tth endoiv  (New England Biolabs)

Journal: Applied and Environmental Microbiology

Article Title: Capillary Electrophoresis-Based Functional Genomics Screening to Discover Novel Archaeal DNA Modifying Enzymes

doi: 10.1128/AEM.02137-21

Figure Lengend Snippet: Evaluating the efficiency of functional genomic screening by identification of a known uracil cleavage enzyme. (A) A 5′-FAM labeled DNA substrate containing a dU was incubated with a control fosmid lysate (neg), UDG, or fosmid lysate followed by Tth EndoIV to cleave AP sites. Examples of fosmid lysates that contained uracil cleavage activities include Well H4, I9, O9, and P16. (B) A 384-well fosmid plate was screened for UDG activity yielding 12 positive fosmid lysate hits. Colored squares represent 14 fosmid wells that contain the UDG gene and green squares confirm that UDG activity was observed, while red squares lacked detectable UDG activity. (C) Fosmids containing UDG activity mapped to a region in the T. kodakarensis genome containing the UDG gene.

Article Snippet: To confirm conversion of all dU sites to AP sites, AP site DNA was incubated with Thermus thermophilius Endonuclease IV 1 (Tth EndoIV, New England Biolabs) at 37°C for 5 min. For DNA ligation assays, 1 μM 5′-VIC-DNA ligation acceptor oligonucleotide was annealed with 1.25 μM ligation donor and 1.5 μM DNA ligation template as described above.

Techniques: Functional Assay, Labeling, Incubation, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens

doi: 10.3390/ijms19020524

Figure Lengend Snippet: TEC-LAMP mechanism. ( A ) TEC-LAMP oligonucleotide components, Tth endonuclease IV enzyme and dsDNA template with oligonucleotide targets highlighted. ( B ) Temperature enabled dsDNA dissociation followed by primer and TEC primer/probe hybridization to corresponding targets. ( C ) Inner primer strand displacement extension, via Bst polymerase, forms dsDNA. Outer primer strand displacement extension dissociates this newly formed dsDNA, forming inner primer linked ssDNA. ( D ) The complementary sections of this newly formed inner primer linked ssDNA hybridize, forming loop structures. The abasic site of the TEC primer/probe is now in dsDNA form, and thus, cleaved by the Tth endonuclease IV enzyme. TEC primer/probe, inner and outer primers hybridize upstream of the stem loop structures. ( E ): Inner primer strand displacement extension forms dsDNA and displaces the downstream stem loop structures, fully dissociating the TEC primer/probe fluorophore and quencher, producing fluorescence. Outer primer strand displacement extension displaces the newly formed dsDNA, producing inner primer linked ssDNA. ( F ) The complementary sections at each end of the newly formed inner primer linked ssDNA hybridize and form loop structures. These double looped DNA templates are targeted by the TEC primer/probe, inner and loop primers, leading to rapid self-primed exponential amplification with increased cleavage and fluorescence events.

Article Snippet: The final TEC-LAMP reaction contained 1× Isothermal Amplification Buffer (New England Biolabs, Hitchin, UK), 6 mM MgSO4 (Roche Diagnostics), 1.4 mM deoxynucleotide triphosphate set (New England Biolabs), S. pneumoniae oligonucleotides [2.6 µM reverse inner, 1.3 µM forward inner and TEC primer/probe, 0.65 µM forward and reverse loop, 0.325 µM forward and reverse outer], N. meningitis oligonucleotides [1 µM reverse inner, 0.5 µM forward inner and TEC primer/probe, 0.25 µM forward and reverse loop, 0.125 µM forward and reverse outer], H. influenzae oligonucleotides [1.2 µM reverse inner, 0.6 µM forward inner and TEC primer/probe, 0.3 µM forward and reverse loop, 0.15 µM forward and reverse outer], IAC oligonucleotides [0.6 µM reverse inner, 0.3 µM forward inner and TEC primer/probe, 0.15 µM forward and reverse loop, 0.075 µM forward and reverse outer], 8 U Bst 2.0 WarmStart DNA polymerase (New England Biolabs), 15 U Tth endonuclease IV (New England Biolabs), 1 µL IAC template (50 copies), 1 µL DNA template (1–3 templates) or 1 µL molecular grade water for no template control (NTC) reactions, and molecular grade water to give a final volume of 25 µL.

Techniques: Hybridization, Fluorescence, Amplification