Integration of multiple PCR amplifications and DNA mutation analyses by using oligonucleotide microchip

We have developed a method for parallel independent on-chip amplification a nd the following sequence variation analysis of multiple DNA regions direct ly using microchip with an array of nanoliter gel pads containing specific sets of tethered primers. The method has three key features, First, DNA to be amplified is enriched at gel pads by its hybridization with immobilized primers, Second, different sets of specific primers are immobilized within various gel pads, and primers are detached within gel pads just before poly merase chain reaction to enhance the amplification. A gel pad may contain a n additional permanently immobilized dormant primer that is activated to ca rry out the allele-specific primer extension reaction to detect mutations. Third, multiple polymerase chain reactions are confined within nanoliter ge l pads covered and separated from each other with mineral oil. The method w as applied to simultaneously identify several abundant drug-resistant mutat ions in three genes of Mycobacterium tuberculosis. (C) 2001 Academic Press.