Direct amplification of the entire ITS region from poorly preserved plant material using recombinant PCR

Abstract

Sequences of the internal transcribed spacers (ITS) of the nuclear ribosomal DNA are important molecular markers in phylogenetic analyses. To obtain sequences from herbarium material in which DNA often is severely degraded, the ITS region has to be amplified in two steps. Two methods that reduce bench time and reagents used are described. (i) Separately amplified preparations of subunits ITS-1 and ITS-2 are combined before purification. The presence of two fragments in the sequencing reaction does not impair the quality of sequences. (ii) Newly designed internal primers amplify partly overlapping regions of the two subunits. A combination of these internal primers with the external primers in one PCR allows the amplification of the entire ITS region even when degraded DNAs are used. This recombinant PCR approach, taking into account the +A bases added by several Taq DNA polymerases, will also be useful with other marker regions used in molecular phylogenetics.