Self-reporting PNA/DNA primers for PCR analysis.

We report a new fluorogenic method for sealed-tube PCR analysis using a quencher-labeled peptide nucleic acid (Q-PNA) probe. The Q-PNA hybridizes to a complementary tag sequence located at the 5' end of a 5' fluorophore-labeled oligonucleotide primer, quenching the primer's fluorescence. Incorporation of the primer into a doublestranded amplicon causes displacement of the Q-PNA such that the fluorescence of the sample is a direct indication of the amplicon concentration. The Q-PNA is able to quench multiple primers bearing distinct 5' fluorophores in a single reaction. We show realtime quantitative detection of a single-copy gene, K-ras, from human genomic DNA, as well as an endpoint multiplex assay for Chlamydia trachomatis and Neisseria gonorrhoeae targets. Because the Q-PNA may be used to quench any primer that contains the 5' tag sequence, it is possible to inexpensively adapt an existing primer set for use in a self-reporting fluorescent assay by including the tag sequence in one of the primers.

Peptide nucleic acid pre-gel hybridization: an alternative to southern hybridization.

We have found that it is possible to use labeled peptide nucleic acid (PNA)-oligomers as probes in pre-gel hybridization experiments, as an alternative for Southern hybridization. In this technique, the PNA probe is hybridized to a denatured DNA sample at low ionic strength and the mixture is loaded directly on to an electrophoresis system for size separation. Ensuing gel electrophoresis separates the single-stranded DNA fragments by length. The neutral backbone of PNA allows for hybridization at low ionic strength and imparts very low mobility to excess PNA. Detection of the bound PNA is possible by direct fluorescence detection with capillary electrophoresis, or the DNA/PNA hybrids can be blotted onto a membrane and detected with standard chemiluminescent techniques. Efficient single bp discrimination was achieved routinely using both capillary and slab-gel electrophoresis.

Biochemical evidence that a D-loop is part of a four-stranded PNA-DNA bundle. Nickel-mediated cleavage of duplex DNA by a Gly-Gly-His bis-PNA.

A peptide nucleic acid (PNA) with improved strand-displacement capability and a site-specific DNA cleavage function is a novel reagent for probing the structure of PNA-DNA complexes in solution. By linking two PNAs in tandem with an aliphatic linker, the bis-PNA forms a bis-PNA-DNA triple-stranded complex having a higher stability to thermal denaturation than conventional monomeric PNAs. When a Gly-Gly-His tripeptide is placed on either the Watson-Crick or Hoogsteen bis-PNA strand, nickel-mediated cleavage is detected at specific sites on the displaced and hybridized DNA strands. Because the displaced strand is cleaved when GGH is placed on either PNA strand, the D-loop must be close to the backbone of the bis-PNA-DNA triplex. Furthermore, the pattern of cleavage on the displaced strand suggests the nickel-tripeptide complex lies in a groove formed by the displaced DNA strand and both PNA strands. These observations suggest that the D-loop is a part of a four-stranded bis-PNA-DNA2 bundle.

Functionalized membrane supports for covalent protein microsequence analysis.

Methods were developed for high yield covalent attachment of peptides and proteins to isothiocyanate and arylamine-derivatized poly(vinylidene difluoride) membranes for solid-phase sequence analysis. Solutions of protein or peptide were dried onto 8-mm membrane disks such that the functional groups on the surface and the polypeptide were brought into close proximity. In the case of the isothiocyanate membrane, reaction between polypeptide amino groups and the surface isothiocyanate moieties was promoted by application of aqueous N-methylmorpholine. Attachment of proteins and peptides to the arylamine surface was achieved by application of water-soluble carbodiimide in a pH 5.0 buffer. Edman degradation of covalently bound polypeptides was accomplished with initial and repetitive sequence yields ranging from 33 to 75% and 88.5 to 98.5%, respectively. The yields were independent of the sample load (20 pmol to greater than 1 nmol) for either surface. Significant loss of material was not observed when attachment residues were encountered during sequence runs. Application of bovine beta-lactoglobulin A chain, staphylococcus protein A, or the peptide melittin to the isothiocyanate membrane allowed for extended N-terminal sequence identification (35 residues from 20 pmol of beta-lactoglobulin). A number of synthetic and naturally occurring peptides were sequenced to the C-terminal residue following attachment to the arylamine surface. In one example, 10 micrograms of bovine alpha-casein was digested with staphylococcal protease V8 and the peptides were separated by reverse-phase chromatography. Peptide fractions were then directly applied to arylamine membrane disks for covalent sequence analysis. From as little as 2 pmol of initial signal it was possible to determine substantial sequence information (greater than 10 residues).

Identification of phosphorylated sites in the mouse glucocorticoid receptor.

Glucocorticoid receptors in vivo are phosphorylated in the absence of hormone and become hyperphosphorylated in the presence of glucocorticoid agonist but not antagonists (Ortí, E., Mendel, D.B., Smith, L.I., and Munck, A. (1989) J. Biol. Chem. 264, 9728-9731). As a preliminary step to elucidating the functional significance of receptor phosphorylation, we have identified seven phosphorylated sites on the mouse receptor. Tryptic phosphopeptides from 32P-labeled receptors were purified from glucocorticoid-treated mouse thymoma cells (WEHI-7) and from stably transfected Chinese hamster ovary cells (WCL2) that express large numbers of mouse receptors. Phosphopeptide maps of receptors from these two cell types were almost indistinguishable. Solid phase sequencing revealed phosphorylation at serines 122, 150, 212, 220, 234, and 315 and threonine 159. Serines 122, 150, 212, 220, and 234 and the sequences surrounding them are conserved in the homologous regions of the rat and human receptors, but threonine 159 and serine 315 have no homologues in the human receptor. The seven phosphorylated sites are in the amino-terminal domain of the receptor. All but serine 315 are within transactivation domains identified in the human and/or rat receptors. Serines 212, 220, and 234 are in a highly acidic region that in the mouse receptor is necessary for full transcription initiation activity and reduces nonspecific DNA binding. Serines 212, 220, and 234 and threonine 159 are in consensus sequences for proline-directed kinase and/or p34cdc2 kinase. Serine 122 is in a consensus sequence for casein kinase II whereas serines 150 and 315 do not appear to be in any known kinase consensus sequence. The location of many of these sites suggests a role of phosphorylation in transactivation.

Solid-phase sequence analysis of proteins electroblotted or spotted onto polyvinylidene difluoride membranes.

Electroblotted proteins noncovalently bound to polyvinylidene difluoride (PVDF) membranes are typically sequenced using adsorptive sequencer protocols (gas-phase or pulsed-liquid) that do not require a covalent linkage between protein and surface. We have developed simple chemical protocols where proteins are first electroblotted onto unmodified PVDF membranes, visualized with common protein stains, and then immobilized for solid-phase sequence analysis. Adsorbed, stained proteins are first treated with phenylisothiocyanate (PITC) to modify alpha and epsilon amines. The protein is then overlayed with a solution of 1,4-phenylene di-isothiocyanate (DITC), followed by a few microliters of a basic solution containing a poly(alkylamine). As the polymer dries onto the surface both polymer and remaining protein amino groups are crosslinked by DITC. The protein is thus immobilized to the membrane surface by entrapment in a thin polymer coating. The coating is transparent to the degradation chemistry, and extensive enough to remain immobilized even in the absence of any covalent link between polymer and surface. Partial modification with PITC allows for identification of N-terminal and internal lysine residues during sequencing. The process was tested with a variety of poly(alkylamines), linear and branched, with molecular weights ranging from 600 to over 100,000. Proteins bound in this manner were successfully sequenced using covalent (solid-phase) sequencer protocols with cycle times as short as 26 min.

Chemiluminescent detection of DNA: application for DNA sequencing and hybridization.

A non-radioactive DNA detection chemistry is described and its application is shown for DNA hybridization and standard dideoxy DNA sequencing. The method employes a biotin-streptavidin system which binds an enzyme specifically to a target DNA and upon exposure to substrate, the enzyme catalyzes a chemiluminescent reaction. The image is captured within seconds by a Polaroid or X-ray film. The method is capable of detecting DNA in the hundred attomol range.

Introduction of 5'-terminal functional groups into synthetic oligonucleotides for selective immobilization.

Oligodeoxyribonucleotides terminating in a 5'-primary amine group are synthesized using solid-phase supported phosphoramidite chemistry. The 5'-terminal amine group in the deprotected oligomers is further derivatized with either succinic anhydride to give 5'-carboxylic acid or with dithiobis(succinimidylpropionate) followed by treatment with dithioerythritol to produce 5'-thiol-terminated oligonucleotides. The 5'-thiol-terminated oligonucleotides are selectively immobilized on solid supports containing either p-chloromercuribenzoate or 2,2'-dithiobis(5-nitropyridine) activated thiol groups.

Determination of impurities in nucleoside 3'-phosphoramidites by fast atom bombardment mass spectrometry.

Negative-ion fast atom bombardment mass spectrometry is quite useful for the identification of products and by-products formed during the synthesis of nucleoside 3'-phosphoramidites. The data show that detritylation and oxidation are side reactions which occur during the synthesis of monomeric units used in the construction of oligodeoxyribonucleotides by the phosphite triester method.