A PCR-based method for site-specific domain replacement that does not require restriction recognition sequences.
A PCR-based method for site-specific domain replacement that does not require restriction recognition sequences.
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作者：
D. Zhong;S. Bajaj
我们描述了一种基于PCR的结构域替换方法，不需要限制性位点序列。我们举例说明的技术，其中第一个表皮生长因子（EGF1）样结构域的因子IX（FIX）被替换的EGF1样结构域的蛋白C。该方法采用四种寡核苷酸引物。两种为外部引物（正向引物A和反向引物B），含有FIX cDNA核苷酸侧翼序列。另外两个引物（正向引物C和反向引物D）指导蛋白C的EGF 1样结构域的PCR扩增，并且它们是在3'端含有蛋白C基因序列和在5'端含有FIX基因序列的杂交引物。因此，蛋白C的EGF 1样结构域的扩增片段（PCEGF 1片段）在两端侧接FIX基因序列。当该片段与FIX cDNA混合并进行一个PCR循环时，获得两种产物：一种含有连接至FIX cDNA序列上游的PCEGF 1片段，另一种含有连接至其EGF 1样结构域下游的FIX cDNA序列的PCEGF 1片段。使用引物A和D扩增第一产物，使用引物B和C扩增第二产物。两种产物均含有重叠序列，其允许在混合时退火。使用引物A和B通过PCR扩增退火产物。最终产物含有FIX cDNA，其中其EGF 1序列已被PCEGF 1序列取代。
We describe a PCR-based method for domain replacement that does not require restriction site sequences. We illustrate the technique in which the first epidermal growth factor (EGF1)-like domain of factor IX (FIX) is replaced by the EGF1-like domain of protein C. The method employs four oligonucleotide primers. Two are external primers (forward primer A and inverse primer B) and contain sequences flanking the FIX cDNA nucleotides. The other two primers (forward primer C and inverse primer D) direct the PCR amplification of the EGF1-like domain of protein C, and they are hybrid primers that contain sequences of protein C gene at the 3' end and of FIX gene at the 5' end. Thus the amplified fragment of EGF1-like domain of protein C (PCEGF1 fragment) is flanked by FIX gene sequences on both ends. When this fragment is mixed with FIX cDNA and subjected to one cycle of PCR, two products are obtained: one containing PCEGF1 fragment linked to FIX cDNA sequence upstream and the other containing PCEGF1 fragment linked to FIX cDNA sequence downstream of its EGF1-like domain. The first product is amplified using primers A and D, and the second product is amplified using primers B and C. Both products contain overlapping sequences, which allow annealing upon mixing. The annealed product is amplified by PCR using primers A and B. The final product contains FIX cDNA in which its EGF1 sequence has been replaced by the PCEGF1 sequence.