Publication details

The effect of different DNA isolation protocols and AFLP fingerprinting optimizations on error rate estimates in the bryophyte Campylopus introflexus



Year of publication 2012
Type Article in Periodical
Magazine / Source Lindbergia : journal of bryology
MU Faculty or unit

Faculty of Science

Field Botany
Keywords AFLP optimization; bryophytes; Campylopus introflexus; DNA isolation; error rate assessment; PCR;
Attached files
Description Amplified fragment length polymorphism (AFLP) has become a standard method for investigating genetic variation in plants. Nevertheless, only a few applications in bryophytes have been published and there is still a need to optimize the method. We optimized DNA isolation and AFLP protocols for Campylopus introflexus (Hedw.) Brid. As DNA quality is crucial for successful AFLP analysis, three different DNA extraction protocols were compared and the Invisorb Plant Mini Kit produced the highest DNA amount and purity. Newly grown stems gave the purest DNA (absorbance at the wavelengths of 260 nm and 280 nm was 1.86). However, banding patterns obtained from dry herbarium specimens (up to two years old) corresponded with those from fresh material at a similarity level of 94.71%. The replicability of AFLP profiles was not dependent on the way plants were stored. We compared commercial kits and the influence of modifications of the protocols for obtaining reliable results. We tested the reproducibility of AFLP fingerprints produced by the final optimized protocol. An increased amount of restriction enzymes and prolonged restriction and ligation of up to 10 h were the most important modifications for improving results. The modified protocol was applied to 30 samples and four selective primer combinations, and gave an average genotyping error rate of 0.0451.
Related projects:

You are running an old browser version. We recommend updating your browser to its latest version.

More info