PMC:99051 / 14873-18762 JSONTXT

{"project":"2_test","denotations":[{"id":"11882253-6300841-10250602","span":{"begin":1312,"end":1314},"obj":"6300841"},{"id":"11882253-2057365-10250603","span":{"begin":2152,"end":2154},"obj":"2057365"},{"id":"11882253-461182-10250604","span":{"begin":2155,"end":2157},"obj":"461182"}],"text":"2 – obtaining patterns from a single clone analysis\nWe randomly picked single clones of 96 colonies from a murine wounded skin library and compared the analyzed restriction fingerprints with the corresponding, experimentally determined cDNA sequence of the vectors. Vectors containing no cDNA inserts produced no signals in the detection window of 45 to 900 base pairs and incomplete or short cDNA inserts yielded only signals of fragments derived from restrictions within the cDNA sequence. This demonstrated that the restriction free region next to the cDNA cloning site fulfills its function in repressing misleading fragments and the corresponding signals.\nA thorough comparison of the analyzed fragment lengths with the corresponding sequences of the clones resulted in a good agreement of theoretical and experimental fragment length for the 6 restriction enzymes. In general, only those fragments were detected, that were derived from the nearest restriction site of the corresponding enzyme as judged by sequencing. However, in several cases the first Alu I site was not recognized by the enzyme despite the use of an at least 4-fold excess of enzyme units compared to the amount of cDNA, indicating site preferences of this restriction enzyme that may depend on the context of the recognition sequence [22]. This did not affect the analysis of fingerprints in mixed clone analyses reported below as the same site preference was also observed under these conditions.\nAnother inherent discrepancy is the integer character of the predicted values versus the non-integer values stemming from the comparison to the size marker. The average of the difference of the experimentally analyzed length to the calculated length was 0.258 bp (median 0.115 bp) which means that the analyzed values were generally a little longer than the predicted ones. About 95% of the analyzed lengths differed less than 1 bp and about 50% differed less than 0.5 bp from the predicted lengths which by far exceeded the experimental deviations in repeated experiments (standard deviation of less than 0.2 bp, see below) and is in accordance to results from other authors [23,24]. These data demonstrated, that there is a reproducible but not yet predictable shift of the fragments within the polyacrylamide gel run. Together with the above mentioned site preference of AluI and additional problems like the not well defined polyadenylation-site in public database entries, this hinders the construction of fingerprints solely based on sequence data. Attempts to predict these shifts based on the sequence composition of the fragments were not successful in the first trials.\nExperimentally determined fingerprints from single clone analysis could be used for the analysis without any of the above mentioned hindrances. The fragment lengths from single clone analyses that by chance contained cDNAs from the same gene reproduced very accurately. The standard deviation from the average value was below 0.2 bp for fragments below 500 bp and increased with larger fragment size. The range in which a band was accepted as being the same as the search value was determined empirically. Colonies of specific genes were mixed into different pools of defined clone compositions and the detection width was adjusted to be the best compromise between not missing any real hits and counting false ones. As assumed from the increasing peak width of the analyzed fragments and the internal size marker, the detection width varied with the length of the fragments (see material and methods). The detection range was increased above the determined 0.2 bp standard deviation especially for the longer fragments. The broader detection width that could in principle lead to false positive identification of a fingerprint was counterbalanced by the fact that the peak density is much lower towards the longer fragments (Fig. 4)."}