Estimation of DNA fragment size and generation of DNA restriction endonuclease maps using linear models (original) (raw)
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The polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for PCR have been extended significantly by the development of "long" PCR, a technique that makes it possible to amplify DNA fragments up to 40 kb in length. This article describes two novel applications of the long PCR technique, one which simplifies restriction mapping and another which enhances amplification specificity and yield. The same primers used to perform the long PCR amplification can be used as probes to perform restriction mapping of the DNA fragment amplified. Restriction digestion performed prior to long PCR amplification can be used to selectively suppress the amplification of members of families of closely related DNA sequences, thereby making it possible to selectively amplify one of a group of highly homologous sequences. These two complimentary techniques, both involving use of the long PCR paired with restriction digestion, have potential application in any laboratory in which PCR is performed. Current Issues Molec. Biol. (1999) 1(2): 77-88. 78 Her and Weinshilboum ORDER FROM www.caister.com UK/Europe: Caister
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Restriction enzymes have proven to be among the most valuable tools in molecular biology. In this work, we demonstrate that the cleavage of Xuorescently labeled, PCR-ampliWed DNA can be used as a simple and highly sensitive technique for detection of sequences present in a percentage as low as 0.6% in a DNA pool. Due to the fact that Xuorescent labeling of DNA fragments enables such sensitive detection and quantiWcation of restriction enzyme cleavage, the method was further exploited in monitoring of the enzymatic digestion completeness and in determination of factors that inXuence restriction enzyme eVectiveness. We analyzed the activity of six restriction endonucleases; the percentage of uncleaved DNA fragments predominantly ranged between 2.0 and 2.5 and the highest value was 8.00%. We conclude that, since the enzymatic digestion completeness may not always be assured, each assay based on restriction enzyme cleavage that is intended to be used in investigations of heterogeneity in a DNA pool should be constructed so that the presence of cleaved sequences is the indication of pool nonuniformity. When the presence of uncleaved sequences indicates pool heterogeneity, the results could be misleading due to possible incompleteness of enzymatic cleavage.