dna barcoding procedure  (Thermo Fisher)

Journal: Biophysical Journal

Article Title: Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay

doi: 10.1016/j.bpj.2018.08.013

Figure Lengend Snippet: DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

Article Snippet: Barcoding procedure Immobilized target DNA was incubated with 50 nM of each upstream and 50 nM of each downstream barcode (independent of the number of different barcode sequences used) and 14 Weiss units/mL of T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA) in freshly prepared ligation buffer (40 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 10 mM dithiothreitol, 0.5 mM ATP) for 1 h at 25°C.

Techniques: Microscopy, Labeling, Ligation, Binding Assay, Imaging

Journal: Biophysical Journal

Article Title: Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay

doi: 10.1016/j.bpj.2018.08.013

Figure Lengend Snippet: Enzymatic restriction confirms specificity of DNA barcoding. The number of barcode pairs detected in four-color single-target and four-target experiments is shown, indicated with the sequence at the ligation site (“GA,” “GC,” “GG,” and “GT”) and with “All,” respectively. Hatched bars show barcode pair counts after the addition of a restriction enzyme specific to the bound Cy3-Cy3 barcode pair. To see this figure in color, go online.

Article Snippet: Barcoding procedure Immobilized target DNA was incubated with 50 nM of each upstream and 50 nM of each downstream barcode (independent of the number of different barcode sequences used) and 14 Weiss units/mL of T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA) in freshly prepared ligation buffer (40 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 10 mM dithiothreitol, 0.5 mM ATP) for 1 h at 25°C.

Techniques: Sequencing, Ligation