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Application of probes having 2′-deoxyinosine for typing of single nucleotide polymorphisms (SNPs) using DNA microarray
Analytica Chimica Acta 561 (2006) 25–31
Application of probes having 2-deoxyinosine for typing of single nucleotide polymorphisms (SNPs) using DNA microarray Hideki Kinoshita a , Shigemasa Ishiwata a , Yasuhiko Tsuji a , Makiko Dejima c , Kazuyoshi Yano b,∗ , Ichiro Takase c , Isao Karube b a
Katayanagi Institute, Tokyo University of Technology, 1404-1 Katakura, Hachioji-City, Tokyo 192-0982, Japan b School of Bionics, Tokyo University of Technology, 1404-1 Katakura, Hachioji-City, Tokyo 192-0982, Japan c Toppan Printing Company, Limited, 4-2-3 Takanodaiminami, Sugito-machi, Saitama 345-8508, Japan Received 1 August 2005; received in revised form 25 November 2005; accepted 5 December 2005 Available online 24 January 2006
Abstract The principle of hybridizing oligonucleotides is employed with single nucleotide polymorphisms (SNPs) typing, but that typing frequently failed. We investigated the SNPs typing of multi-alleles on a chip. Two bases adjacent to the SNPs on the immobilized oligonucleotide probes were substituted for the 2 -deoxyinosine (dI) residues. The biotinylated probes containing the partial sequence of CYP2C9, CYP2C19, GLC1A and ApoE alleles were applied to the streptavidin-embedded plasma-polymerized (PP) DNA microarrays. For the probes containing dI, up to a 2–5-fold difference in the match to a mismatch oligonucleotide fluorescence intensity was found as the probes without dI were 1.5-fold or less. According to the substitution for dI, the hybridization accuracy was enhanced, and then several single-base mismatches were detected on a chip. © 2006 Elsevier B.V. All rights reserved. Keywords: Single nucleotide polymorphisms; Oligonucleotide array; Gene chips; 2 -Deoxyinosine
1. Introduction Single nucleotide polymorphisms (SNPs) have gained widespread interest as markers for biomedical and pharmacogenetic research. Certain genetic polymorphisms may cause significantly different responses among individuals upon exposure to a particular drug [1–3]. A number of genotyping assays have been successful, which are based on one or more of the followings schemes: allele specific hybridization [4], flap end nuclease discrimination [5], primer extension [5,6], allele specific digestion [7–9], and oligonucleotide ligation [8]. However, these methods are complicated and the cost per SNPs typing is higher than the oligonucleotide microarray. TaqManTM system (Applied Biosystems) has been successfully used to genotype SNP. However, the present methods are still laborious, timeconsuming and not rigid enough for clinical applications [10]. The significant importance is to establish high-throughput and reliable methods for the genotyping of SNPs.
∗
Corresponding author. Tel.: +81 426 37 4516; fax: +81 426 37 4516. E-mail address: [email protected] (K. Yano).
0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.12.009
Oligonucleotide microarrays are widely used for highthroughput SNP screening. The hybridization-based microarray has driven the development of accessible and relatively inexpensive tools for arraying nucleic acids onto solid supports, hybridizing to probes of choice and scoring the results automatically [11]. For example, in a high-throughput study using the Affymetrix Variation Detection ArrayTM , ∼80% of all the haploid and diploid sites were readable [11]. However, the study failed to genotype 20% of the sites, and the need for further advancement of the technology is widely recognized. It is hard to distinguish many SNPs on a microarray at one time. The variety of SNPs detectable in parallel is restricted by the melting temperature resulting from the probe sequence [12]. The hybridization conditions are determined by temperature and composition of the buffer. The conditions for SNPs detection can be partly optimized by cationic counterions (usually Na+ from added NaCl), because nucleic acids are strong polyanionic electrolytes. The addition of cationic counterions shields the repulsion and results in a strong increase in the double-helix (duplex) thermal stability. Vainrub and Pettitt examined several concentrations of the immobilized probe on the surface of an array and the melting temperature, and found that the range of
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screened SNPs with different melting temperatures is quite independent of the surface probe density [13]. Moreover, the melting temperatures calculated from the GC contents in the nucleotides sequence were not matched with those experimentally obtained. We propose that the oligonucleotide probes containing the 2 deoxyinosine (dI) (its nucleoside form) residue can be applied to the SNP typing. dI residues match with each of the four normal DNA bases [14], and the stability of the matched base is I:C > I:A > I:T = I:G [15]. However, the neighboring-base effects on the stability are greater than the stability effect [15]. The melting temperatures of duplexes containing dI vary widely depending on the base to which the dI is paired and on the neighboring sequence. It was expected that the SNP typing using the oligonucleotide probe, which substituted the nucleosides adjacent to the SNP site for the dI residues, increases the allelic differentiation compared to the standard method. Miyachi et al. suggested that plasma-polymerized (PP) thin film-modified arrays are generally useful in the hybridization analyses, resulting in a greater sensitivity and lower backgrounds [16]. Furthermore, SNP typing on a DNA macroarray using the plasma-polymerization method has been demonstrated [17]. In this study, we fabricated microarrays by employing the hexamethyldisiloxane (HMDS) plasma-polymerized layers with hydrophobic characteristics for immobilization of probe DNA. Performance of the microarrays together with improved methods were examined by using a number of target DNA. Moreover, the oligonucleotide probes containing the dI residue were immobilized on glass substrates with a PP layer.
peroxide/ammonia solution/H2 O (approximately 1:1:18, by volume) and boiling for 1 h. The glasses were then washed in H2 O and dried. Before using the glass substrates, they were wiped with Clean-wipe (Asahi Kasei Co., Japan) containing isopropanol. 2.4. Apparatus for plasma-polymer deposition Plasma-polymerized thin films (PPFs) were prepared using a plasma deposition system (model BP-1, Samco International Laboratories, Inc., Japan). Two electrodes were horizontally placed 15 cm above the sample stage (external electrode reactors). An RF generator (Model RFG-300, Samco), coupled to an automatching network (to minimize reflected power), was also employed. The working frequency of the power supply was 13.56 MHz. HMDS was introduced from a reservoir above the equipment, and a plasma glow discharge treatment was carried out. The glass support was then placed on the sample stage. The needle valve remained fixed during plasma deposition to maintain a stable monomer flow rate. The thickness of the PPFs was measured by ellipsometry (ESM-1AT, ULVAC, Japan). 2.5. Preparation of DNA microarray
HMDS was provided by the Shin-Etsu Chemical Co. (Japan). Streptavidin was purchased from the Sigma Co. (US). The other reagents were of analytical grade from Wako Pure Chemical Industries (Japan).
The plasma-polymerization was performed as previously described [16]. A 6 nm thick HMDS-PPF was deposited (the first PP layer) on the glasses substrates using the model BP1. One nanoliter of 1.6 fmol/nl streptavidin (dissolved in H2 O) was spotted (BioChip Arrayer, Perkin-Elmer Co., US) at the dew-point temperature, and then dried. The entire area of the array was then covered with another 2 nm of HMDS-PPF (the second PP layer). Two nanoliter of the 1.6 fmol/nl 5 biotinylated DNA probe was spotted onto the immobilized streptavidin. The glasses were washed to remove any unimmobilized probe, first by soaking in 2× SSC [21] for 5 min and then by shaking 0.2× SSC for 25 min.
2.2. Oligodeoxyribonucleotides
2.6. DNA hybridization and fluorescence detection
The oligonucleotides used in this study are shown in Table 1. These sequences were derived from positions within the clarified human SNPs. These oligonucleotides were synthesized by Sigma Genosys (US). Some polymorphisms in GLC1A and ApoE may cause glaucoma and atherosclerosis, respectively [18]. CYP2C9*3, CYP2C19*2 and CYP2C19*3 encode a member of the cytochrome P450 superfamily [19]. These are polymorphic enzymes responsible for the metabolism of a large number of clinically important drugs such as S-warfarin, phenytoin, tolbutamide, iosartan and nonsteroidal anti-inflammatory drugs [20].
DNA hybridization on the streptavidin-embedded PP DNA arrays was performed at 60–70 ◦ C for 15 min using 50 l of the 0.1 M 5 Cy5-labeled targets DNA (Table 1) dissolved in 0.2× SSC. The excess target DNA was washed out using 0.5× SSC at 37–42 ◦ C for 5 min at 60 rpm using a slide-washer (Juji Field Inc., Japan), followed by 0.05× SSC at the same temperature for 5 min at 60 rpm. The fluorescence measurements were performed using a ScanArray (Perkin-Elmer Co.). The fluorescence intensities were analyzed using ScanArray Software (Perkin-Elmer Co.).
2. Experimental 2.1. Reagents
3. Results and discussion 2.3. Preparation of glass substrate 3.1. The discrimination of CYP2C9*3 SNPs Glass substrates (Dow Corning 7059, thickness 0.7 mm) were cut into 76 mm × 24.5 mm pieces. All experiments were performed after cleaning the substrates by immersion in hydrogen
First, we fabricated microarrays by spotting 2 nl of probe solution each using BioChip Arrayer (Perkin-Elmer) whereas
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Table 1 Oligonucleotides used in this study Name
Reference
Oligonucleotide
Sequence (5 –3 )
CYP2C19*3
Ibeanu et al. [19]
C19(G)-Cy5 C19(C)-B C19(C)-3 B C19-Fd(G)-3 B C19(A)-B C19(T)-B C19(G)-B C19(C)-3T-B C19(C)-9T-B C19(C)-12T-B C19(A)-12T-B C19(T)-12T-B C19(G)-12T-B C19-Fd(G)-12T-B C19(C)L-B
F-ACCCCCTGgATCCAGAT B-ATCTGGATcCAGGGGGT ATCTGGATcCAGGGGGT-B ACCCCCTGgATCCAGAT-B B-ATCTGGATaCAGGGGGT B-ATCTGGATtCAGGGGGT B-ATCTGGATgCAGGGGGT B-TTTATCTGGATcCAGGGGGT B-TTTTTTTTTATCTGGATcCAGGGGGT B-TTTTTTTTTTTTATCTGGATcCAGGGGGT B-TTTTTTTTTTTTATCTGGATaCAGGGGGT B-TTTTTTTTTTTTATCTGGATtCAGGGGGT B-TTTTTTTTTTTTATCTGGATgCAGGGGGT B-TTTTTTTTTTTTACCCCCTgGATCCAGAT B-TTGCATATCTGGATcCAGGGGGTGCTTAC
CYP2C9*3
rs1057910
C9(A)-Cy5 C9(T)-B C9(G)-B C9(T)-IB C9(G)-IB
F-GTCCAGAGATACaTTGACCTTCTCC B-TTTTTTTTTTTTGGAGAAGGTCAAtGTATCTCTGGAC B-TTTTTTTTTTTTGGAGAAGGTCAAgGTATCTCTGGAC B-TTTTTTTTTTTTGGAGAAGGTCAItITATCTCTGGAC B-TTTTTTTTTTTTGGAGAAGGTCAIgITATCTCTGGAC
CYP2C19*2
rs4244285
C19*2(G)-Cy5 C19*2(T)-B C19*2(A)-B C19*2(G)-B C19*2(C)-B
F-TATTTCCCgGGAACCCA B-TGGGTTCCtGGGAAATA B-TGGGTTCCaGGGAAATA B-TGGGTTCCgGGGAAATA B-TGGGTTCCcGGGAAATA
GLC1A 361
Stone et al. [18]
G361(C)-Cy5 G361(G)-B G361(A)-B G361(G)-IB G361(A)-IB
F-CTACCACGGAcAGTTCCCGTA B-TTTTTTTTTTTTTACGGGAACTgTCCGTGGTAG B-TTTTTTTTTTTTTACGGGAACTaTCCGTGGTAG B-TTTTTTTTTTTTTACGGGAACIgICCGTGGTAG B-TTTTTTTTTTTTTACGGGAACIaICCGTGGTAG
GLC1A 430
Stone et al. [18]
G430(T)-Cy5 G430(C)-Cy5 G430(A)-B G430(G)-B G430(A)-IB G430(G)-IB
F-TGGCACCTTGtACACCGTCAG F-TGGCACCTTGcACACCGTCAG B-TTTTTTTTTTTTCTGACGGTGTaCAAGGTGCCA B-TTTTTTTTTTTTCTGACGGTGTgCAAGGTGCCA B-TTTTTTTTTTTTCTGACGGTGIaIAAGGTGCCA B-TTTTTTTTTTTTCTGACGGTGIgIAAGGTGCCA
ApoE112
rs429358
A112(T)-Cy5 A112(A)-B A112(G)-B
F-AGGACGTGtGCGGCCGC B-TTTTTTTTTTTTGCGGCCGCaCACGTCCT B-TTTTTTTTTTTTGCGGCCGCgCACGTCCT
ApoE158
rs7412
A158(C)-Cy5 A158(G)-B A158(A)-B
F-TGCAGAAGcGCCTGGCA B-TTTTTTTTTTTTTGCCAGGCgCTTCTGCA B-TTTTTTTTTTTTTGCCAGGCaCTTCTGCA
B and F represent the probe DNA labeled with biotin and the target DNA labeled with fluorescence Cy5, respectively. Lower case shows SNPs site. The references showed the reference SNP (refSNP) ID number (belonging to dbSNP on National Center for Biotechnology Information).
Miyachi et al. spotted 1 l manually using a micropipetter [17]. The thickness of PP layers and wash conditions for hybridization were also changed accordingly, as described in Section 2. These minor improvements in protocol were inevitable for high reproducibility. The streptavidin-embedded PP microarrays were used for the CYP2C19*3 SNPs typing. The target oligonucleotide (C19(G)-Cy5) was a complementary sequence of a normal type on CYP2C19 from human SNPs (Table 1). The 2 nl of the probes were spotted on the streptavidin-embedded PP microarrays. The immobilized probes, C19(C)-B, C19(C)3 B, C19(C)-3T-B, C19(C)-9T-B, C19(C)-12T-B and C19(C)LB contained a complementary sequence with the target oligonu-
cleotide (Table 1). The C19(C) L-B probe sequence was longer than the other probes. C19(A)-B, C19(T)-B and C19(G)-B contained an A, T or G base mutation in the center, respectively. The sequences of C19-Fd(G)-3 B and C19-Fd(G)-12T-B without the poly(T) spacer probes were the same as target oligonucleotide sequences. When the target oligonucleotide was hybridized with these probes at 62 ◦ C, a matched type produced strongly fluorescing Cy5 spots which immobilized the C19(C)-B, C19(C)3 B, C19(C)-3T-B, C19(C)-9T-B, C19(C)-12T-B and C19(C)LB probes (Fig. 1). In addition, probes with single mutations, i.e., C19(A)-B, C19(T)-B, C19(G)-B, C19(A)-12T-B, C19(T)12T-B and C19(G)-12T-B, yielded weakly fluorescent light; they
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Fig. 1. The discrimination of CYP2C19*3 and the effect of poly(T) linker. C19(C)-B, C19(C)-3 B, C19(C)-3T-B, C19(C)-9T-B, C19(C)-12T-B and C19(C)L-B probes are complementary with C19(G)-Cy5, which are shown as the hatched boxes; the others contain a mutation shown as white boxes. A target oligonucleotide (C19(G)-Cy5) was hybridized with each probe on an array, and then the fluorescence intensity was detected using a ScanArray (Perkin-Elmer Co.). Bars represent standard errors (n = 15).
are immobilized probes. Moreover, the fluorescence intensity increased with the addition of the poly(T) spacer with the length of 9–12 mer (C19(C)-9T-B and C19(C)-12T-B probes). The longer probe, C19(C)L-B, also increased the fluorescence intensity. The detection of the allelic differentiation of CYP2C19*3 was successful. The poly(T) spacer was used in the following experiments.
to adjust uniformly. The sequence of the oligonucleotide probe plays a crucial role in the hybridization [12]. It was supposed that the sequence and 3-dimensional conformation might affect the hybridization. 3.3. Hybridization on the array using mixed target
3.2. Independent genotyping at six SNPs
Fourteen immobilized probes on the chip described above were hybridized with six mixed target oligonucleotides (C9(A)-
The detection of six types of SNPs (CYP2C9*3, CYP2C19*2, GLC1A361, GLC1A430, ApoE 112 and ApoE 158 are shown in Table 1) were examined using the above method. The lengths of each probe coding the six alleles were adjusted so that their melting temperatures were calculated to be approximately 60 ◦ C. Fourteen probes (C9(T)-B, C9(G)-B, C19*2(T)-B, C19*2(A)-B, C19*2(G)-B, C19*2(C)B, G361(G)-B, G361(A)-B, G430(A)-B, G430(G)-B, A112(A)B, A112(G)-B, A158(G)-B and A158(A)-B, shown in Table 1) were immobilized in an array on one chip, and then the microarray was hybridized with a target oligonucleotide in each (C9(A)Cy5, C19*2(G)-Cy5, G361(C)-Cy5, G430(T)-Cy5, A112(T)Cy5 and A158(C)-Cy5), as shown in Fig. 2. These optimal temperatures of hybridization and washing to remove a mismatch target were different. In the case of the G361(C)-Cy5, A112(T)-Cy5, A158(C)-Cy5 and C19*2(G)-Cy5 targets, the temperature of the hybridization and washing were 64 ◦ C and 37 ◦ C, respectively. On the other hand, the washing temperature of the G430(T)-Cy5 target was 42 ◦ C. Moreover, the C9(A)-Cy5 target was not under optimal conditions. The temperature of the optimal hybridization were different, therefore, it was difficult
Fig. 2. Allele-specific hybridization for distinguishing a single base mismatch. The immobilized probes were complementary consensus sequences taken from six wild types of human SNP sites. The hatched boxes and white boxes represent the fluorescence intensity from the matched and mismatched probes, respectively. The G361(G)-B and G361(A)-B, C9(T)-B and C9(G)-B, A112(A)B and A112(G)-B, A158(G)-B and A158(T)-B, and C19*2(T,A,G or C)-B were reacted with each target (G361(C)-Cy5, C9(A)-Cy5, A112(T)-Cy5, A158(C)Cy5 and C19*2(G)-Cy5) at 64 ◦ C, and then washed at 37 ◦ C, respectively. The G430(A)-B and G430(G)-B were reacted with G430(T)-Cy 5 at 64 ◦ C, and then washed at 42 ◦ C. Bars represent standard errors (n = 15).
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Fig. 3. The discrimination of alleles using target mixed. The constructed microarray was the same as the one used in Fig. 2. The hatched boxes and white boxes represent the fluorescence intensity of the immobilized matched and mismatched probes, respectively. The six target oligonucleotides (G361(C)-Cy5, G430(T)-Cy5, C9(A)-Cy5, A112(T)-Cy5, A158(C)-Cy5 and C19*2(G)-Cy5) were mixed at 35 pmol each, added onto a chip, and incubated at 64 ◦ C, and then washed at 37 ◦ C. Bars represent standard errors (n = 15).
Cy5, C19*2(G)-Cy5, G361(C)-Cy5, G430(T)-Cy5, A112(T)Cy5 and A158(C)-Cy5) at 62 ◦ C. As shown in Fig. 3, genotyping of CYP2C19*2, ApoE 112 and ApoE 158 SNPs could be clear. On the other hand, the mismatch discrimination of GLCA361, GLCA430 and CYP2C9*3 was difficult. The ratio of the fluorescence intensity from the perfectly matching probes (G361(G)-B, G430(A)-B and C9(T)-B) to that from the mismatching probes (G361(A)-B, G430(G)-B and C9(G)-B) was less than 2-fold. The each target hybridized to other probes nonspecifically to some extent, and the presence of mixed six targets might further increase those noises (Fig. 3), resulting in failure in clear discrimination of SNPs. Because the melting temperatures calculated from the GC contents in the nucleotides sequence were not matched with those experimentally obtained, the melting temperatures of these probes were different. Moreover, the hybridization condition was unified on a chip, and then the conditions between the perfect matching and single mismatching probes were almost the same. It was suggested that there were no optimal temperatures for hybridization to detect the six genotypes at once on a chip. 3.4. Genotyping with probe involving 2 -deoxyinosine Because the Tm of a perfect matching probe with a target was almost same as its mutated probe, it was difficult to optimize the hybridization condition of some of the SNPs, simultaneously. Because the melting temperatures of the duplexes containing dI widely vary depending on the neighboring sequence [15], it was expected that if the nucleotides of an adjacent mismatched nucleotide were substituted for dI, the probe involving dI cannot form a stable double helix with the target. On the other hand, if the nucleotides of the adjacent matched nucleotide
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were substituted for dI, the probe involving dI may pair with its target. Probes, G430(A)-B, G430(G)-B, G430(A)-IB and G430(G)IB, were immobilized on a single chip (Fig. 4 (I, II, III and IV, respectively). The G430(A)-IB and G430(G)-IB contained two dIs adjacent to a mismatch nucleotide. A Cy5 modifying target (G430(T)-Cy5) was added to the chip (Fig. 4), but the ratio of the fluorescence intensity of G430(A)-B to G430(G)-B, which was formed from the four natural DNA bases, was about 1.2-fold. Also, the ratio of its G430(A)-B to G430(G)-B was almost the same as the hybridization temperature at 64 ◦ C and 60 ◦ C (Fig. 4 (1 and 2 (I and II)), and the washing temperature at 37 ◦ C and 42 ◦ C (higher stringency) (Fig. 4 (2 and 3 (I and II)). However, when the hybridization temperatures were both at 64 ◦ C and 60 ◦ C, a greater fluorescence intensity from the probes involving dI (G430(A)-IB and G430(G)-IB) were obtained than that from the mismatched probes (Fig. 4 (1 and 2 (III and IV)). The fluorescent intensity of the fully matched probe (G430(A)-IB) to a mismatched probe (G430(G)-IB) ratio was >2-fold. When the washing temperature increased to 42 ◦ C, the fluorescent intensity were weak for both probes (G430(A)-IB and G430(G)-IB) (Fig. 4 (3 (III and IV))). It was indicated that the dI pair were less stable than a natural base pair. It was suggested that the substitution of a neighboring base of the SNPs with dI significantly increased its discrimination. On the other hand, a Cy5 modifying target (G430(C)-Cy5) was added to the chip (Fig. 4 (4)). As a result of the hybridization, GLC1A 430 SNPs could be clearly distinguished. Moreover, the probes containing dI were 2-fold clearer than the one without dI. It was concluded that the dI incorporating probes into an adjacent mismatch nucleotide enhanced the dissociation compared to one without dI. 3.5. Genotyping with probe involving 2 -deoxyinosine using mixed targets Because the range of Tm for discrimination of the alleles has been wide, it was postulated that a lot of genotypes on an array could be found using this method. An array immobilizing six oligonucleotides (G361(G)-B, G361(A)-B, G430(A)B, G430(G)-B, C9(T)-B and C9(G)-B) and six oligonucleotides containing dI (G361(G)-IB, G361(A)-IB, G430(A)IB, G430(G)-IB, C9(T)-IB and C9(G)-IB) was prepared, and three mixed targets (G361(C)-Cy5, G430(T)-Cy5 and C9(A)Cy5) were added to the chip (Fig. 5). As the probes without dI, the ratio of the fluorescent intensity of the fully complementary probe (G361(G)-B, G430(A)-B and C9(T)-B) to a mismatched probe (G361(A)-B, G430(G)-B and C9(G)-B) was <1.5-fold. On the other hand, for the probes containing dI, the ratio was >2.5. The stability of base pairing cannot be determined by only the number of hydrogen bonds [15], so that it is important to measure the melting point. However, its temperature was different between the immobilized probes and free probes in the liquid phase, and it was difficult to estimate with regard to the wash steps (temperature, shaking and buffer component). Although it was possible to use the surface plasmon resonance technology, it is difficult to reconstruct of conditions in this study.
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Fig. 4. Temperature effect on hybridization using probes containing dI. The chips constructed in Fig. 4 were used. The #1, 2 and 3 chips were reacted with G430(T)Cy5 at 64 ◦ C or 60 ◦ C, and then washed at 37 ◦ C or 42 ◦ C. The I (G430(A)-B) and II (G430(A)-B) row in the arrays were the natural oligonucleotides, and the III and IV row in it were two bases substituted for dI. The #4 chip was reacted with G430(C)-Cy5 at 64 ◦ C, and then washed at 37 ◦ C. The hatched boxes and white boxes represent the fluorescence intensity of the immobilized matched and mismatched probes, respectively. Bars represent standard errors (n = 15).
According to the substitution for dI of the bases adjacent to an allele, the allelic discrimination was clear. If a base adjacent to dI on a probe is paired with a complementary base on a target oligonucleotide, it was postulated that a dI is paired with a target. On the other hand, a base adjacent to dI is not a complementary base, and then a dI could not pair with the target. 4. Conclusion The results of this study suggested that the substitution for dI significantly increased the allelic discrimination compared to the normal DNA bases. By substitution for dI of the oligonucleotide adjacent to an allele, CYP2C9*3, CYP2C19*2, CYP2C19*3, GLC1A361, GLC1A430, ApoE112 and ApoE158 allele typing was successful on a chip. Moreover, these results may be applied to not only other microarray immobilized oligonucleotides, but also non-microarray types. We suggested that this method can detect a number of SNPs on one chip at once and can be applied to high-density microarrays for SNPs. References
Fig. 5. Three types of allele discriminations with three target mixed oligonucleotides a, b, e, f, i and j were without dI, and c, d, g, h, k and l contained dI. The a, e and i probes were complementary consensus sequences with the G361(C)-Cy5, G430(T)-Cy5 and C9(A)-Cy5 targets, respectively, and the c, g and k probes were substituted for two dI adjacent to an SNP on a, e and i, respectively. Moreover, d, h and l, or b, f and j involved an SNP between two dIs or not, respectively. The array was reacted with the target oligonucleotide G361(C)-Cy5, G430(T)-Cy5 and C9(A)-Cy5 mixed at 64 ◦ C, and then washed at 37 ◦ C.
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[16] H. Miyachi, A. Hiratsuka, K. Ikebukuro, K. Yano, H. Muguruma, I. Karube, Biotechnol. Bioeng. 69 (2000) 323–329. [17] H. Miyachi, K. Ikebukuro, K. Yano, H. Aburatani, I. Karube, Biosens. Bioelectron. 20 (2004) 184–189. [18] E.M. Stone, J.H. Fingert, W.L. Alward, T.D. Nguyen, J.R. Polansky, S.L. Sunden, D. Nishimura, A.F. Clark, A. Nystuen, B.E. Nichols, D.A. Mackey, R. Ritch, J.W. Kalenak, E.R. Craven, V.C. Sheffield, Science 275 (1997) 668–670. [19] G.C. Ibeanu, J.A. Goldstein, U. Meyer, S. Benhamou, C. Bouchardy, P. Dayer, B.I. Ghanayem, J. Blaisdell, J. Pharmacol. Exp. Ther. 286 (1998) 1490–1495. [20] S.Y. Wen, H. Wang, O.J. Sun, S.Q. Wang, World J. Gastroenterol. 9 (2003) 1342–1346. [21] J. Sambrook, E.F. Fritsch, T. Maniatis (Eds.), Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, Cold Spring, NY, 1989.
Application of probes having 2-deoxyinosine for typing of single nucleotide polymorphisms (SNPs) using DNA microarray Hideki Kinoshita a , Shigemasa Ishiwata a , Yasuhiko Tsuji a , Makiko Dejima c , Kazuyoshi Yano b,∗ , Ichiro Takase c , Isao Karube b a
Katayanagi Institute, Tokyo University of Technology, 1404-1 Katakura, Hachioji-City, Tokyo 192-0982, Japan b School of Bionics, Tokyo University of Technology, 1404-1 Katakura, Hachioji-City, Tokyo 192-0982, Japan c Toppan Printing Company, Limited, 4-2-3 Takanodaiminami, Sugito-machi, Saitama 345-8508, Japan Received 1 August 2005; received in revised form 25 November 2005; accepted 5 December 2005 Available online 24 January 2006
Abstract The principle of hybridizing oligonucleotides is employed with single nucleotide polymorphisms (SNPs) typing, but that typing frequently failed. We investigated the SNPs typing of multi-alleles on a chip. Two bases adjacent to the SNPs on the immobilized oligonucleotide probes were substituted for the 2 -deoxyinosine (dI) residues. The biotinylated probes containing the partial sequence of CYP2C9, CYP2C19, GLC1A and ApoE alleles were applied to the streptavidin-embedded plasma-polymerized (PP) DNA microarrays. For the probes containing dI, up to a 2–5-fold difference in the match to a mismatch oligonucleotide fluorescence intensity was found as the probes without dI were 1.5-fold or less. According to the substitution for dI, the hybridization accuracy was enhanced, and then several single-base mismatches were detected on a chip. © 2006 Elsevier B.V. All rights reserved. Keywords: Single nucleotide polymorphisms; Oligonucleotide array; Gene chips; 2 -Deoxyinosine
1. Introduction Single nucleotide polymorphisms (SNPs) have gained widespread interest as markers for biomedical and pharmacogenetic research. Certain genetic polymorphisms may cause significantly different responses among individuals upon exposure to a particular drug [1–3]. A number of genotyping assays have been successful, which are based on one or more of the followings schemes: allele specific hybridization [4], flap end nuclease discrimination [5], primer extension [5,6], allele specific digestion [7–9], and oligonucleotide ligation [8]. However, these methods are complicated and the cost per SNPs typing is higher than the oligonucleotide microarray. TaqManTM system (Applied Biosystems) has been successfully used to genotype SNP. However, the present methods are still laborious, timeconsuming and not rigid enough for clinical applications [10]. The significant importance is to establish high-throughput and reliable methods for the genotyping of SNPs.
∗
Corresponding author. Tel.: +81 426 37 4516; fax: +81 426 37 4516. E-mail address: [email protected] (K. Yano).
0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.12.009
Oligonucleotide microarrays are widely used for highthroughput SNP screening. The hybridization-based microarray has driven the development of accessible and relatively inexpensive tools for arraying nucleic acids onto solid supports, hybridizing to probes of choice and scoring the results automatically [11]. For example, in a high-throughput study using the Affymetrix Variation Detection ArrayTM , ∼80% of all the haploid and diploid sites were readable [11]. However, the study failed to genotype 20% of the sites, and the need for further advancement of the technology is widely recognized. It is hard to distinguish many SNPs on a microarray at one time. The variety of SNPs detectable in parallel is restricted by the melting temperature resulting from the probe sequence [12]. The hybridization conditions are determined by temperature and composition of the buffer. The conditions for SNPs detection can be partly optimized by cationic counterions (usually Na+ from added NaCl), because nucleic acids are strong polyanionic electrolytes. The addition of cationic counterions shields the repulsion and results in a strong increase in the double-helix (duplex) thermal stability. Vainrub and Pettitt examined several concentrations of the immobilized probe on the surface of an array and the melting temperature, and found that the range of
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screened SNPs with different melting temperatures is quite independent of the surface probe density [13]. Moreover, the melting temperatures calculated from the GC contents in the nucleotides sequence were not matched with those experimentally obtained. We propose that the oligonucleotide probes containing the 2 deoxyinosine (dI) (its nucleoside form) residue can be applied to the SNP typing. dI residues match with each of the four normal DNA bases [14], and the stability of the matched base is I:C > I:A > I:T = I:G [15]. However, the neighboring-base effects on the stability are greater than the stability effect [15]. The melting temperatures of duplexes containing dI vary widely depending on the base to which the dI is paired and on the neighboring sequence. It was expected that the SNP typing using the oligonucleotide probe, which substituted the nucleosides adjacent to the SNP site for the dI residues, increases the allelic differentiation compared to the standard method. Miyachi et al. suggested that plasma-polymerized (PP) thin film-modified arrays are generally useful in the hybridization analyses, resulting in a greater sensitivity and lower backgrounds [16]. Furthermore, SNP typing on a DNA macroarray using the plasma-polymerization method has been demonstrated [17]. In this study, we fabricated microarrays by employing the hexamethyldisiloxane (HMDS) plasma-polymerized layers with hydrophobic characteristics for immobilization of probe DNA. Performance of the microarrays together with improved methods were examined by using a number of target DNA. Moreover, the oligonucleotide probes containing the dI residue were immobilized on glass substrates with a PP layer.
peroxide/ammonia solution/H2 O (approximately 1:1:18, by volume) and boiling for 1 h. The glasses were then washed in H2 O and dried. Before using the glass substrates, they were wiped with Clean-wipe (Asahi Kasei Co., Japan) containing isopropanol. 2.4. Apparatus for plasma-polymer deposition Plasma-polymerized thin films (PPFs) were prepared using a plasma deposition system (model BP-1, Samco International Laboratories, Inc., Japan). Two electrodes were horizontally placed 15 cm above the sample stage (external electrode reactors). An RF generator (Model RFG-300, Samco), coupled to an automatching network (to minimize reflected power), was also employed. The working frequency of the power supply was 13.56 MHz. HMDS was introduced from a reservoir above the equipment, and a plasma glow discharge treatment was carried out. The glass support was then placed on the sample stage. The needle valve remained fixed during plasma deposition to maintain a stable monomer flow rate. The thickness of the PPFs was measured by ellipsometry (ESM-1AT, ULVAC, Japan). 2.5. Preparation of DNA microarray
HMDS was provided by the Shin-Etsu Chemical Co. (Japan). Streptavidin was purchased from the Sigma Co. (US). The other reagents were of analytical grade from Wako Pure Chemical Industries (Japan).
The plasma-polymerization was performed as previously described [16]. A 6 nm thick HMDS-PPF was deposited (the first PP layer) on the glasses substrates using the model BP1. One nanoliter of 1.6 fmol/nl streptavidin (dissolved in H2 O) was spotted (BioChip Arrayer, Perkin-Elmer Co., US) at the dew-point temperature, and then dried. The entire area of the array was then covered with another 2 nm of HMDS-PPF (the second PP layer). Two nanoliter of the 1.6 fmol/nl 5 biotinylated DNA probe was spotted onto the immobilized streptavidin. The glasses were washed to remove any unimmobilized probe, first by soaking in 2× SSC [21] for 5 min and then by shaking 0.2× SSC for 25 min.
2.2. Oligodeoxyribonucleotides
2.6. DNA hybridization and fluorescence detection
The oligonucleotides used in this study are shown in Table 1. These sequences were derived from positions within the clarified human SNPs. These oligonucleotides were synthesized by Sigma Genosys (US). Some polymorphisms in GLC1A and ApoE may cause glaucoma and atherosclerosis, respectively [18]. CYP2C9*3, CYP2C19*2 and CYP2C19*3 encode a member of the cytochrome P450 superfamily [19]. These are polymorphic enzymes responsible for the metabolism of a large number of clinically important drugs such as S-warfarin, phenytoin, tolbutamide, iosartan and nonsteroidal anti-inflammatory drugs [20].
DNA hybridization on the streptavidin-embedded PP DNA arrays was performed at 60–70 ◦ C for 15 min using 50 l of the 0.1 M 5 Cy5-labeled targets DNA (Table 1) dissolved in 0.2× SSC. The excess target DNA was washed out using 0.5× SSC at 37–42 ◦ C for 5 min at 60 rpm using a slide-washer (Juji Field Inc., Japan), followed by 0.05× SSC at the same temperature for 5 min at 60 rpm. The fluorescence measurements were performed using a ScanArray (Perkin-Elmer Co.). The fluorescence intensities were analyzed using ScanArray Software (Perkin-Elmer Co.).
2. Experimental 2.1. Reagents
3. Results and discussion 2.3. Preparation of glass substrate 3.1. The discrimination of CYP2C9*3 SNPs Glass substrates (Dow Corning 7059, thickness 0.7 mm) were cut into 76 mm × 24.5 mm pieces. All experiments were performed after cleaning the substrates by immersion in hydrogen
First, we fabricated microarrays by spotting 2 nl of probe solution each using BioChip Arrayer (Perkin-Elmer) whereas
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Table 1 Oligonucleotides used in this study Name
Reference
Oligonucleotide
Sequence (5 –3 )
CYP2C19*3
Ibeanu et al. [19]
C19(G)-Cy5 C19(C)-B C19(C)-3 B C19-Fd(G)-3 B C19(A)-B C19(T)-B C19(G)-B C19(C)-3T-B C19(C)-9T-B C19(C)-12T-B C19(A)-12T-B C19(T)-12T-B C19(G)-12T-B C19-Fd(G)-12T-B C19(C)L-B
F-ACCCCCTGgATCCAGAT B-ATCTGGATcCAGGGGGT ATCTGGATcCAGGGGGT-B ACCCCCTGgATCCAGAT-B B-ATCTGGATaCAGGGGGT B-ATCTGGATtCAGGGGGT B-ATCTGGATgCAGGGGGT B-TTTATCTGGATcCAGGGGGT B-TTTTTTTTTATCTGGATcCAGGGGGT B-TTTTTTTTTTTTATCTGGATcCAGGGGGT B-TTTTTTTTTTTTATCTGGATaCAGGGGGT B-TTTTTTTTTTTTATCTGGATtCAGGGGGT B-TTTTTTTTTTTTATCTGGATgCAGGGGGT B-TTTTTTTTTTTTACCCCCTgGATCCAGAT B-TTGCATATCTGGATcCAGGGGGTGCTTAC
CYP2C9*3
rs1057910
C9(A)-Cy5 C9(T)-B C9(G)-B C9(T)-IB C9(G)-IB
F-GTCCAGAGATACaTTGACCTTCTCC B-TTTTTTTTTTTTGGAGAAGGTCAAtGTATCTCTGGAC B-TTTTTTTTTTTTGGAGAAGGTCAAgGTATCTCTGGAC B-TTTTTTTTTTTTGGAGAAGGTCAItITATCTCTGGAC B-TTTTTTTTTTTTGGAGAAGGTCAIgITATCTCTGGAC
CYP2C19*2
rs4244285
C19*2(G)-Cy5 C19*2(T)-B C19*2(A)-B C19*2(G)-B C19*2(C)-B
F-TATTTCCCgGGAACCCA B-TGGGTTCCtGGGAAATA B-TGGGTTCCaGGGAAATA B-TGGGTTCCgGGGAAATA B-TGGGTTCCcGGGAAATA
GLC1A 361
Stone et al. [18]
G361(C)-Cy5 G361(G)-B G361(A)-B G361(G)-IB G361(A)-IB
F-CTACCACGGAcAGTTCCCGTA B-TTTTTTTTTTTTTACGGGAACTgTCCGTGGTAG B-TTTTTTTTTTTTTACGGGAACTaTCCGTGGTAG B-TTTTTTTTTTTTTACGGGAACIgICCGTGGTAG B-TTTTTTTTTTTTTACGGGAACIaICCGTGGTAG
GLC1A 430
Stone et al. [18]
G430(T)-Cy5 G430(C)-Cy5 G430(A)-B G430(G)-B G430(A)-IB G430(G)-IB
F-TGGCACCTTGtACACCGTCAG F-TGGCACCTTGcACACCGTCAG B-TTTTTTTTTTTTCTGACGGTGTaCAAGGTGCCA B-TTTTTTTTTTTTCTGACGGTGTgCAAGGTGCCA B-TTTTTTTTTTTTCTGACGGTGIaIAAGGTGCCA B-TTTTTTTTTTTTCTGACGGTGIgIAAGGTGCCA
ApoE112
rs429358
A112(T)-Cy5 A112(A)-B A112(G)-B
F-AGGACGTGtGCGGCCGC B-TTTTTTTTTTTTGCGGCCGCaCACGTCCT B-TTTTTTTTTTTTGCGGCCGCgCACGTCCT
ApoE158
rs7412
A158(C)-Cy5 A158(G)-B A158(A)-B
F-TGCAGAAGcGCCTGGCA B-TTTTTTTTTTTTTGCCAGGCgCTTCTGCA B-TTTTTTTTTTTTTGCCAGGCaCTTCTGCA
B and F represent the probe DNA labeled with biotin and the target DNA labeled with fluorescence Cy5, respectively. Lower case shows SNPs site. The references showed the reference SNP (refSNP) ID number (belonging to dbSNP on National Center for Biotechnology Information).
Miyachi et al. spotted 1 l manually using a micropipetter [17]. The thickness of PP layers and wash conditions for hybridization were also changed accordingly, as described in Section 2. These minor improvements in protocol were inevitable for high reproducibility. The streptavidin-embedded PP microarrays were used for the CYP2C19*3 SNPs typing. The target oligonucleotide (C19(G)-Cy5) was a complementary sequence of a normal type on CYP2C19 from human SNPs (Table 1). The 2 nl of the probes were spotted on the streptavidin-embedded PP microarrays. The immobilized probes, C19(C)-B, C19(C)3 B, C19(C)-3T-B, C19(C)-9T-B, C19(C)-12T-B and C19(C)LB contained a complementary sequence with the target oligonu-
cleotide (Table 1). The C19(C) L-B probe sequence was longer than the other probes. C19(A)-B, C19(T)-B and C19(G)-B contained an A, T or G base mutation in the center, respectively. The sequences of C19-Fd(G)-3 B and C19-Fd(G)-12T-B without the poly(T) spacer probes were the same as target oligonucleotide sequences. When the target oligonucleotide was hybridized with these probes at 62 ◦ C, a matched type produced strongly fluorescing Cy5 spots which immobilized the C19(C)-B, C19(C)3 B, C19(C)-3T-B, C19(C)-9T-B, C19(C)-12T-B and C19(C)LB probes (Fig. 1). In addition, probes with single mutations, i.e., C19(A)-B, C19(T)-B, C19(G)-B, C19(A)-12T-B, C19(T)12T-B and C19(G)-12T-B, yielded weakly fluorescent light; they
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Fig. 1. The discrimination of CYP2C19*3 and the effect of poly(T) linker. C19(C)-B, C19(C)-3 B, C19(C)-3T-B, C19(C)-9T-B, C19(C)-12T-B and C19(C)L-B probes are complementary with C19(G)-Cy5, which are shown as the hatched boxes; the others contain a mutation shown as white boxes. A target oligonucleotide (C19(G)-Cy5) was hybridized with each probe on an array, and then the fluorescence intensity was detected using a ScanArray (Perkin-Elmer Co.). Bars represent standard errors (n = 15).
are immobilized probes. Moreover, the fluorescence intensity increased with the addition of the poly(T) spacer with the length of 9–12 mer (C19(C)-9T-B and C19(C)-12T-B probes). The longer probe, C19(C)L-B, also increased the fluorescence intensity. The detection of the allelic differentiation of CYP2C19*3 was successful. The poly(T) spacer was used in the following experiments.
to adjust uniformly. The sequence of the oligonucleotide probe plays a crucial role in the hybridization [12]. It was supposed that the sequence and 3-dimensional conformation might affect the hybridization. 3.3. Hybridization on the array using mixed target
3.2. Independent genotyping at six SNPs
Fourteen immobilized probes on the chip described above were hybridized with six mixed target oligonucleotides (C9(A)-
The detection of six types of SNPs (CYP2C9*3, CYP2C19*2, GLC1A361, GLC1A430, ApoE 112 and ApoE 158 are shown in Table 1) were examined using the above method. The lengths of each probe coding the six alleles were adjusted so that their melting temperatures were calculated to be approximately 60 ◦ C. Fourteen probes (C9(T)-B, C9(G)-B, C19*2(T)-B, C19*2(A)-B, C19*2(G)-B, C19*2(C)B, G361(G)-B, G361(A)-B, G430(A)-B, G430(G)-B, A112(A)B, A112(G)-B, A158(G)-B and A158(A)-B, shown in Table 1) were immobilized in an array on one chip, and then the microarray was hybridized with a target oligonucleotide in each (C9(A)Cy5, C19*2(G)-Cy5, G361(C)-Cy5, G430(T)-Cy5, A112(T)Cy5 and A158(C)-Cy5), as shown in Fig. 2. These optimal temperatures of hybridization and washing to remove a mismatch target were different. In the case of the G361(C)-Cy5, A112(T)-Cy5, A158(C)-Cy5 and C19*2(G)-Cy5 targets, the temperature of the hybridization and washing were 64 ◦ C and 37 ◦ C, respectively. On the other hand, the washing temperature of the G430(T)-Cy5 target was 42 ◦ C. Moreover, the C9(A)-Cy5 target was not under optimal conditions. The temperature of the optimal hybridization were different, therefore, it was difficult
Fig. 2. Allele-specific hybridization for distinguishing a single base mismatch. The immobilized probes were complementary consensus sequences taken from six wild types of human SNP sites. The hatched boxes and white boxes represent the fluorescence intensity from the matched and mismatched probes, respectively. The G361(G)-B and G361(A)-B, C9(T)-B and C9(G)-B, A112(A)B and A112(G)-B, A158(G)-B and A158(T)-B, and C19*2(T,A,G or C)-B were reacted with each target (G361(C)-Cy5, C9(A)-Cy5, A112(T)-Cy5, A158(C)Cy5 and C19*2(G)-Cy5) at 64 ◦ C, and then washed at 37 ◦ C, respectively. The G430(A)-B and G430(G)-B were reacted with G430(T)-Cy 5 at 64 ◦ C, and then washed at 42 ◦ C. Bars represent standard errors (n = 15).
H. Kinoshita et al. / Analytica Chimica Acta 561 (2006) 25–31
Fig. 3. The discrimination of alleles using target mixed. The constructed microarray was the same as the one used in Fig. 2. The hatched boxes and white boxes represent the fluorescence intensity of the immobilized matched and mismatched probes, respectively. The six target oligonucleotides (G361(C)-Cy5, G430(T)-Cy5, C9(A)-Cy5, A112(T)-Cy5, A158(C)-Cy5 and C19*2(G)-Cy5) were mixed at 35 pmol each, added onto a chip, and incubated at 64 ◦ C, and then washed at 37 ◦ C. Bars represent standard errors (n = 15).
Cy5, C19*2(G)-Cy5, G361(C)-Cy5, G430(T)-Cy5, A112(T)Cy5 and A158(C)-Cy5) at 62 ◦ C. As shown in Fig. 3, genotyping of CYP2C19*2, ApoE 112 and ApoE 158 SNPs could be clear. On the other hand, the mismatch discrimination of GLCA361, GLCA430 and CYP2C9*3 was difficult. The ratio of the fluorescence intensity from the perfectly matching probes (G361(G)-B, G430(A)-B and C9(T)-B) to that from the mismatching probes (G361(A)-B, G430(G)-B and C9(G)-B) was less than 2-fold. The each target hybridized to other probes nonspecifically to some extent, and the presence of mixed six targets might further increase those noises (Fig. 3), resulting in failure in clear discrimination of SNPs. Because the melting temperatures calculated from the GC contents in the nucleotides sequence were not matched with those experimentally obtained, the melting temperatures of these probes were different. Moreover, the hybridization condition was unified on a chip, and then the conditions between the perfect matching and single mismatching probes were almost the same. It was suggested that there were no optimal temperatures for hybridization to detect the six genotypes at once on a chip. 3.4. Genotyping with probe involving 2 -deoxyinosine Because the Tm of a perfect matching probe with a target was almost same as its mutated probe, it was difficult to optimize the hybridization condition of some of the SNPs, simultaneously. Because the melting temperatures of the duplexes containing dI widely vary depending on the neighboring sequence [15], it was expected that if the nucleotides of an adjacent mismatched nucleotide were substituted for dI, the probe involving dI cannot form a stable double helix with the target. On the other hand, if the nucleotides of the adjacent matched nucleotide
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were substituted for dI, the probe involving dI may pair with its target. Probes, G430(A)-B, G430(G)-B, G430(A)-IB and G430(G)IB, were immobilized on a single chip (Fig. 4 (I, II, III and IV, respectively). The G430(A)-IB and G430(G)-IB contained two dIs adjacent to a mismatch nucleotide. A Cy5 modifying target (G430(T)-Cy5) was added to the chip (Fig. 4), but the ratio of the fluorescence intensity of G430(A)-B to G430(G)-B, which was formed from the four natural DNA bases, was about 1.2-fold. Also, the ratio of its G430(A)-B to G430(G)-B was almost the same as the hybridization temperature at 64 ◦ C and 60 ◦ C (Fig. 4 (1 and 2 (I and II)), and the washing temperature at 37 ◦ C and 42 ◦ C (higher stringency) (Fig. 4 (2 and 3 (I and II)). However, when the hybridization temperatures were both at 64 ◦ C and 60 ◦ C, a greater fluorescence intensity from the probes involving dI (G430(A)-IB and G430(G)-IB) were obtained than that from the mismatched probes (Fig. 4 (1 and 2 (III and IV)). The fluorescent intensity of the fully matched probe (G430(A)-IB) to a mismatched probe (G430(G)-IB) ratio was >2-fold. When the washing temperature increased to 42 ◦ C, the fluorescent intensity were weak for both probes (G430(A)-IB and G430(G)-IB) (Fig. 4 (3 (III and IV))). It was indicated that the dI pair were less stable than a natural base pair. It was suggested that the substitution of a neighboring base of the SNPs with dI significantly increased its discrimination. On the other hand, a Cy5 modifying target (G430(C)-Cy5) was added to the chip (Fig. 4 (4)). As a result of the hybridization, GLC1A 430 SNPs could be clearly distinguished. Moreover, the probes containing dI were 2-fold clearer than the one without dI. It was concluded that the dI incorporating probes into an adjacent mismatch nucleotide enhanced the dissociation compared to one without dI. 3.5. Genotyping with probe involving 2 -deoxyinosine using mixed targets Because the range of Tm for discrimination of the alleles has been wide, it was postulated that a lot of genotypes on an array could be found using this method. An array immobilizing six oligonucleotides (G361(G)-B, G361(A)-B, G430(A)B, G430(G)-B, C9(T)-B and C9(G)-B) and six oligonucleotides containing dI (G361(G)-IB, G361(A)-IB, G430(A)IB, G430(G)-IB, C9(T)-IB and C9(G)-IB) was prepared, and three mixed targets (G361(C)-Cy5, G430(T)-Cy5 and C9(A)Cy5) were added to the chip (Fig. 5). As the probes without dI, the ratio of the fluorescent intensity of the fully complementary probe (G361(G)-B, G430(A)-B and C9(T)-B) to a mismatched probe (G361(A)-B, G430(G)-B and C9(G)-B) was <1.5-fold. On the other hand, for the probes containing dI, the ratio was >2.5. The stability of base pairing cannot be determined by only the number of hydrogen bonds [15], so that it is important to measure the melting point. However, its temperature was different between the immobilized probes and free probes in the liquid phase, and it was difficult to estimate with regard to the wash steps (temperature, shaking and buffer component). Although it was possible to use the surface plasmon resonance technology, it is difficult to reconstruct of conditions in this study.
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Fig. 4. Temperature effect on hybridization using probes containing dI. The chips constructed in Fig. 4 were used. The #1, 2 and 3 chips were reacted with G430(T)Cy5 at 64 ◦ C or 60 ◦ C, and then washed at 37 ◦ C or 42 ◦ C. The I (G430(A)-B) and II (G430(A)-B) row in the arrays were the natural oligonucleotides, and the III and IV row in it were two bases substituted for dI. The #4 chip was reacted with G430(C)-Cy5 at 64 ◦ C, and then washed at 37 ◦ C. The hatched boxes and white boxes represent the fluorescence intensity of the immobilized matched and mismatched probes, respectively. Bars represent standard errors (n = 15).
According to the substitution for dI of the bases adjacent to an allele, the allelic discrimination was clear. If a base adjacent to dI on a probe is paired with a complementary base on a target oligonucleotide, it was postulated that a dI is paired with a target. On the other hand, a base adjacent to dI is not a complementary base, and then a dI could not pair with the target. 4. Conclusion The results of this study suggested that the substitution for dI significantly increased the allelic discrimination compared to the normal DNA bases. By substitution for dI of the oligonucleotide adjacent to an allele, CYP2C9*3, CYP2C19*2, CYP2C19*3, GLC1A361, GLC1A430, ApoE112 and ApoE158 allele typing was successful on a chip. Moreover, these results may be applied to not only other microarray immobilized oligonucleotides, but also non-microarray types. We suggested that this method can detect a number of SNPs on one chip at once and can be applied to high-density microarrays for SNPs. References
Fig. 5. Three types of allele discriminations with three target mixed oligonucleotides a, b, e, f, i and j were without dI, and c, d, g, h, k and l contained dI. The a, e and i probes were complementary consensus sequences with the G361(C)-Cy5, G430(T)-Cy5 and C9(A)-Cy5 targets, respectively, and the c, g and k probes were substituted for two dI adjacent to an SNP on a, e and i, respectively. Moreover, d, h and l, or b, f and j involved an SNP between two dIs or not, respectively. The array was reacted with the target oligonucleotide G361(C)-Cy5, G430(T)-Cy5 and C9(A)-Cy5 mixed at 64 ◦ C, and then washed at 37 ◦ C.
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