| abstract |
The invention comprises a two-step process for analysis of polynucleotides by chain extension of multiple oligonucleotide primers attached by 5'-linkages to solid supports by first performing PCR of the sample to be analysed in the presence of multiple oligonucleotides in solution, the nucleotide sequences of both sets of oligonucleotides being similar or identical. This produces immobilised single-stranded polynucleotides containing genetic sequence data derived from sample molecules. In a second step, support-bound polynucleotides are interrogated by hybridisation with a single labelled oligonucleotide probe or by second-strand synthesis with a primer-dependent polymerase using an oligonucleotide primer and nucleotide monomers, in which either or both of the primer and nucleotide monomers are labelled. Incorporation of label demonstrates the presence of two separate defined-sequence primers within the sample polynucleotide. The presence or absence within the sample of multiple combinations of primers are demonstrable in a single experiment by use of suitable apparatus, which may take the form of an oligonucleotide array. Multiple arrays may be accommodated in a 96-well format, allowing all possible combinations of 96 different primers to be generated simultaneously. If sequence data for the sample DNA is available in advance of the analysis, the results may be predicted by computer modelling, allowing design of highly-specific automated DNA-based assay procedures. |