Saliva, diagnostics, and dentistry.

Saliva, a scientific and clinical entity familiar to every oral health researcher and dental practitioner, has emerged as a translational and clinical commodity that has reached national visibility at the National Institutes of Health and the President's Office of Science and Technology. "Detecting dozens of diseases in a sample of saliva" was issued by President Obama as one of the 14 Grand Challenges for biomedical research in the 21(st) Century (National Economic Council, 2010). In addition, NIH's 2011 Government Performance Report Act (GPRA) listed 10 initiatives in the high-risk long-term category (Collins, 2011). The mandate is to determine the efficacy of using salivary diagnostics to monitor health and diagnose at least one systemic disease by 2013. The stage is set for the scientific community to capture these national and global opportunities to advance and substantiate the scientific foundation of salivary diagnostics to meet these goals. A specific calling is to the oral, dental, and craniofacial health community. Three areas will be highlighted in this paper: the concept of high-impact diagnostics, the role of dentists in diagnostics, and, finally, an infrastructure currently being developed in the United Kingdom--The UK Biobank--which will have an impact on the translational and clinical utilizations of saliva.

Human apolipoprotein B: structure of carboxyl-terminal domains, sites of gene expression, and chromosomal localization.

Apolipoprotein (apo-) B is the ligand responsible for the receptor-mediated catabolism of low density lipoproteins, the principal cholesterol-transporting lipoproteins in plasma. The primary structure of the carboxyl-terminal 30 percent (1455 amino acids) of human apo-B (apo-B100) has been deduced from the nucleotide sequence of complementary DNA. Portions of the protein structure that may relate to its receptor binding function and lipid binding properties have been identified. The apo-B100 messenger RNA is about 19 kilobases in length. The apo-B100 gene is expressed primarily in liver and, to a lesser extent, in small intestine, but in no other tissues. The gene for apo-B100 is located in the p24 region (near the tip of the short arm) of chromosome 2.

Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay.

AIM: To determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. METHOD: Two HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. RESULTS: In one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 10(5) copies/ml, and the branched DNA results ranged from 2.6 x 10(4) to 4.2 x 10(4) HIV RNA equivalent/ml. In the other sample the corresponding figures were 6.3 x 10(4) to 5.5 x 10(5) copies/ml and 5.7 x 10(4) to 7.5 x 10(4) HIV RNA equivalents/ml. CONCLUSION: In contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.

Study of cholesterol side-chain cleavage (20,22 desmolase) deficiency causing congenital lipoid adrenal hyperplasia using bovine-sequence P450scc oligodeoxyribonucleotide probes.

Conversion of cholesterol to pregnenolone is mediated by the cholesterol side-chain cleavage (SCC) enzyme, P450scc. Deficient SCC activity causes congenital lipoid adrenal hyperplasia (also known as 20,22 desmolase deficiency), a potentially lethal defect in the synthesis of all steroid hormones. To probe for possible genetic defects causing this disease we synthesized four oligodeoxyribonucleotides containing 63 to 72 bases corresponding to portions of the bovine complementary DNA (cDNA) sequence for P450scc. The bovine oligonucleotides were labeled and used directly to probe Southern blots of normal human genomic DNA, revealing a pattern indicating there is a single P450scc gene in the human genome. Hybridization to Northern blots of normal human and bovine adrenal messenger RNA indicates that P450scc messenger RNA is about 2.0 kilobases long in both species. Hybridizations of the oligonucleotides to genomic DNA from three unrelated patients with SCC deficiency did not detect a deletion in the human P450scc gene. The bovine sequence oligonucleotides were then used to isolate a human P450scc cDNA clone. The isolated P450scc cDNA fragment contains 818 bases encoding 239 amino acids of the protein, the translation termination signal, and 98 bases of the 3' untranslated region. The sequence of this carboxy-terminal half of the human P450scc protein is 72% homologous with the bovine sequence and contains an additional amino acid not found in bovine P450scc; the human and bovine nucleotide sequences are 81% homologous. Repetition of the genomic DNA blotting studies with the cDNA probe gave the same results obtained with the bovine-sequence oligonucleotide probes, confirming that SCC deficiency is not due to a deletion in the regions of the P450scc hybridizing with the probes. Long, chemically synthesized heterologous sequence oligonucleotides containing unknown numbers of base mismatches with human sequences may thus be used to study human genes so that access to a cDNA is not necessary for such studies.

A novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity; application to the analysis of hepatitis B virus in human serum.

The detection of a little as 0.2 pg (60,000 molecules) of hepatitis B viral (HBV) DNA in human serum samples in 4 h has been demonstrated using a solution-hybridization and bead-capture method. An amplification method based on chemically crosslinked oligodeoxyribonucleotides was coupled with a horseradish peroxidase-labeling scheme for the ultimate detection of the analyte. Two sets of HBV complementary synthetic oligodeoxyribonucleotide probes containing one of two types of single-stranded (ss) overhangs were employed. These ss overhangs were used to capture the probe-analyte complex onto a bead and subsequently to label it. Detection was achieved with either a chemiluminescent or colorimetric output substrate for the enzyme. Only in the presence of the virus was label specifically bound to the support. The assay was relatively unaffected by either sample composition or by the presence of heterologous nucleic acids.

High level expression of proinsulin in the yeast, Saccharomyces cerevisiae.

Human proinsulin (PI) has been expressed to a high level (100 mg/liter) as a human superoxide dismutase-PI fusion protein in the yeast, Saccharomyces cerevisiae. At the junction of the two proteins is a methionine residue, allowing PI to be released from the fusion by reaction with cyanogen bromide. The fusion is expressed using a regulated, hybrid promoter containing the regulatory region of the alcohol dehydrogenase II promoter and the 3' end of a glyceraldehyde-3-phosphate dehydrogenase promoter, allowing the recombinant yeast cells to be stably maintained. Production of the fusion protein is induced by growth in medium lacking a fermentable carbon source. The heterologous fusion protein is probably insoluble within the cell, since electron microscopy reveals the presence of 'inclusion bodies'. In a cell-free extract the fusion protein is also insoluble, but can be solubilized with sodium dodecyl sulfate, and cleaved with cyanogen bromide. The PI that is produced contains incorrect disulfide bonds. After sulfitolysis, the product can be easily purified, renatured, and processed to yield insulin.

Quantification of cytokine mRNA in peripheral blood mononuclear cells using branched DNA (bDNA) technology.

Changes in the patterns of cytokine expression are thought to be of central importance in human infectious and inflammatory diseases. As such, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting. In this report, we describe the design and performance of a branched DNA (bDNA) assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). Oligonucleotide target probe sets were designed for several human cytokines, including TNFalpha, IL-2, IL-4, IL-6, IL-10, and IFNgamma. The bDNA assay yielded highly reproducible quantification of cytokine mRNAs, exhibited a broad linear dynamic range of over 3-log10, and showed a sensitivity sufficient to measure at least 3000 molecules. The potential clinical utility of the bDNA assay was explored by measuring cytokine mRNA levels in PBMCs from healthy and immunocompromised individuals. Cytokine expression levels in PBMCs from healthy blood donors were found to remain relatively stable over a one-month period of time. Elevated levels of IFNgamma mRNA were detected in PBMCs from HIV-1 seropositive individuals, but no differences in mean levels of TNFalpha or IL-6 mRNA were detected between seropositive and seronegative individuals. By providing a reproducible method for quantification of low abundance transcripts in clinical specimens, the bDNA assay may be useful for studies addressing the role of cytokine expression in disease.

Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay.

The aim of this study was to establish the performance characteristics of a nonradioisotopic branched DNA (bDNA) signal amplification assay for quantitation of hepatitis B virus (HBV) DNA in human serum. Quantitation was determined from a standard curve and expressed as HBV DNA equivalents/mL (Eq/mL; 285,000 Eq = 1 pg of double stranded HBV DNA). The bDNA assay exhibited a nearly four log dynamic range of quantitation and an analytical detection limit of approximately 100,000 Eq/mL. To ensure a specificity of 99.7%, the quantitation limit was set at 700,000 Eq/mL. The interassay percent coefficient of variance for quantification values ranged from 10% to 15% when performed by novice users with different sets of reagents. Using the bDNA assay, HBV DNA was detected in 94% to 100% of hepatitis B e antigen-positive specimens and 27% to 31% of hepatitis B e antigen-negative specimens from chronic HBV-infected patients. The bDNA assay may be useful as a prognostic and therapy monitoring tool for the management of HBV-infected patients undergoing antiviral treatment.

Quantification of HCV RNA in Clinical Specimens by Branched DNA (bDNA) Technology.

The diagnosis and monitoring of hepatitis C virus (HCV) infection have been aided by the development of HCV RNA quantification assays A direct measure of viral load, HCV RNA quantification has the advantage of providing information on viral kinetics and provides unique insight into the disease process. Branched DNA (bDNA) signal amplification technology provides a novel approach for the direct quantification of HCV RNA in patient specimens. The bDNA assay measures HCV RNA at physiological levels by boosting the reporter signal, rather than by replicating target sequences as the means of detection, and thus avoids the errors inherent in the extraction and amplification of target sequences. Inherently quantitative and nonradioactive, the bDNA assay is amenable to routine use in a clinical research setting, and has been used by several groups to explore the natural history, pathogenesis, and treatment of HCV infection.

Quantification of HCV RNA in Liver Tissue by bDNA Assay.

With this statement, Sherlock and Dooley have described two of the three major challenges involved in quantitatively measuring any analyte in tissue samples: the distribution of the analyte in the tissue; and the standard of reference, or denominator, with which to make comparisons between tissue samples. The third challenge for quantitative measurement of an analyte in tissue is to ensure reproducible and quantitative recovery of the analyte on extraction from tissue samples. This chapter describes a method that can be used to measure HCV RNA quantitatively in liver biopsy and tissue samples using the bDNA assay. All three of these challenges-distribution, denominator, and recovery-apply to the measurement of HCV RNA in liver biopsies.

Solid supported hydrolysis of apurinic sites in synthetic oligonucleotides for rapid and efficient purification on reverse-phase cartridges.

Rapid purification methods for synthetic oligonucleotides involving reverse-phase cartridges (RPC) and enzymatic hydrolysis have been introduced. These methods are based on a discrimination between the desired target fragment protected with a 5'-DMT group and incompletely elongated products possessing a 5'-hydroxyl function. For target products over 60 nucleotides, the rapid methods are of little use as reported to date. We have found that the problem is due to the presence of truncated 5'-DMT fragments generated from apurinic sites within the target product during NH4OH deprotection. These side products are co-purified with the target fragment when the rapid purification procedures are employed. If a step is included during deprotection to cleave the apurinic sites prior to removal of the crude product from the solid support (1 M lysine-HCl, pH 9 for 90 min at 60 degrees C), fragments up to 118 bases can be purified by RPC to near homogeneity.

Use of unpurified synthetic deoxynucleotide primers for rapid dideoxynucleotide chain termination sequencing.

Synthetic oligodeoxyribonucleotides were used as primers for sequencing DNA by the dideoxy chain termination method. We report that the use of unpurified preparations of such oligonucleotides decreases the time involved in preparing the primers and yields a sequencing reaction of comparable quality to that obtained with pure preparations. It is possible to use unpurified primers if the yield per coupling step during manual or machine synthesis of oligonucleotides is greater than 98%. Also, a method for the rapid purification and desalting of synthetic DNAs is presented for cases in which the coupling is less efficient. Unpurified primers have been used to determine the sequence of a 1987-bp fragment with a single M13 recombinant clone. This DNA was first sequenced using the universal M13 primer. Subsequently, a primer homologous to the 3' end of the newly obtained sequence was synthesized and used to extend the sequence. New primers were synthesized until the sequence of the whole fragment was determined. Because only one clone is necessary, this method can be used to obtain the nucleotide sequence of large DNA fragments in a relatively short time and in an orderly fashion, simplifying analysis of the data.

Development of a method for the incorporation of substitution-inert metal ions into proteins. Site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with cobalt(III).

This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.

Enzymatic purification of chemically synthesized oligodeoxyribonucleotides prior to removal from a solid-support.

A novel combined solid-phase DNA synthesis and solid-phase DNA purification strategy is described. Removal of all N-, cap-, and phosphate protecting groups while maintaining a 5'-blocking group on the final product followed by a 5'- specific exonuclease digestion of failure sequences with the synthetic DNA still attached to the solid phase yields an essentially pure oligomer.

A chemical 5'-phosphorylation of oligodeoxyribonucleotides.

To circumvent the use of T4 polynucleotide kinase and ATP for the 5'-phosphorylation of synthetic oligodeoxyribonucleotides after deprotection and purification, we have developed a new chemical phosphorylation reagent that can be used on automated DNA synthesis instruments. The phosphite-derived compound, bis(beta-cyanoethoxy)-N,N-diisopropylaminophosphine, is coupled to a solid-supported fragment and oxidized under the same conditions as nucleoside phosphoramidites. The resultant 5'-phosphate oligomer is then fully deprotected in standard ammonium hydroxide solution at elevated temperature. Reverse-phase, high-pressure liquid chromatography, silica thin-layer chromatography, and 31P-nuclear magnetic resonance spectra of chemically phosphorylated thymidine were compared with a variety of phosphorus derivatives of thymidine. All evidence suggested that the nucleoside was converted to the 5'-phosphate form quantitatively with the reagent. Moreover, an excellent enzymatic ligation reaction was obtained with a chemically phosphorylated oligonucleotide.

Forks and combs and DNA: the synthesis of branched oligodeoxyribonucleotides.

Nucleoside phosphoramidite derivatives containing two protected primary hydroxyl functions have been incorporated into synthetic oligonucleotides as 'branching monomers'. With selective deprotection, multiple identical copies of an additional oligonucleotide can be incorporated to form fork- or comb-like structures for use as signal amplification materials in nucleic acid hybridization assays.