Performance of two-layer and four-layer linear CHA cascades that contain enzymatically synthesized substrate hairpins. (A) The performance of the two-layer linear cascade consisting of enzymatically synthesized eM1 and eA1 and chemically synthesized DN-pure eM2 and eA2. (B and C) Scheme and performance of the two-stage reaction of the four-layer linear cascade consisting of eM1, eA1, eM2, eA2, eM3, eA3, eM4, and eA4. Note that the two stages of reaction were carried out sequentially in different test tubes (main text).