Authenticase™는 이중 가닥 DNA의 단일 염기와 2개 이상(1~10 bp)의 염기쌍 DNA가 불일치하는 영역을 인식하고 절단하는 structure-specific nucleases mixture입니다. Authenticase™는 PCR 증폭 단계 전에 오류를 제거하여 oligo-based PCR gene assembly의 오류 수정 시약으로 사용할 수 있습니다.
- C/C, T/C, A/C, T/G, G/G, T/T 및 A/A와 같은 단일 염기 불일치 인식 (G/A 제외)
- Insertions 및/또는 deletions 인식
- Error-correction in oligonucleotide synthesis
- Mismatch Detection Assay
|NEB #||Component Name||Stored at (°C)||Amount||Concentration|
|M0689S||Authenticase™||-20||1 x 0.025 ml||25 reactions|
|Authenticase DNA Annealing Buffer||1 x 0.7 ml||5 X|
|Authenticase Reaction Buffer||1 x 0.5 ml||10 X|
1X Authenticase Reaction Buffer
Incubate at 42°C
Figure 1. Mechanism of Authenticase
Authenticase cleaves dsDNA carrying mismatches or indels, leaving either 3′ or 5′ overhangs.
Figure 2. Applications of Authenticase
Authenticase is a mixture of structure-specific nucleases capable of recognizing and cleaving outside mismatch and indel (insertion and/or deletion) regions, ranging from 1–10 basepairs (bp) on double-stranded DNA. The formulation has limited non-specific activity on homoduplex regions of DNA. Authenticase can be used as an error-correction reagent in oligo-based PCR gene assembly by enzymatically removing “mistakes” prior to the final renaturation and amplification step. Alternatively, Authenticase can replace T7 Endonuclease I in the mismatch detection assay used to assess the efficiency of genome editing (S1 is the starting material. P1 and P2 are products of Authenticase digestion.).
A reaction is defined as 1 µl of Authenticase in a 20 µl reaction containing 1X Authenticase Reaction Buffer, a mixture of 0.33 pmol of 60-mer heteroduplex of A/C, T/G and 2 bp indel (insertion and/or deletion) mismatches, respectively. After incubation at 42°C for 30 min, >90% of the substrate DNA was digested, as determined by 4% agarose E-gel. More details of the Authenticase protocol and usage can be found in the product manual.
Authenticase is a proprietary mixture of structure-specific nucleases capable of recognizing and cleaving outside mismatch and indel (insertion and/or deletion) regions, ranging from 1-10 base pairs (bp) on double-stranded DNA. By enzymatically removing “mistakes” prior to the final renaturation and amplification step, Authenticase can reduce errors in oligo-based PCR gene assembly.
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