Formation of template-switching artifacts by linear amplification.
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.
Chakravarti
Dhrubajyoti
D
Eppley Institute for Research in Cancer and Allied Diseases, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA. dchakrav@unmc.edu
Mailander
Paula C
PC
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D017931:DNA Primers / Q000235:genetics; D004277:DNA, Single-Stranded / Q000138:chemical synthesis* / Q000235:genetics; D011905:Genes, ras; D051379:Mice; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*
D017931:DNA Primers; D004277:DNA, Single-Stranded