We have constructed a three-step platform technology called MultiCode ® PLx that eliminates the most technically challenging steps involved in multiplexed genetic analysis and compared it with the line probe assay in a clinical setting. MultiCode PLx uses one additional nucleobase pair constructed from the complementary nucleobases 2Ј-deoxy-isoguanosine (diG) 5 and 5-Me-iso-cytosine (iC). These nucleobases specifically recognize each other based on a different pattern of hydrogen bondings. We chose this pair because no other such pair is available commercially and their chemistries have been well explored. For example, iG and iC have been successfully used for both molecular recognition (13) (14) (15) and for site-specific incorporation (16 -18 ) . In addition, because iG:iC recognition is "orthogonal" to the naturally occurring nucleobase pairs, a strand of DNA that contains several iC/iG components can be constructed so that it will not hybridize to natural DNA. In complex reactions in which high concentrations of natural DNAs of known or unknown sequence exist, this orthogonal attribute allows specific molecular recognition to take place without interference. These additional nucleobases are used in each step of the PLx process: PCR, extension labeling, and liquid decoding. PCR primers are first designed to be target-specific and contain single iCs. The amplicons act as labeling templates for the target-specific extension (TSE) step. During that step, labels attached to diG triphosphate (diGTP) are incorporated site specifically into coded target-specific extenders (Fig. 1 ). The tags are short sequences (typically 10 nucleotides in length) assembled by use of a mixture of natural and nonnatural bases. The tags are designed to hybridize only to their perfect complements encoded on color-addressed microspheres. In the final step, decoding of the extension reactions is accomplished at room temperature by capture of the coded extenders on the addressed microspheres and reading of the reporter signal on a Luminex 100 instrument. All steps are carried out in the same reaction vessel without transfers or washings.