We show that archaebacterial DNA polymerases are strongly inhibited by the presence of small amounts of uracil-containing DNA. Inhibition appears to be competitive, with the DNA polymerase exhibiting ϳ6500-fold greater affinity for binding the inhibitor than a DNase I-activated DNA substrate. All six archaebacterial DNA polymerases tested were inhibited, while no eubacterial, eukaryotic, or bacteriophage enzymes showed this effect. Only a small inhibition resulted when uracil was present as the deoxynucleoside triphosphate, dUTP. The rate of DNA synthesis was reduced by ϳ40% when dUTP was used in place of dTTP for archaebacterial DNA polymerases. Furthermore, an incorporated dUMP served as a productive 3-primer terminus for subsequent elongation. In contrast, the presence of an oligonucleotide containing as little as a single dUrd residue was extremely inhibitory to DNA polymerase activity on other primer-template DNA.During the last few years, several DNA polymerases have been identified from thermophilic archaebacteria (1-4). The small number of representatives available thus far have shared sequence similarities (5), and the purified enzymes have had several notable properties in common. All are thermostable as anticipated. Each is associated with a 3Ј35Ј proofreading exonuclease activity, and none have a 5Ј33Ј exonuclease activity. Archaea is a third kingdom distinct from eubacteria and eukaryotes (6, 7) and is thought to be evolutionarily closer to the eukaryotes. Some archaebacterial proteins share strong homology with eukaryotic counterparts such as RNA polymerase; however, some transcription-associated genes are organized in clusters resembling those of eubacteria (8). The archaebacterial DNA polymerases share significant homology with the family B DNA polymerases (5), which include eukaryotic cell DNA polymerases as well as Escherichia coli pol 1 II and bacteriophage T4 DNA polymerase.We report here an unusual property that is apparently unique to the archaea. The presence of uracil in DNA results in a dramatic increase in the binding affinity of the DNA polymerase. We first observed this effect while attempting to carry out polymerase chain reaction protocols that included dUrdcontaining oligonucleotides. These oligonucleotides appeared to block all DNA synthesis, suggesting a direct action on the DNA polymerase. Evidence is presented that suggests that the DNA polymerase forms a tight nonproductive complex with dUrd-containing DNA. We speculate on the possibility that this effect is related in some way to the extreme temperatures at which these thermophiles live and that it may serve in a biological function.
By incorporating dUMP residues into the 5' end of PCR primers, one can generate products which, after treatment with uracil DNA glycosylase (UDG), contain 3' overhangs. These overhangs can be annealed to vector molecules with complementary overhangs generated in a similar fashion and transformed directly into Escherichia coil without the need for ligase. We have tested this method of ligation-independent cloning by using UDG to create complementary single-stranded sticky ends between vector and Alu-PCR products generated from cosmid clones containing DNA from human chromosome 21. Using a single primer, Alu-PCR amplifies the sequence between appropriately oriented, repetitive (Alu) sequences in human DNA that are no more than 2 to 3 kb apart. Nineteen Alu-PCR products were observed in four human chromosome 21 cosmids. Thirteen of these products were detected among 48 subclones picked at random after cloning of the Alu-PCR products using UDG. The size or abundance of an Alu-PCR product did not appear to affect significantly the efficiency of cloning. Eight of the subclones were tested and all hybridized to human chromosome 21 DNA. UDG cloning should prove to be a general PCR cloning method that allows one to rapidly subclone small fragments from human genomic DNA.
Polymerase chain reaction has been applied to the amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs. However, polymerase chain reaction amplification from cDNA templates produced by reverse transcription has generally been restricted to products of less than 10 kilobases. In this paper, we report a system to effectively amplify fragments up to 20 kilobases from human coronavirus 229E genomic RNA. We demonstrate that the integrity of the RNA template and the prevention of false priming events during reverse transcription are the critical parameters to achieve the synthesis of long cDNAs. The optimization of the polymerase chain reaction conditions enabled us to improve the specificity and yield of product but they were not definitive. Finally, we have shown that the same reverse transcription polymerase chain reaction technology can be used for the amplification of extended regions of the dystrophin mRNA, a cellular RNA of relatively low abundance.
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