The Effects Of Single Primer PCR And The Cloning Of Promoter Of Toc33 Gene In Brassica Napus

Single primer PCR was a method of chromosome walking to isolate sequences flanking a known DNA sequence with only one primer. It utilized the character that mismatch occurred between the primer and the template under low stringency. And a primer was employed in PCR where a low annealing temperature allowed this primer to bind with a known sequence perfectly on one strand and partially homologous sequences on another strand flanking the target sequence. In order to research how the length of primer affect the efficiency of amplification, three groups of short primers (6-, 8- and 10-mer primers) and a group of long primers (19- and 22-mer primers) were designed to amplify the target sequence in Brassica napus. Then the PCR products were assayed by electrophoresis and the bright bands were excised from the agarose gel. Afterwards, the PCR products and the recovered products were identified with a pair of nested primers. In despite of long primer PCR reaction performed under low stringency or high stringency or by two steps, the assay showed that the PCR products which were clear and bright did not include the target sequence. Some products amplified by 6-mer or 8-mer primers were dispersive and faint, and the rest were not assayed by electrophoresis at all. On the contrary, the PCR products, amplified by the use of eight out of the ten 10-mer primers, demonstrated comparatively clear and various bands, and then the products of seven primers were identified as positive. So the total positive rate was 70 percent.Moreover, 17 out of 34 recovered bands were positive. It implied that the average...