GENERATION OF ENTIRE HUMAN PAPILLOMAVIRUS GENOMES BY LONG PCR-FREQUENCY OF ERRORS PRODUCED DURING AMPLIFICATION

Recently, several improvements of traditional PCR techniques have faci litated the amplification of significantly longer DNA target sequences . Here we report an improved method for amplification of entire human papillomavirus (HPV) genomes. Using rTth DNA polymerase, XL (Perkin-El mer, Foster City, CA), and the accompanying XL PCR buffer system, we h ave successfully amplified 8-kb genomes from -10 copies of input refer ence strain HPV16 DNA. This long PCR (LPCR) method was subsequently us ed to amplify the entire HPV16 genome from clinical specimens. The fid elity with which the rTth DNA polymerase XL. amplifies target sequence s under our chosen amplification conditions was estimated by partial s equencing of cloned LPCR products generated from cloned reference stra in HPV16 genomes. A region spanning the HPV16 E6, E7, and part of the E1 open reading frames (ORFs) was sequenced in 29 clones. A total of 3 3 nucleotide substitutions were observed in the 23.5 kb sequenced. Thi s corresponds to an error frequency of -one error per 700 bases. Final ly, LPCR methods were used to amplify entire, novel HPV genomes from c linical specimens. LPCR primer pairs were designed for amplification o f seven potentially novel HPV types. Amplicons of -8 kb were generated from five of the seven HPV types targeted. One of the LPCR-generated novel genomes, CP141, was subsequently cloned and a partial sequence w as determined.