Parallel sequencing of several nucleic acids, useful e.g. in gene expression analysis, using irreversibly immobilized amplification primers

摘要

Parallel sequencing of at least two different nucleic acids (NA), present in a mixture, is new. Parallel sequencing of at least two different nucleic acids (NA), present in a mixture. At least one pair of primers is immobilized irreversibly on a surface and treated with an NA mixture containing molecules that can hybridize to both primers. The immobilized primers are extended, in complementary fashion, to form a counter strand, resulting in formation of secondary NA (sNA). The surface is freed of NA that is not irreversibly bound and the sNA amplified to form tertiary nucleic acid (tNA). Counter-strands (gtNA) of tNA are prepared and extended by a single nucleotide (nt) in which the 2'- or 3'-hydroxy is protected, and which is detectably labeled. The incorporated nt is identified, the protecting group removed and the label either removed or altered. The single-nt extension procedure is repeated until the required sequence information has been obtained. Independent claims are also included for the following: (1) similar method in which tNA is treated so that it is bound to the surface only through the 5'-end of one strand, then cut with a type IIS restriction enzyme (RE) to generate 3' or 5' overhangs, determining one or more bases in these overhangs, ligating linkers to the free ends (these linkers include a recognition site for type IIS RE), treating again with RE that recognizes the site introduced in the linker and repeating the process as required; (2) apparatus for performing the new process; (3) method for localized amplification of NA, comprising the new process as far as amplification to produce tNA; and (4) surface-bound library of NA produced by the method for localized amplification of NA.