Journal: Nucleic Acids Research
Article Title: Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments
Figure Lengend Snippet: Selector construct and circularization reaction principles. ( A ) The selector consists of two oligonucleotides, one selector probe of ∼70 nt and one general vector oligonucleotide of 34 nt. The selector probe has two target-specific ends (black) of ∼18 nt complementary to the selected target and a primer-pair motif (gray) of 34 nt. The vector oligonucleotide is complementary to the primer-pair motif (gray). ( B ) The circularization procedure starts with digestion of the DNA to generate targets with defined ends. The digested DNA is then combined with selectors and the mixture is denaturated, allowing the selectors to hybridize to their respective targets. This can be performed in two different ways: (i) a selector probe hybridizes to both ends of the selected target and the ends are connected to the vector by ligation, or (ii) a selector probe hybridizes with one end to the 3′ end of the selected target and the other end to an internal sequence of the target, forming a branched structure that can be cleaved by the endonucleolytic activity of Taq DNA polymerase. Both ends of the selected single-stranded target are now ready for ligation to the vector oligonucleotide, forming a closed circular molecule. The circularization mixture is then exonuclease-treated and finally PCR-amplified using a common primer pair.
Article Snippet: To enrich for circularized DNA by degrading linear strands including selectors, 10 μl of the circularization mixtures (0.4 μg DNA) were then added to a 10 μl mixture of 5 U Exonuclease I (New England Biolabs), 110 mM Tris–HCl, pH 9.0, 3 mM MgCl2 and 0.2 μg BSA and incubated for 2 h at 37°C, followed by 95°C for 10 min. Amplification was performed using 4 μl of each exonuclease-treated circularization reaction (80 ng DNA each) added to 17 μl mixture of 1× PCR buffer (Invitrogen), supplemented with 0.5 U Platinum Taq DNA polymerase (Invitrogen), 0.25 mM dNTP, 0.4 μM Cy-3-labeled forward and reverse primer, respectively, and 2 mM MgCl2 .
Techniques: Construct, Plasmid Preparation, Ligation, Sequencing, Activity Assay, Polymerase Chain Reaction, Amplification