PubMed · 5358936
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J Peachey. 1969. Automatic peritoneal dialyser.. https://pubmed.ncbi.nlm.nih.gov/5358936/
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This paper demonstrates the procedure of sequencing DNA restriction fragments isolated by a recently developed fraction collector after CE separation. In particular, using pBr 322 plasmid as a model system, a double digest was performed with Eco RI and Pst 1 restriction enzymes to produce two fragments of 749 base pairs (bp) and 3612 bp, both with cohesive ends. Prinkers, specific linkers complementary to the cohesive ends, were then ligated to both fragments (increasing the size by 59 bp each). These Prinker-modified fragments were separated by CE and collected. The success of the collection was demonstrated by reinjection of each isolated fraction with laser-induced fluorescence detection, using ethidium bromide as intercalater. The 808 bp isolated fragment was then polymerase chain reaction-amplified with appropriate primers for the Prinker ends, followed by cycle sequencing. Both strands of the fragment were run on an ABI 373, sequencing 427 bases and 450 bases, respectively, with a read accuracy of 99.3%. This approach with Prinker-modified restriction fragment and automated CE fraction collection can be used as a general procedure for sequencing unknown genomic DNA as well as mutated DNA mixtures.
The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised due to the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column; hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time and cost. One particularly effective clean-up system, membrane dialysis, was automated using a robotic workstation.
Loss of heterozygosity (LOH) and replication error (RER) are important phenomena in tumor development, with diagnostic and prognostic relevance. Therefore, screening for LOH and RER is a desirable first step in the molecular analysis of tumors. We used semiautomated procedures based on multicolor fluorescently labeled microsatellite markers and an automated sequencer for PCR amplification, electrophoresis of PCR products, and allele detection with a set of 16 microsatellites in 56 colorectal tumors. We improved existing software for computer-assisted assessment of LOH and RER. A comparison of these results with those of a conventional, radioactive technique and visual interpretation shows a high degree of correlation between the two methods. The detection rates of LOH and RER are similar to those reported previously. The main advantages of the semiautomated fluorescence-based typing are in the objective, observer-unrelated, easy, and rapid computer-based scoring, and the resulting quantitative assessment of RER.