Formation of template-switching artifacts by linear amplification

Abstract

Linear amplification is a method of synthesizing single-stranded DNA from either a single-stranded DNA or one strand of a double-stranded DNA. In this protocol, molecules of a single primer DNA are extended by multiple rounds of DNA synthesis at high temperature using thermostable DNA polymerases. Although linear amplification generates the intended full-length single-stranded product, it is more efficient over single-stranded templates than double-stranded templates. We analyzed linear amplification over single- or double-stranded mouse H-ras DNA (exon 1-2 region). The single-stranded H-ras template yielded only the intended product. However, when the double-stranded template was used, additional artifact products were observed. Increasing the concentration of the double-stranded template produced relatively higher amounts of these artifact products. One of the artifact DNA bands could be mapped and analyzed by sequencing. It contained three template-switching products. These DNAs were formed by incomplete DNA strand extension over the template strand, followed by switching to the complementary strand at a specific Ade nucleotide within a putative hairpin sequence, from which DNA synthesis continued over the complementary strand.

FIGURE 1

Structure of pWT1.

FIGURE 2

Synthesis of single-stranded (ss) H-ras DNA by linear amplification of BamHI-digested pWT1. The indicated DNA band indicated was extracted for use as single-strand H-ras template.

FIGURE 3

Formation of artifact DNA products by linear amplification. Single-stranded H-ras template yielded the desired products with primers C61F or C61R (a), whereas double-stranded H-ras template yielded the desired products as well as artifact DNAs (Art 1–4) (B, c). D : The sites of annealing of the primers used in this study. The top strand was designated as the Watson strand (W) and the bottom strand was designated the Crick strand (C). An alternative definition also exists in which the antisense or the transcription template strand is designated as the Watson strand and the sense strand is designated as the Crick strand. Art, artifact.

FIGURE 4

Linear amplification primer walking analysis of artifact 1. The artifact product generated by linear amplification of double-stranded H-ras DNA with the C61R primer was subjected to linear amplification with a number of primers that anneal to the H-ras sequence. Only C61R and C61Rv yielded linear amplification products with artifact 1.

FIGURE 5

Sequence analysis of artifact 1. Results of sequence analysis from two experiments are joined together. The electropherogram showed overlapping peaks in sequences previous to nt 134, but none at the downstream sequences. The indicated sequences were readable within the overlapping peaks. The nucleotide 133 should have been a g, which is not observed in the electropherogram.

FIGURE 6

Illustration of the template-switched products of artifact 1. At 80ºC, the H-ras gene segment may contain two hairpin-type secondary structures (the locations shown with segmented columns, and the sequences indicated on top).