Cross‐species amplification of glutamine synthetase cDNA by polymerase chain reaction with degenerate primers

Abstract

We have developed and optimized a consistent polymerase chain reaction (PCR)‐based strategy to quickly obtain specific sequence information on novel plant glutamine synthetase (GS, EC 6.3.1.2) cDNAs. Two sets of degenerate primer pairs were designed to discriminate regions conserved in either any kind of GS messenger or exclusively in those for the chloroplastic GS. Novel GS cDNA sequences were successfully amplified from total RNA obtained from 14 different monocotyledonous and dicotyledonous plants. The procedure, coupled with a further restriction analysis, allowed us to uncover the presence of GS cDNA polymorphism, which most likely stems from the different GS gene family members within a single species. Contrary to previously reported strategies in other systems, GS cDNA oligonucleotide primers were designed keeping the degeneracy level to a minimum, together with a high melting temperature. This approach proved to be particularly effective, generating high yields of the expected products without requiring extra nested amplification steps or time‐consuming optimization steps for each species GS cDNA amplification. Different clones containing sequence information from either the coding or the 3′‐untranslated regions were further sequenced and characterized, confirming the high sequence identity and size uniformity the of GS cDNAs across higher plant species. Therefore, this approach is proposed as a stand‐alone procedure to quickly determine the sequence of unknown GS cDNAs, as well as to speed up and complement classical molecular cloning methodologies.