic frac∗

Corresponding author. Tel.: +81-337848194; fax: +81-337848247. E-mail address: [email protected] (H. Arakawa).

ture by approximately two times at various skeletal sites. Recently, Morrison et al. demonstrated the Vitamin D receptor (VDR) gene to be a major locus for genetic influences on BMD; moreover, polymorphisms in the 3 -end region of the VDR gene (as determined by the restriction enzyme Bsm I) appeared to predict spinal and femoral BMD and bone turnover markers [1,2]. Subjects homozygous (bb, wild type) for the presence of the Bsm I restriction endonuclease site were reported to have a BMD approximately one standard deviation within the normal range higher than that of subjects homozygous (BB, mutant type) for the absence of the site [2]. Matsuyama et al. noted that

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analysis of VDR alleles may prove useful in selection of optimum dose in active Vitamin D therapy for osteoporosis or optimum therapy for osteoporosis management [3]. Thus, allelic variation of Bsm I polymorphism in intron 8, which is digested with Bsm I and is represented as bb type (wild type), is correlated with serum osteocalcin concentration and BMD. Furthermore, Tokita et al. reported that Bsm I polymorphism (bb type, wild type) correlated well with the presence of a Taq I site (TT type, wild type), which cannot be digested with Taq I [4]. These two polymorphic types (bb and TT) were found to have higher BMD than those displaying the BB genotype, which is not digested with Bsm I, and tt genotype (mutant type), which is digested with Taq I due to a change of base (in exon 94), respectively. Therefore, analysis of these DNA polymorphisms can be applicable to the prediction of osteocalcin concentration and BMD. Additionally, it is preferable to determine these polymorphism types simultaneously in order to obtain a precise diagnosis. Generally, DNA polymorphism can be detected with a number of techniques, including allele-specific PCR [5], PCR-restriction fragment length polymorphism [6], hybridization with DNA probe, Taq man [7] and single-stranded conformation polymorphisms (SSCP) [8]. SSCP analysis is the simplest among these techniques because other methods except SSCP need primers and probes labeled with a fluorescent dye, restriction enzyme, preparation of the electrophoresis gel and dyeing, respectively. SSCP analysis is based on the conformational change of single-stranded DNA caused by mutation resulting in a mobility shift on non-denaturing gel electrophoresis [8]. Therefore, detection of a slight conformational difference with conventional electrophoresis requires strict running conditions, such as low temperature, and extends for several hours. Consequently, SSCP analysis by conventional electrophoresis cannot be performed easily in the clinical setting. Recently, analysis of SSCP by CE has been conducted as this method possesses several advantages, including high resolution, reproducibility, high sensitivity, speed and ease of analysis [9]. P53 gene [10], ras oncogene [11,12], cystathiamine ␤-synthase gene [13], factor V gene [14], methylenetetrahydrofolate reductase [15] and LDL receptor gene [16] have thus far been analyzed by CE employing laser-induced fluorescence as the de-

tection method and gel or polymer solution as the sieving media. In the current study, SSCP analysis of the VDR gene by CE was developed. The effects of DNA fragment size amplified by PCR, an intercalation dye and the use of entangled polymer solution were investigated in order to develop a specific and sensitive method for CE SSCP analysis of the VDR gene. This method permits promptly identification of VDR gene polymorphism. Moreover, the technique can be readily applied to clinical samples in order to predict serum osteocalcin levels.

2. Experimental 2.1. Procedure 2.1.1. Apparatus A Model P/ACE 5010 CE system and Laser Module 488 (Beckman, Fullerton, CA, USA) were utilized. The system generated a signal wavelength (488 nm) of argon ion laser (3–4 mW) with detection set at 520 nm. 2.2. Materials ␥-Methacryloxypropyltrimethoxysilane was purchased from Sigma (St. Louis, MO, USA). Acrylamide, bis-N,N,N,N-tetramethylethylenediamine (TEMED) and ammonium peroxydisulfate (APS) for coating were obtained from Wako (Osaka, Japan). Acridine orange was acquired from Merck (USA). Polyacrylamide (MW 700,000–1,000,000, 10%) was purchased from Polysciences Inc. (Warmington, USA). Silica capillary tubing (37 cm (effective length = 30 cm) × 100 ␮m i.d., 375 ␮m o.d.) was obtained from GL Sciences (Tokyo, Japan). Gene Amp PCR reagent kit with Amplitaq DNA polymerase was purchased from Perkin-Elmer Cetus (Norwalk, CT, USA). Primers used for PCR were synthesized by Takara Shuzo Co. (Kyoto, Japan). Other chemicals were reagent grade. 2.2.1. Capillary coating procedure Fused-silica capillary tubing was utilized for CE. Coating of capillary inner walls was accomplished according to the methods of Paulus and Ohms [17]. Acrylamide was covalently attached to the walls

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of the fused silica capillary with ␥-methacryloxypropyltrimethoxysilane. An amount of 3% acrylamide (5 ml), dissolved in 100 mM Tris–250 mM borate buffer (pH 7.8), was polymerized by addition of 2 ␮l of TEMED solution and 50 ␮l of 1% ammonium persulfate solution. The polymerization solution was quickly introduced into the treated capillary via pressure injection using a P/ACE 5010 instrument for 10 min and left for at least 2 h. After coating with acrylamide, the capillary was sequentially washed with H2 O and the following polymer solution. The polymer solution was prepared as follows: 11% linear acrylamide polymer (MW 700,000–1,000,000) was diluted with 0.1 M Tris–borate (pH 7.8) containing 0.1 ␮g/ml acridine orange.

Table 2 Conditions of PCR for the VDR gene

2.2.2. Capillary electrophoresis In a CE experiment, the electrode of the injection side was attached to the negative pole of the power supply. Sample injections were effected via pressure for 40 s. Electrophoresis was performed with 3% polyacrylamide and 0.1 ␮g/ml acridine orange in 0.1 M Tris–borate (pH 7.8) at 7.4 kV and 20–30◦ C. DNA fragments were detected by a LIF detector employing 488 and 520 nm wavelengths for excitation and emission, respectively. CE with UV detection was conducted under the conditions described above. Exceptions to these conditions included an absorbance wavelength of 254 nm and the absence of acridine orange.

method involving digestion with proteinase K and extraction with phenol–chloroform, followed by ethanol precipitation. The harvested genomic DNA was dissolved in TE buffer and stored at −40◦ C until use.

2.2.3. Specimen preparation DNA from peripheral blood mononuclear cells collected from 32 subjects was obtained by a standard

Condition H2 O (␮l) 10 × buffer (␮l) 2.5 mM dNTPs (␮l) 10 ␮mol/l R primer (␮l) 10 ␮mol/l F primer (␮l) DMSO (␮l)

Cycle 10.8 2.0 2.0

One step, 95◦ C, 3 min Two steps, 62◦ C, 1 min Three steps, 74◦ C, 2 min

7

1.0 1.0 1.0

Two steps, 62◦ C, 20 s One step, 95◦ C, 20 s Three steps, 74◦ C, 1 min

5

Template DNA (␮l) 2.0 5 U/␮l Taq polymerase (␮l) 0.2

One step, 95◦ C, 5 s Two steps, 62◦ C, 5 s Three steps, 74◦ C, 30 s

Total volume (␮l)

One step, 74◦ C, 3 min

20.0

40

2.2.4. SSCP of VDR gene A DNA fragment of 155 bp of Taq I site in the VDR gene was amplified by PCR with Taq primers F and R. DNA fragments of 75, 133, 215 and 273 bp including the Bsm I site were amplified with various combinations of Bsm primers F and R. Primers and sequences used in this experiment are shown in Table 1. PCR was conducted according to the standard method. The PCR reaction mixture consisted of 50 mM KCl, 10 mM Tris–HCl (pH 8.7), 1.5 mM MgCl2 , 2.5 mM dNTPs, 1.25 ␮M each of amplification primer sense and antisense, dimethyl sulfoxide (DMSO), 100 ng of

Table 1 Primers and sequences used in PCR experimentsa Bsm I site

PCR product 5 -TAGATAAGCAGGGTTCCTGG-3

Primer F1; Primer R1; 5 -AGTTCACGCAAGAGCAGAGC-3

75

Primer F2; 5 -CCCTTAGCTCTGCCTTGCAGA-3 Primer R1; 5 -AGTTCACGCAAGAGCAGAGC-3

133

Primer F1; 5 -TAGATAAGCAGGGTTCCTGG-3 Primer R2; 5 -AACCAGCGGAAGAGGTCAAGGG-3

215

Primer F2; 5 -CCCTTAGCTCTGCCTTGCAGA-3 Primer R2; 5 -AACCAGCGGAAGAGGTCAAGGG-3

273

a

All values are in bp.

Taq I site

PCR product 5 -CGTTGAGTCTGTGTGTGGGT-3

Primer F; Primer R; 5 -TGTGTTGGACAGGCGGTCCT-3

155

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extracted DNA and 1.25 U of Taq DNA polymerase in a total volume of 20 ␮l. PCR reaction was conducted according to the conditions described in Table 2. PCR products generated from genes were then precipitated separately with ethanol. Precipitated PCR products were dissolved in 30 ␮l of H2 O. Sample solutions were heated to 99◦ C for 6 min and cooled in ice for 6 min. Subsequently, samples were analyzed by CE with LIF detection.

3. Results and discussion Previously, we reported SSCP analysis by CE and its application in the detection of single point mutations in K-ras [11] and N-ras genes [18]. The results indicated that SSCP analysis by CE was more rapid and sensitive than traditional electrophoresis; however, several problems, including the preparation of gel in the capillary tube, preparation of the 5 -end labeled oligonucleotide primer with fluorescent dye and difficulty in clinical application, remain to be resolved. In this study, intercalation dye and a polyacrylamide polymer solution were utilized in order to develop a simple, highly sensitive, and rapid CE-SSCP method. Furthermore, various conditions for SSCP analysis were examined in the analysis of two polymorphisms in the VDR gene. 3.1. Selection of fluorescent dye Various intercalating dyes, including monomeric species thiazole orange (TO), oxazole yellow (YO)-PRO-I and SYBER Green I and dimeric species TOTO-1, YOYO-1 and YOYO-3, have been used for sensitive fluorescence detection of double-stranded DNA [19]. In previous SSCP analysis studies [11], PCR products were labeled with Texas red and

FITC. However, this procedure made necessary the preparation of 5 -end primers labeled with a fluorescent dye as described above. Furthermore, PCR and SSCP specificity are thought to be influenced by the dye selected. In this study, the use of acridine orange, a fluorescent RNA dye, was examined for SSCP analysis. A preliminary experiment evaluated the length of single-stranded DNA required for emission of fluorescence. Strong fluorescence intensity was observed with single-stranded DNA of 30 bases or more; moreover, fluorescence signals from 20 bases were very weak (data not shown). This result indicated that sense and antisense primers of approximately 20 bases generally used for PCR would emit nearly no fluorescence and thus would not interfere with analysis of SSCP on electropherograms. Utility of an intercalation reagent for doublestranded DNA is known to increase separation resolution. This effect is believed to result from structural changes in the DNA due to its unfolding as a consequence of intercalation reagent binding and a reduction in the effective electric potential of the DNA itself [20,21]. The effects of acridine orange on separation were examined for both double-stranded DNA of 72–1353 bp (RF DNA fragment/Hae III digest) and synthesized oligonucleotide of 60 bases in the current study. Results are displayed in Table 3. Resolution of double-stranded DNA (271 bp/281 bp) improved by 2.2-fold in comparison with the UV method in the absence of acridine orange. Furthermore, plate numbers of double-stranded DNA (310 bp) and synthesized oligonucleotide (60 bases) increased by 1.2-fold. Therefore, addition of acridine orange improved resolution of separation of oligonucleotide of 60 bases and double-stranded DNA. Subsequently, precision of migration time and peak height were examined. Results are shown in Table 4. The CV (%) for migration time and peak height were 0.18–0.2 and 8.2–16.1%,

Table 3 Comparison of plate numbers and resolution obtained by LIF and UV detection CE with UV detection

CE with LIF detection using acridine orangea

Plate numbers

310 bp 60 bases

2.8 × 105 2.8 × 105

3.5 × 105 3.5 × 105

Resolution

271 bp/281 bp

1.11

2.53b

a b

Acridine orange: 1 ␮g/ml. Rs: 1.83 for 0.1 ␮g/ml.

H. Arakawa et al. / Analytica Chimica Acta 445 (2001) 197–204 Table 4 Precision of migration time and peak height for double-stranded DNA and oligonucleotide of 60 bases obtained by CE-LIF Migration time (CV%)

Peak height (CV%)

Oligonucleotide of 60 bases

0.2 (n = 6)

13.3 (n = 6)

Double-stranded DNA (bp) 72 234 603

0.18 (n = 9) 0.19 (n = 9) 0.18 (n = 9)

8.2 (n = 9) 11.6 (n = 9) 16.1 (n = 9)

respectively. These findings indicated that the separation of double-stranded DNA and oligonucleotide of 60 bases employing acridine orange was highly reproducible and qualitative. Sensitivity using acridine orange was compared with that of UV detection. Peak heights of 603 bp and 60 bases obtained with LIF and UV detection were plotted against concentration. The limits of detection of double-stranded DNA and oligonucleotide

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of 60 bases obtained from LIF were 20 and 100 ng/ml, respectively. This result indicated that the fluorescence detection system employing acridine orange was ca. 100-fold more sensitive than UV detection. Thus, the limit of DNA detection by LIF was well suited to SSCP analysis of a specific gene amplified from a small amount of human DNA. Previously, we described the effects of gel concentration, running temperature and fragment size obtained by PCR on the separation of SSCP [18]. Our results indicated that the determination of optimum conditions for individual DNA polymorphism is of paramount importance. These factors were investigated in order to develop a method for SSCP analysis of VDR gene polymorphism in the current study. Consequently, the following experimental parameters, including running temperature and DNA size, were examined with respect to the development of methods for SSCP analysis of the Taq I and Bsm I sites of the VDR gene.

Fig. 1. Electropherogram of SSCP obtained from Taq I site. TT, Tt and tt types represent a higher bone density polymorphism, heterozygous carrier and a lower bone density polymorphism, respectively. CE condition: electrophoresis was performed with 3% polyacrylamide and 0.1 ␮g/ml acridine orange in 0.1 M Tris–borate (pH 7.8) at 7.4 kV and 20◦ C. DNA fragments were detected by a LIF detector employing 488 and 520 nm wavelengths for excitation and emission, respectively.

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3.2. SSCP analysis of the Taq I site in the VDR gene 3.2.1. PCR products and running temperature for SSCP separation of the Taq I site A PCR product of 155 bp was generated using the sense and antisense primers shown in Table 1. PCR cycle and conditions are presented in Table 2. These conditions were selected so as to enhance the specificity and sensitivity of PCR. This PCR product included the polymorphic Taq I site of the VDR gene, i.e. a T-to-C substitution in exon 9 in subjects exhibiting lower bone density. The effects of capillary temperatures between 20 and 40◦ C were initially studied for the separation of SSCP. Our findings indicated that SSCPs of the Taq I site could be completely separated at 20◦ C as shown in Fig. 1. Thus, the TT (higher bone density polymorphism, wild) and tt (lower bone density polymorphism, mutant) types displayed two and three peaks, respectively. The two peaks corresponding to TT type are either sense or anti-sense SSCP. The tt type based on the presence of three peaks

demonstrated a more complex SSCP than did the TT type. This observation may be due to self-annealing of either the sense or antisense SSCP. Tt type (heterozygous carrier) revealed four peaks, indicating that one SSCP of TT and tt overlaps and that one SSCP of TT and two SSCP of tt display different migration time. As a result, these polymorphisms (TT, Tt, tt) could be clearly distinguished. 3.2.2. Effects of acridine orange on separation of SSCP The effects of acridine orange on separation of SSCP for the Taq I site polymorphism were examined utilizing samples of TT and Tt types. In cases of SSCP separation in the absence of acridine orange, PCR products of TT and Tt type polymorphisms were generated using FITC-labeled sense primer in lieu of the sense primer described above. The PCR products obtained were treated in a manner identical to that described for the SSCP method and were separated employing the identical polymer solution lacking acridine orange. SSCP electropherograms with and

Fig. 2. Electropherograms of SSCP of FITC-labeled PCR products obtained from TT, Tt and tt types. CE condition: electrophoresis was performed with 3% polyacrylamide in 0.1 M Tris–borate (pH 7.8) at 7.4 kV and 20◦ C. DNA fragments were detected by a LIF detector employing 488 and 520 nm wavelengths for excitation and emission in the absence of acridine orange as an intercalating agent.

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without acridine orange were compared. The findings indicated that SSCP patterns of TT and tt with acridine orange consisted of two and three peaks, respectively, as shown in Fig. 1. Consequently, the SSCP of the Taq I site could be clearly determined. In contrast, SSCP patterns of TT, Tt and tt of fluorescein isothiocyanate (FITC)-labeled PCR products, which is detected by FITC fluorescence in the absence of acridine orange, could not be distinguished as illustrated in Fig. 2. The results described above indicated that the technique employing acridine orange as the detection method allowed not only sensitive detection of SSCP, but also improved SSCP separation. 3.3. SSCP of the Bsm I site in the VDR gene 3.3.1. DNA size of PCR products and running temperature for SSCP separation of Bsm I type DNA fragments (75, 133, 215 and 273 bp) including a Bsm I site were amplified using the

203

combinations of various primers described in Table 1. These PCR products of differing size were separated at various running temperatures of 20, 25, 30, 35 and 40◦ C following dissociation of double-stranded DNA. The SSCP of BB (mutant), Bb (heterozygous carrier) and bb (wild) types in the Bsm I type could be clearly separated under the conditions employed for a 273 bp PCR product and 20–25◦ C running temperature. However, PCR products of 75, 133 and 215 bp displayed no differences in SSCP for Bsm I at 20–40◦ C (data not shown). Therefore, the following conditions for the Bsm I type were chosen: base size of 273 bp and running temperature of 25◦ C. Although it is generally believed that shorter aplicon base size increases SSCP sensitivity, the largest fragment of 273 bases (in 75–273 bases) exclusively demonstrated SSCP differences in the case of the Bsm I type. The reason remains unknown. The typical SSCP for Bsm I polymorphism exhibited two peaks of BB, two peaks of bb and three peaks of Bb, respectively. These data are presented in Fig. 3.

Fig. 3. Electropherogram of SSCP obtained from the Bsm I site. BB, Bb and bb types represent a lower bone density polymorphism, heterozygous carrier and a higher bone density polymorphism, respectively. CE condition: electrophoresis was performed with 3% polyacrylamide and 0.1 ␮g/ml acridine orange in 0.1 M Tris–borate (pH 7.8) at 7.4 kV and 25◦ C.

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3.3.2. Identification of SSCP of the Bsm I type SSCP electropherograms of BB and bb polymorphisms revealed two peaks. In addition, the hetero type of the B and b genes displayed three peaks. This result signifies identical migration times in sense or anti-sense SSCP in BB and bb genes; moreover, SSCP electropherogram of the Bsm I type is more simple than that of the Taq I type. However, migration time distinctions of another sense or anti-sense SSCP, which was used to distinguish SSCP, were slight as presented in Fig. 3. This finding indicated that SSCP analysis for the Bsm I type is more difficult than the determination of the Taq SSCP. Therefore, we utilized the following method in order to perform precise assignment of Bsm I polymorphism. Thus, the last peaks (19 min) observed in BB, Bb and bb were used as an internal standard as they demonstrated constant migration time (designated as α). In contrast, earlier peaks observed in BB and bb types exhibited clearly different migration times (designated as β and γ , respectively). β/α and γ /α values were then calculated from these migration times; the ratios of β/α and γ /α were 0.979 and 0.957, respectively. The reliability of these ratios was determined by examination of reproducibility (n = 10) based on the Bb type; CV were 0.15% for β/α, and 0.3% for γ /α, respectively. These results indicated that this identification method for polymorphism was highly reliable. Consequently, SSCP analysis for BB, bb and Bb types could be performed with greater precision and ease by comparison of β/α and γ /α ratios. 3.4. Application of clinical sample The effectiveness of the proposed method was examined via determination of VDR gene polymorphism in 32 clinical samples. VDR gene polymorphism was also analyzed by the RFLP technique using agarose gel electrophoresis according to the procedure described by Morrison et al.; moreover, the findings were compared with those of the proposed method. The results of the TT (wild), Tt (heterozygous carrier) and tt (mutant) types as well as the BB (mutant), Bb (heterozygous carrier) and bb (wild) types obtained by the present method were in complete agreement with those determined by the traditional technique involving agarose gel electrophoresis (data not shown).

In conclusion, a novel SSCP method by CE-LIF employing entangled polymer solution and intercalation dye was developed for analysis of VDR gene polymorphism. The proposed method is sensitive, simple and rapid (<16 min for Taq I type, <20 min for Bsm I type) for SSCP analysis. Furthermore, the reproducibility and specificity of SSCP analysis for the DNA polymorphism of the VDR gene were high. Therefore, the proposed method can be applied clinically for diagnosis of VDR gene polymorphism. References [1] N.A. Morrison, R. Yeoman, P.J. Kelly, J.A. Eisman, Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 6665. [2] N.A. Morrison, J.C. Qi, A. Tokita, P.J. Kelly, L. Crofts, T.V. Nguen, P.N. Sambrook, J.A. Eisman, Nature 367 (1994) 284. [3] T. Matsuyama, S. Ishii, A. Tokita, K. Yabuta, S. Yamamori, N.A. Morrison, J.A. Eisman, Lancet 345 (1995) 1238. [4] A. Tokita, H. Matsumoto, N.A. Morrison, J. Bone Miner. Res. 11 (1996) 1003. [5] C.R. Newton, A. Graham, L.E. Heptinstall, S.J. Powell, C. Summers, N. Kalsheker, J.C. Smith, A.F. Markham, Nucl. Acids Res. 17 (1989) 2503. [6] A. Haliassos, J.C. Chomel, S. Grandjouan, J. Kruh, J.C. Kaplan, A. Kitzis, Nucl. Acids Res. 17 (1989) 8093. [7] Livak, PCR Methods Appl. 4 (1995) 357. [8] M. Orita, H. Iwahana, H. Kanazawa, K. Hayashi, T. Sekiya, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 2766. [9] J. Ren, J. Chromatogr. B 741 (2000) 115. [10] D.H. Atha, H.M. Wenz, H. Morehead, J. Tian, C.D. O’Connell, Electrophoresis 19 (1998) 172. [11] H. Arakawa, A. Tsuji, M. Maeda, M. Kamahori, H. Kambara, J. Pharm. Biomed. Anal. 15 (1997) 1537. [12] A. Nishimura, M. Tsuhako, Chem. Pharam. Bull. 48 (2000) 774. [13] J. Ren, A. Ulvik, H. Refsum, P.M. Ueland, Anal. Biochem. 276 (1999) 188. [14] J. Ren, P.M. Ueland, Hum. Mutat. 13 (1999) 458. [15] J. Ren, A. Ulvik, P.M. Ueland, H. Refsum, Anal. Biochem. 245 (1997) 79. [16] J. Geisel, T. Walz, M. Bodis, M. Nauck, K. Oette, W. Herrmann, J. Chromatogr. B 724 (1999) 239. [17] A. Paulus, J.I. Ohms, J. Chromatogr. 507 (1990) 113. [18] H. Arakawa, S. Nakashiro, M. Maeda, A. Tsuji, J. Chromatogr. A 722 (1996) 359–368. [19] Y. Baba, J. Chromatogr. B 687 (1996) 271. [20] A. Guttman, N. Cooke, Anal. Chem. 63 (1991) 2038. [21] H.E. Schwartz, K. Ulfelder, F.J. Sunzeri, M.P. Busch, R.G. Brownlee, J. Chromatogr. 559 (1991) 267.