耐热性碱性磷酸酯酶作为非放射性标记物的研究

Study on Thermostable Alkaline Phosphatase as Non radioactive Label

通过生物素与亲和素－酶复合物系统或地高辛与抗地高辛－酶复合物系统可把酶间接标记到探针上．Ｒｅｎｚ等通过不同的化学方法直接把酶标记到探针上［１～３］．耐热性碱性磷酸酯酶ＦＤ－ＴＡＰ（ｔｈｅｒｍｏｓｔａｂｌｅａｌｋａｌｉｎｅｐｈｏｓｐｈａｔａｓｅ）具有耐...

Using glutaraldehyde as the crosslinking agent the thermostable alkaline phosphatase could be crosslinked to single stranded DNA. The optimal crosslinking pH was about 6.5, the optimal concentration of glutaraldehyde was about 0.25%～0.45% and the optimal mass ratio of enzyme to DNA was 80. However, only 25% of the added enzyme was cross linked to DNA, therefore the actual mass ratio of crosslinked enzyme to DNA was about 20. Such DNA enzyme complex was tested for spot hybridization performed at higher temperature. After color development 5 pg of complementary target DNA could be visualized. 0.2 μg of foreign DNA (λ phage,4×10 4 fold of the amount of the target) did not interfere with the hybridization. The procedure is simple, convenient and rapid and can be applied to routine dot blot.