“GFP-Display,” an Easy Detection Method for Single Amino Acid Changes in a Target Polypeptide: Application to Random Mutagenesis
✍ Scribed by Takashi Aoki; Toshiyuki Tahara; Hiroyoshi Fujino; Hiroyuki Watabe
- Publisher
- Elsevier Science
- Year
- 2002
- Tongue
- English
- Weight
- 179 KB
- Volume
- 300
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
ecules which partially or completely lack the GC clamp (data not shown). PCR amplicons using these primers are indistinguishable from others produced with HPLC-purified oligonucleotides (Fig. 1A, compare lanes 1-4 with 5-8). However, the heterogeneity of PCR products produced with contaminated primers impairs TGGE analysis. The resulting poor resolution of the different amplicons in a temperature gel gradient is probably due to a different GC clamp length for identical 16S rDNA fragments (Fig. 1B).
In summary, purification of the oligonucleotides (using HPLC for example) is absolutely required for a correct TGGE analysis of their PCR products.
Acknowledgments. The analysis presented here was performed in projects supported by Grant BIO4-CT98-0483 from the Biotechnology Programme of the EU and from the Comisio ´n Asesora de Investigacio ´n Cientı ´fica y Te ´cnica Grant BIO99-0905. We thank Antonio Me ´rida and Antonio Lario for their assistance in the synthesis of oligonucleotides, Miss Nuria Mun ˜oz for technical assistance, and Pieter van Dillewijn and Jose Ignacio Jime ´nez-Zurdo for critical reading of the manuscript.