Linear amplification followed by single primer polymerase chain reaction to amplify unknown DNA fragments: complete nucleotide sequence of Oropouche virus M RNA segment

The whole nucleotide sequence of Oropouche virus medium (M) RNA, Orthobunyavirus genus, Bunyaviridae family, was obtained using a new genomic amplification method. This method is based on the use of a single and specific primer of high melting temperature in a linear amplification (LA), followed by a single primer polymerase chain reaction (LASP-PCR). The LASP-PCR was used to walk along the Oropouche M RNA completing the sequence in seven successive walks. The amplicons obtained in each walking step ranged from 300 to I 100 bp; however, amplicons of up to 3970 bp was obtained when the extension time of the LASP-PCR was increased from 120 to 270 s. This method was tested successfully for Escherichia coli and cytomegalovirus obtaining amplicons of up to 2130 and 6500 bp, respectively, indicating that it can be applied to amplify unknown DNA sequences adjacent to a short stretch of known sequence of more complex genomes. (C) 2003 Elsevier B.V. All rights reserved.