A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaql, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent

We have used the polymerase chain reaction (PCR) to amplify up to 22 kb of the 3-globin gene cluster from human genomic DNA and up to 42 kb from phage A DNA. We have also amplified 91 human genomic inserts of 9-23 kb directly from recombinant A plaques. To do this, we increased pH, added glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and used a secondary thermostable DNA polymerase that possesses a 3'-to-5'-exonuclease, or "proofreading," activity. Our "long PCR" protocols maintain the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing. The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics.PCR (1, 2) and molecular cloning are powerful tools for the amplification of genetic sequences; yet PCR can be quicker, simpler, and less costly to perform. As a result, PCR has been widely applied in molecular biology, molecular evolution, genetics, and forensic biology (3). PCR has also had broad impact on genome mapping and sequencing projects (4, 5). PCR would have an even greater role, however, if sequences longer than 10 kb-sequences currently cloned with phage A or cosmid vectors-could be amplified reliably.Although recent reports (6-11) described amplifications of 5-15 kb, reported yields were low. Our goals were to amplify targets of at least 20 kb with high yields, even from singlecopy genes within complex genomes, and to better understand the most critical parameters for longer amplifications.We surveyed various thermostable DNA polymerases, reaction buffers and additives, and thermal cycling profiles, guided by the following likely requirements for a reliable "long PCR": (i) complete denaturation of target sequences, as longer targets may become increasingly difficult to denature; (ii) extension times sufficient for complete strand synthesis in each PCR cycle; (iii) protection of template DNA against damage [e.g., depurination (12)] during thermal cycling; and (iv) retention of specificity necessary for singlecopy gene amplifications from genomic DNA.One of us (W.M.B.) has also hypothesized that misincorporated nucleotides reduce the efficiency of amplifying long targets. A mismatched 3'-terminal base may cause prematurely terminated strand synthesis (13). Even the low levels of nucleotide misincorporation estimated for Taq DNA polymerase [<1 in 10-50,000 bases (14)] will affect sequences longer than 10 kb. A small amount of thermostable 3'-to-5'-exonuclease activity removes such mismatched nucleotides and permits the predominant polymerase activity to complete strand synthesis (15). The use of such proofreading activity, combined with conditions identified in our survey, resulted in the highest yields of the longest products. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked...

The nucleotide sequence of the lac promoter-operator region has been determined. The 122 base pairs comprising this region include the recognition sites for RNA polymerase, the positive regulatory protein, CAP, and the negative regulatory protein, the repressor. Identification of mutant variants of the sequence combined with the in vitro biochemical studies of others has allowed us to tentatively identify the recognition site for each of these proteins, and to suggest how CAP might act at a distance to affect the interaction of RNA polymerase with the promoter.

Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is some 10–100-fold inhibition resistant to whole blood compared to full-length, wild-type (w.t.) Taq, which is strongly inhibited by 0.1–1% blood. Further mutations at codon 708, both in Klentaq 1 and Taq, confer enhanced resistance to various inhibitors of PCR reactions, including whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid, as well as high concentrations of intercalating dyes. Blood PCR inhibitors can predominantly reduce the DNA extension speed of the w.t. Taq polymerase as compared to the mutant enzymes. Single-copy human genomic targets are readily amplified from whole blood or crude soil extract, without pretreatment to purify the template DNA, and the allowed increase in dye concentration overcomes fluorescence background and quenching in real-time PCR of blood.

ETT2 is a second cryptic type III secretion system in Escherichia coli which was first discovered through the analysis of genome sequences of enterohemorrhagic E. coli O157:H7. Comparative analyses of Escherichia and Shigella genome sequences revealed that the ETT2 gene cluster is larger than was previously thought, encompassing homologues of genes from the Spi-1, Spi-2, and Spi-3 Salmonella pathogenicity islands. ETT2-associated genes, including regulators and chaperones, were found at the same chromosomal location in the majority of genome-sequenced strains, including the laboratory strain K-12. Using a PCR-based approach, we constructed a complete tiling path through the ETT2 gene cluster for 79 strains, including the well-characterized E. coli reference collection supplemented with additional pathotypes. The ETT2 gene cluster was found to be present in whole or in part in the majority of E. coli strains, whether pathogenic or commensal, with patterns of distribution and deletion mirroring the known phylogenetic structure of the species. In almost all strains, including enterohemorrhagic E. coli O157:H7, ETT2 has been subjected to varying degrees of mutational attrition that render it unable to encode a functioning secretion system. A second type III secretion systemassociated locus that likely encodes the ETT2 translocation apparatus was found in some E. coli strains. Intact versions of both ETT2-related clusters are apparently present in enteroaggregative E. coli strain O42.The species Escherichia coli contains a wide range of commensal strains and pathogenic varieties (pathotypes) in addition to the model laboratory organism, E. coli K-12 (16). At least six pathotypes are associated with human intestinal disease: they are enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli, enteroaggregative E. coli (EAEC), and diffusely adherent E. coli. Two pathotypes are associated with extraintestinal disease in humans, namely uropathogenic E. coli (UPEC) and neonatal meningitic E. coli (NMEC). In addition, it is now clear that on phylogenetic grounds, all members of the genus Shigella belong within the species of E. coli (50). Furthermore, this dazzling phenotypic variety is matched by remarkable variations in genome size, with the largest E. coli genomes possessing more than a megabase more DNA than the smallest ones (43).Initial studies of UPEC, and later of other pathotypes, suggested that E. coli strains often acquire new complex pathogenic phenotypes in a single step by the acquisition of pathogenicity islands, which contain virulence genes clustered on the chromosome and which are acquired en bloc by horizontal gene transfer (21). Similar studies with the related bacterium Salmonella enterica have delineated several Salmonella pathogenicity islands (Spi-1, Spi-2, Spi-3, etc.) (2, 22). The horizontal transfer of DNA by mobile elements such as bacteriophages and plasmids is also known to play a role in the evolution of virulence in E. coli and S...