Bruce P. Neri

Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.

Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

A Comparison of Eubacterial and Archaeal Structure-specific 5′-Exonucleases

The 5′-exonuclease domains of the DNA polymerase I proteins of Eubacteria and the FEN1 proteins of Eukarya and Archaea are members of a family of structure-specific 5′-exonucleases with similar function but limited sequence similarity. Their physiological role is to remove the displaced 5′ strands created by DNA polymerase during displacement synthesis, thereby creating a substrate for DNA ligase. In this paper, we define the substrate requirements for the 5′-exonuclease enzymes from Thermus aquaticus, Thermus thermophilus,Archaeoglobus fulgidus, Pyrococcus furiosus,Methanococcus jannaschii, and Methanobacterium thermoautotrophicum. The optimal substrate of these enzymes resembles DNA undergoing strand displacement synthesis and consists of a bifurcated downstream duplex with a directly abutted upstream duplex that overlaps the downstream duplex by one base pair. That single base of overlap causes the enzymes to leave a nick after cleavage and to cleave several orders of magnitude faster than a substrate that lacks overlap. The downstream duplex needs to be 10 base pairs long or greater for most of the enzymes to cut efficiently. The upstream duplex needs to be only 2 or 3 base pairs long for most enzymes, and there appears to be interaction with the last base of the primer strand. Overall, the enzymes display very similar substrate specificities, despite their limited level of sequence similarity.

Clinical, genetic, and pharmacogenetic applications of the invader assay*

The Invader technology has been developed for the detection of nucleic acids. It is a signal amplification system able to accurately quantify DNA and RNA targets with high sensitivity. Exquisite specificity is achieved by combining hybridization with enzyme recognition, which provides the ability to discriminate mutant from wild-type at ratios greater than 1/1000 (mutant/wt). The technology is isothermal and flexible and incorporates a homogeneous fluorescence readout. It is therefore readily adaptable for use in clinical reference laboratories, as well as high-throughput applications using 96-, 384-, and 1,536-well microtiter plate formats. The molecular mechanism of the system and specific applications for use in clinical and research laboratories are described. These include direct analysis of unamplified human genomic DNA to detect mutations and single-nucleotide polymorphisms associated with factor V Leiden, factor II, cystic fibrosis, and apolipoprotein E, and gene expression assays that quantify messenger RNA levels in cells using direct lysates.

RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases.

DNA replication and repair require a specific mechanism to join the 3′- and 5′-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5′ nucleases with substrates containing an RNA template strand. By comparing the eubacterial and archaeal 5′ nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5′ nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus(TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the “thumb” and in the “palm” subdomains, critical for RNA-dependent 5′ nuclease activity. There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5′ nuclease activity 4–10-fold. Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5′ nuclease activity. The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5′ nuclease mode, that is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5′ nuclease activity for RNA detection using the invasive signal amplification assay is discussed.

Comparison of detection platforms and post‐polymerase chain reaction DNA purification methods for use in conjunction with Cleavase fragment length polymorphism analysis

The removal of impurities and contaminants from PCR‐amplified fragments is important for mutation detection methods which identify mutations based on shifts in electrophoretic mobility. This is particularly critical for assays and detection methods which use target DNA that is labeled prior to analysis and electrophoretic detection. We examined several procedures for purifying DNA amplified by the polymerase chain reaction (PCR) and their use in conjunction with a novel DNA scanning method, the Cleavase fragment length polymorphism (CFLP)* assay. In this study, a 480 bp DNA fragment, fluorescently labeled on the 5′‐end of one strand, was amplified and subjected to various widely used purification procedures, including several commercially available clean‐up kits. We demonstrate that visualization of the fluorescent label, as opposed to simple ethidium bromide staining, reveals the presence of considerable levels of labeled, truncated, amplification products. The various procedures were evaluated on the basis of their ability to remove these unwanted DNA fragments as well as on the degree to which they inhibited or promoted the CFLP reaction. Several procedures are recommended for use with CFLP analysis, including isopropanol precipitation, gel excision, and several commercially available spin columns. Concurrently, we evaluated (compared) a number of commonly used visualization platforms, including fluorescence imaging, chemiluminescence, and post‐electrophoretic staining, for the ability to detect CFLP pattern changes. The advantages and disadvantages of different methods are discussed and amounts of DNA to be used for CFLP analysis on different detection platforms are recommended.