Mutation detection by ligation to complete n-mer DNA arrays

A new approach to comparative nucleic acid sequence analysis is described that uses the ligation of DNA targets to high-density arrays containing complete sets of covalently attached oligonucleotides of length eight and nine. The combination of enzymatic or chemical ligation with a directed comparative analysis avoids many of the intrinsic difficulties associated with hybridization-based de novo sequence reconstruction methods described previously. Double-stranded DNA targets were fragmented and labeled to produce quasirandom populations of 5' termini suitable for ligation and detection on the arrays. Kilobase-size DNA targets were used to demonstrate that complete n-mer arrays can correctly verify known sequences and can determine the presence of sequence differences relative to a reference. By use of 9-mer arrays, sequences of 1.2-kb targets were verified with >99.9% accuracy. Mutations in target sequences were detected by directly comparing the intensity pattern obtained for an unknown with that obtained for a known reference sequence. For targets of moderate length (1.2 kb), 100% of the mutations in the queried sequences were detected with 9-mer arrays. For higher complexity targets (2.5 and 16.6 kb), a relatively high percentage of mutations (90% and 66%, respectively) were correctly identified with a low false-positive rate of <0.03 percent. The methods described provide a general approach to analyzing nucleic acid samples on the basis of the interpretation of sequence-specific patterns of hybridization and ligation on complete n-mer oligonucleotide arrays.