tth dna polymerase  (New England Biolabs)

Journal: Journal of Translational Medicine

Article Title: Novel serum small extracellular vesicle miRNAs with multi-target RCA-CRISPR sensor for liver cancer detection

doi: 10.1186/s12967-025-07628-3

Figure Lengend Snippet: The miRNA detection principle of RCA-CRISPR. The circular probe consists of sequences complementary to both the target miRNA and the crRNA. The miRNAs hybridize with the phosphorylated circular probe, and T4 DNA ligase catalyzes the covalent binding of the 3’ and 5’ ends, forming a closed DNA loop. This loop serves as a template for the RCA reaction, which is initiated by phi 29 DNA polymerase. The RCA product, containing multiple crRNA-complementary sequences, activates the nucleolytic activity of Cas12a upon hybridization with the Cas12a-crRNA complex. Activated Cas12a cleaves adjacent FAM-BHQ1-labeled ssDNA reporters, separating the fluorophore from the quencher and producing a quantifiable fluorescent signal that reflects the presence and abundance of the target miRNA

Article Snippet: Subsequently, 2 μL of dNTPs (10 mM each) (#4043, TaKaRa, Japan), 0.3 μL of phi 29 DNA polymerase (10 U/μL) (#M0269L, New England Biolabs, USA), 3 μL of 10 × phi 29 DNA polymerase buffer (#M0269L, New England Biolabs, USA), 1 μL of BSA solution (100 μg/mL) (#M0269L, New England Biolabs, USA), and 3.7 μL of DEPC-treated water were added to initiate the RCA reaction, which was carried out at 30 °C for 1.5 h. For the cleavage reaction, 0.5 μL of Cas12a enzyme (2 μM) (#M0653T, New England Biolabs, USA), 1 μL of crRNA (3 μM), 2 μL of Fluorophore-quencher (F-Q) reporter (10 μM), 5 μL of 10 × NEB buffer (#B7202S, New England Biolabs, USA), and 11.5 μL of DEPC-treated water were added to the mixture and incubated at 37 °C for 30 min.

Techniques: CRISPR, Binding Assay, Activity Assay, Hybridization, Labeling