NONREPRESENTATIVE PCR AMPLIFICATION OF VARIABLE GENE-SEQUENCES IN CLINICAL SPECIMENS CONTAINING DILUTE, COMPLEX-MIXTURES OF MICROORGANISMS

PCR amplification and DNA sequencing of the expression locus from Neis seria gonorrhoeae contained in urine sediments collected from experime ntally infected human subjects produced two observations. First, diffe rent pilin sequences were obtained when separate aliquots of the same sample were amplified and sequenced. In contrast, the same pilin seque nce was obtained when repeated amplifications were performed on indivi dual colonies grown from the clinical samples. Second, mixed sequences (i.e., more than one nucleotide at variable positions in the pilin ge ne sequence) were observed in both the direct clinical isolates and in dividual cultures grown from the isolates. These results suggest that when clinical samples are directly examined by PCR amplification and s equencing, multiple amplifications may be required to detect sequence variants in the sample and minority variant sequences will not always be detected.