tth endonuclease iv  (New England Biolabs)

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