Abstract:

【Objective】In this experiment, Junzao was used as materials based on previous studies, to establish a SON-PCR technique for isolation unknown flanking sequence of Chinese jujube. 【Method】 The original two-stage second round of the SON-PCR reaction was improved to two-step: firstly, the PCR results of primary reaction were used as template, single nested primer was used to drive the 5′selected linear amplification; then, a small amount of the primer designed for primary reaction and primer designed for secondary reaction was added to specially amplificating secondary reaction. The specificity and efficiency were further enhanced, and the dilution of the original secondary reaction PCR template was adjusted to 10ng?μL-1 of the primary reaction result. 【Result】 Finally, the test data using the system to a 626bp fragment of 3 'flanking sequences were amplified to obtain a 934bp encoding leucine-rich repeat sequence related fragment. The universality of this technique was proved by using two other fragments.【Conclusion】The method can efficiently amplify unknown sequence flanking sequence.

Key words: Chinese jujube, gene-related, SON-PCR, flanking sequence