Amplification Success Rate Research Articles

Transcriptional profiling on all open reading frames of Saccharomyces cerevisiae.

Open reading frames (6116) of the budding yeast Saccharomyces cerevisiae were PCR-amplified from genomic DNA using 12,232 primers specific to the ends of the coding sequences; the success rate of amplification was 97%. PCR-products were made accessible to hybridization by being arrayed at very high density on solid support media using various robotic devices. Probes made from total RNA preparations were hybridized for the analysis of the transcriptional activity of yeast under various growth conditions and of different strains. Experimental factors that proved critical to the performance, such as different RNA isolation procedures and the assessment of hybridization results, for example, were investigated in detail. Various software tools were developed that permit convenient handling and sound analysis of the large data quantities obtained from transcriptional profiling studies. Comprehensive arrays are being distributed within the European Yeast Functional Analysis Network (EUROFAN) and beyond.

A comparison of methods for DNA extraction from paraffin‐embedded tissue for microsatellite instability analysis by polymerase chain reaction

AbstractIn a prospective study, nuclear DNA was extracted from colorectal tumours and normal mucosa which had been fixed in buffered formalin and embedded into paraffin. DNA‐extraction was performed using three different methods: a commercial kit which was not especially created for this use; a known fast procedure without DNA‐cleaning steps; and a more conventional DNA‐preparation protocol with DNA‐cleaning. Using the polymerase chain reaction (PCR), DNA was amplified by being targeted onto two β‐globin fragments with different lengths (536 bp and 989 bp) and (CA)n repeats localized on chromosome 5q (D5S346) and chromosome 17p (TP53CA) with a length of about 100 bp for detection of microsatellite instability. The success rate of microsatellite amplification was 100% with all methods. The 536 bp β‐globin fragment could be amplified with a success rate ranging from 40% to 100%. The amplification of the 989 bp β‐globin fragment was unsuccessful. Significant differences were observed between the three methods in the final DNA concentration and DNA yield. In microsatellite instability studies of paraffin‐embedded tissues, the investigator can expect a high success rate of nearly 100% using any of the described methods.

Characterization of the host genotype and fungal diversity in Scots pine ectomycorrhiza from natural humus microcosms using isozyme and PCR‐RFLP analyses

SUMMARYThe early development of indigenous and introduced Scots pine (Pinus sylvestris L.) ectomycorrhiza in natural forest humus was examined using molecular fingerprinting techniques. Non‐mycorrhizal or mycorrhizal (Suillus bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch ex Fr.) Fr.) seedlings were placed in transparent two‐dimensional microcosms on a thin layer of sieved humus to allow observation of ectomycorrhizal development and fungal growth. Twelve ectomycorrhizal morphotypes, mainly based on colour, gross morphology and outer mantle structure, were identified over a 3‐month period. No successional trends in mycorrhiza formation were observed. For further characterization, individual ectomycorrhiza representing the different morphotypes were subjected to analyses of esterase (EST) isozyme profiles and RFLPs of the internal transcribed spacer (ITS) from fungal rRNA genes following amplification using PCR. All 10 morphotypes analysed displayed different esterase isozyme profiles, and the characteristic s. bovinus species diagnostic band (S. b. EST8) was detected in two white morphotypes. Ten Pink and Black ectomycorrhiza were all separated into one and three groups, respectively. A fast‐running plant‐specific polymorphic locus (Z) was also confirmed in most ectomycorrhizal morphotypes. Diagnostic species‐specific (S. bovinus and Pink) EST bands were detected in intact external mycelium colonizing soil. Successful amplification of the ITS from individual ectomycorrhiza, of eight different morphotypes, was found to be mainly influenced by the DNA template concentration. Different RFLPs (Hinf I, Mbo I and Hha I) of the ITS placed the white morphotypes into two groups, one corresponding to S. bovinus, in agreement with the esterase fingerprinting. By contrast, Pink and Beige morphotypes displayed different EST profiles but nearly identical RFLP fingerprints. The ITS amplification success rate of standardized DNA template concentrations from 10 individual Pink and Black ectomycorrhiza was 90 and 50%, respectively. In all successful amplifications, the black morphotype yielded the smallest ITS fragment, similar to earlier reports for Cenococcum geophilum, that gave identical RFLPs. The ecological significance of the observed mycorrhizal diversity and combined application of these two identification methods is discussed.