Microarray-based method to evaluate the accuracy of restriction endonucleases HpaII and MspI.
A double-strand DNA (ds DNA) microarray was fabricated to analyze the structural perturbations caused by methylation and the different base mismatches in the interaction of the restriction endonucleases HpaII and MspI with DNA. First, a series of synthesized oligonucleotides were arrayed on the aldehyde-coated glass slides. Second, these oligonucleotides were hybridized with target sequences to obtain ds DNA microarray, which includes several types of double strands with the fully methylated, semi-methylated, and unmethylated canonical recognition sequences, semi-methylated and unmethylated base mismatches within the recognition sequences. The cleavage experiments were carried out under normal buffer conditions. The results indicated that MspI could partially cleave methylated and semi-methylated canonical recognition sequences. In contrast, HpaII could not cleave methylated and semi-methylated canonical recognition sequences. HpaII and MspI could both cleave the unmethylated canonical recognition sequence. However, HpaII could partially cleave the sequence containing one GG mismatch and not cleave other base mismatches in the corresponding recognition site. In contrast, MspI could not recognize the base mismatches within the recognition sequence. A good reproducibility was observed in several parallel experiments. The experiment indicates that the microarray technology has great potentials in high-throughput identifying important interactions between protein and DNA.
Hou
Peng
P
Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing, China. zhlu@seu.edu.cn
Ji
Meiju
M
He
Nongyue
N
Lu
Zuhong
Z
D020137:Base Pair Mismatch; D001665:Binding Sites; D004262:DNA Restriction Enzymes / Q000032:analysis / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry; D015257:DNA-Cytosine Methylases / Q000032:analysis* / Q000737:chemistry*; D019003:Deoxyribonuclease HpaII / Q000032:analysis* / Q000737:chemistry*; D019544:Equipment Failure Analysis; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D015203:Reproducibility of Results; D015183:Restriction Mapping / Q000295:instrumentation / Q000379:methods; D012680:Sensitivity and Specificity
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D004262:DNA Restriction Enzymes; D019003:Deoxyribonuclease HpaII