MIKULÁŠKOVÁ, Eva, Tomáš FÉR and Veronika KUČABOVÁ. The effect of different DNA isolation protocols and AFLP fingerprinting optimizations on error rate estimates in the bryophyte Campylopus introflexus. Lindbergia : journal of bryology. Copenhagen: Nordic bryological society, 2012, vol. 35, No 1, p. 7-17. ISSN 0105-0761.
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Basic information
Original name The effect of different DNA isolation protocols and AFLP fingerprinting optimizations on error rate estimates in the bryophyte Campylopus introflexus
Name in Czech Efekt rozdílných protokolů pro izolaci DNA a optimalizací protokolu pro AFLP fingerprinting na hodnocení chybovosti u mechu Campylopus introflexus
Authors MIKULÁŠKOVÁ, Eva (203 Czech Republic, guarantor, belonging to the institution), Tomáš FÉR (203 Czech Republic) and Veronika KUČABOVÁ (203 Czech Republic).
Edition Lindbergia : journal of bryology, Copenhagen, Nordic bryological society, 2012, 0105-0761.
Other information
Original language English
Type of outcome Article in a journal
Field of Study 10600 1.6 Biological sciences
Country of publisher Denmark
Confidentiality degree is not subject to a state or trade secret
WWW URL
RIV identification code RIV/00216224:14310/12:00059386
Organization unit Faculty of Science
Keywords in English AFLP optimization; bryophytes; Campylopus introflexus; DNA isolation; error rate assessment; PCR;
Tags AKR, bryophytes, invazive, rivok
Tags Reviewed
Changed by Changed by: Ing. Andrea Mikešková, učo 137293. Changed: 11. 4. 2013 19:12.
Abstract
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.
Links
MSM0021622416, plan (intention)Name: Diverzita biotických společenstev a populací: kauzální analýza variability v prostoru a čase
Investor: Ministry of Education, Youth and Sports of the CR, Diversity of Biotic Communities and Populations: Causal Analysis of variation in space and time
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