Chemical cleavage of mismatch (CCM) is a highly specific and sensitive method to induce mismatch-cleavages 33. However, the major disadvantage of CCM is the use of toxic chemicals during the process in the reactions or buffers, which could be avoided by using enzymatic mismatch cleavage. The celery enzyme is a member of a family of plant endonucleases that recognizes mismatches in heteroduplex DNA and cleaves both strands on the 3' side of the mismatch distortion 27. To investigate the diagnostic value of this approach, the exons of HBB genes were amplified by PCR using primers labeled at the 5' end with fluorescent dyes (Figure 1). We also employed a simple method to detect mutations in HBB genes using the mismatch-specific endonuclease cleavage and separation of the PCR products by capillary electrophoresis. The approach is based on mismatch-specific selective reactions of mismatched DNA cleavage by endonuclease and separation of the PCR product fragments by capillary electrophoresis. This method is also adaptable to automated capillary electrophoresis which could increase its speed, sensitivity, and reproducibility, making it suitable for large-scale and high-throughput mutation screening.

We detected all the DNA variants of the HBB gene including single base substitutions and deletions of TCTT. The different mutation sites and the expected sizes of PCR products cleaved by mismatch-specific endonuclease are described in Table 1. Samples of variant DNA fragments obtained with different base pairs were analyzed using capillary electrophoresis and the results are shown in Figure 2.

The PCR fragment amplified by the B1 and B2 primers was 337 bp; this fragment was cut into fragments of 107 bp and 230 bp by mismatch-specific endonuclease indicatingthe presence of c.-78 A>G mutation (Fig. 2A). Similar to the c.-78 A>G variation, the common c.9 C>T single nucleotide polymorphism (SNP) of the PCR fragment was cleaved to 193 bp and 144 bp (Fig. 2C).

The FAM- and HEX-labeled probes had the same sensitivity and specificity and could be used for double confirmation of the results. The formation of new cleavage products indicated the presence of variation, while their size provided some information about their location in the entire gene (Figure 3). The two variations could be also determined by the mismatch-specific endonuclease method as shown in Figure 2. The 107 bp, 230 bp, 186 bp, and 151 bp cutting by mismatch-specific endonuclease represented the presence of compound heterozygous variations (c.-78 A>G with c.2 T>A) (Fig. 2H). Similarly, the 193 bp, 144 bp, 236 bp, and 101 bp due to c.9 C>T polymorphism combined with the c.52 A>T variation (Fig. 2I). The other DNA substitutions were identified by following this principle to predict the locations of the mutation sites. This technique could be the gold standard for the identification of healthy carriers, while for the diagnosis of affected patients direct sequencing probably remains the best approach, also considering the small size of HBB gene.

DHPLC is a promising tool for mutations detection and gene quantification 343536. Previously, we developed a powerful and rapid PCR-DHPLC assay for detection of HBB mutations 37. However, DHPLC had better sensitivity and specificity in the analysis of heteroduplexes in PCR fragments shorter than 500 bp. In an attempt to increase sensitivity, we modified and optimized the PCR primers and DHPLC conditions for this study (Table 2), providing a significant advantage to this technique. The modified assay was used for identification of various genotypes of the HBB gene (Figure 4) which were confirmed by direct sequencing.

HBB genes could contain one or more mutations that could affect the results of DHPLC. This results in complex chromatography as shown in Figure 4, and failure to easily determine the genotype.

DNA samples were obtained from a total of 50 subjects, including 20 carriers, 20 patients, and 10 normal individuals at National Taiwan University Hospital. Genomic DNA was collected from peripheral whole blood using the Chemagic DNA Blood Kit (Chemagen) according to the manufacturer's instructions. The different genotypes of the DNA samples used in the mismatch-specific endonuclease study are shown in Table 1.

PCR primers and conditions for each exon to amplify the HBB gene were modified according to previously reported methods 37 and are described in Table 2. The PCR techniques for the provided DNA fragments were performed in a total volume of 25 μl containing 50 ng of genomic DNA; 0.12 μM of each primer; 100 μM dNTPs; 0.5 units of AmpliTaq Gold enzyme (Applied Biosystems); 2.5 μl of GeneAmp 10 × buffer II (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl), in 2 mmol/L MgCl2 as provided by the manufacturer. Amplification was performed in a multiblock system (MBS) thermocycler (ThermoHybaid). PCR amplification began with denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at72°C for 45 s, and a final extension step at 72°C for 10 min.

Mutations screening was performed using the Transgenomic Wave Nucleic Acid Fragment Analysis System (Transgenomic Inc.) with a C₁₈ reversed-phase column, which was based on 2-μm nonporous poly (styrene/divinylbenzene) particles (DNASep column, Transgenomic Inc.). PCR products were analyzed in a linear acetonitrile gradient with triethylammonium acetate as the mobile phase, using buffer A (0.1 M TEAA) and buffer B (0.1 M TEAA with 25% acetonitrile) (WAVE Optimized, Transgenomic Inc.). Heteroduplex analyses were performed according to the manufacturer's protocol and previous studies 374445.

In the mismatch-specific endonuclease process, the Microcon YM-100 system (Millipore Corp.) was used to remove excess primers and unincorporated dNTPs to purify the PCR products.

After PCR amplification and purification, the PCR product was digested by mismatch-specific endonuclease using the SURVEYOR kit with the fluorescent capillary electrophoresis system (Transgenomic). The PCR products were assayed in a total volume of 60 μl containing 15 μl of PCR product, 6 μl of 10 × Surveyor Nuclease reaction buffer, 1 μl of Surveyor Nuclease Enhancer W, and 1 μl of Surveyor Nuclease W (Surveyor) as provided by the manufacturer. Mismatch-specific endonuclease digestion was performed at 42°C for 20 min, followed by adding 6 μl of stop solution as provided by the manufacturer.

The final mismatch-specific endonuclease digestion product (2 μl) was diluted with 10 μl of Hi-Di formamide (Applied Biosystems) and 0.25 μl of ROX size standard (Applied Biosystems). Samples were heated at 95°C for 5 min and cooled on ice for 5 min. Each mixture was injected into the ABI PRISM 310 Genetic Analyzer (Applied Biosystems) and was separated across a capillary containing POP-4 polymer (Applied Biosystems). The results were analyzed using GeneScan application software (Applied Biosystems) according to the manufacturer's protocol 46.

For sequencing, the PCR products were purified by solid-phase extraction and bi-directionally sequenced with the Applied Biosystems Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions. Sequencing reactions were separated on a PE Biosystems 373A/3100 sequencer.