Reproducibility of random amplified polymorphic DNA (RAPD) analysis among laboratories.

Abstract

Random amplified polymorphic DNA (RAPD) analysis appears to offer a cost-and time-effective alternative to restriction fragment-length polymorphlsm (RFLP) analysis. However, concerns about the ability to compare RAPD results from one laboratory to another have not been addressed effectively. DNA fragments that were amplified by five primers and shown to be reproducibly polymorphic between two oat cultivars (within the Ottawa laboratory) were tested in six other laboratories in North America. Four of the six participants amplified very few or no fragments using the Ottawa protocol. These same participants were able to generate a considerable number of amplified fragments by using their own protocols. The reproducibility of results among laboratories was affected by two factors. First, different laboratories amplified different size ranges of DNA fragments, and, consequently, small and large polymorphic fragments were not always reproduced. Second, although reproducible results were obtained with four of the primers, reproducible resuits were not obtained with the fifth primer, using the same reaction conditions. It is suggested that if the overall temperature profiles (especially the annealing temperature) in-MATERIALS AND METHODS RAPD primers were obtained from Dr.

Assessment of genomic instability in breast cancer and uveal melanoma by random amplified polymorphic DNA analysis

“…For many, it is also confusing that the several groups have acquired different experiences with random priming during their optimization procedures. Most researchers agree that the type of thermocycler 12,13 and the type of polymerase 14 used in RAPD assays contribute to the variation in the PCR results. The underlying reasons are suboptimal thermocycler performance and the differences between the various strains/sources of the polymerases, the variable properties of the enzymes (e.g., proofreading activity, processitivity and Mg 2ϩ optimum), the different unit definitions, the variations in the concentrations of buffer ingredients and pH and the presence or absence of additives (e.g., the various stabilizers).…”

Randomly amplified polymorphic DNA typing: a useful tool for rapid epidemiological typing ofKlebsiella pneumoniae

“…a band is only considered significantly if it is present in a duplicate profile [22]. In a recent study, seven laboratories across North America produced varied RAPD profiles from oat cultivar DNA despite using a standardized protocol [23]. If RAPD typing of K. pneumoniae is to have widespread use, its inter-laboratory reproducibility will therefore require further investigation.…”

DNA analyses and their applications in plant breeding

“…RAPD markers are in general dominant, thereby they have a lower information content than codominant markers in the linkage analysis of F 2 populations (WIL- LIAMS et al 1990). PENNER et al (1993) reported on difficulties in obtaining identical band patterns from the same set of primers and materials among different laboratories. In their study the type of thermocycler used for RAPD analysis seemed to be a key determinant of the reproducibility of band patterns.…”