Reducing the number of microlocations in oligonucleotide microchip matrices by the application of degenerate oligonucleotides

The application of degenerate oligonucleotides to DNA Sequencing by Hybridi sation with Oligonucleotide Matrix (SHOM) is proposed. The use of degenerat e oligonucleotides is regarded as an example of pooling methods that are su itable for various laboratory procedures requiring numerous samples to be a ssayed. As each DNA sequence coded by four letters (A, G, C, T) may be defi ned by two sequences: a sequence coded by W and S (W-weak-A or T, S-strong- G or C) and a sequence coded by R and Y (R-purine-A or G, Y-pirymidine-T or C), n(4) n-nucleotide sequences may be defined with the help of 2 x n(2) s equences. In the place of the originally described microchip matrix compose d of all possible unambiguous octanucleotides (4(8) = 65 536) attached to t he equal number of 65 536 microlocations a matrix composed of 512 microloca tions containing 256 2(8)-degenerate octanucleotides is proposed. The matri x contains all 256 possible octanucleotides coded by W and S variations and ail 256 possible octanucleotides coded by R and Y variations. The 512 256- degenerate octanucleotides allows to retrieve the same information as 65 53 6 unambiguous octanucleotides. A variant of the DNA sequence reconstruction method applicable to this system is presented. The use of degenerate oligo nucleotides also gives the possibility to apply matrices composed of longer oligonucleotides without increasing the number of microlocations in matric es, which would enable increasing the length of unambiguously reconstructed sequence, e.g. a matrix comprising 131 072 16-mer oligonucleotides i.e. 65 536 65 536-fold degenerate oligonucleotide coded by W and S Variations and 65 536 65 536-fold degenerate oligonucleotide coded by R and Y variations could replace one matrix comprising all possible unambiguous 16-mer oligonu cleotides (ca. 4.3 x 10(9)). (C) 1999 Academic Press.