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Biomedical subjects

E Lusby

Publications and source records attributed to E Lusby.

5 recordsLinked to original sources
PubMed2002

Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence.

Here we describe the properties of a novel class of oligonucleotide probes capable of sensitive hybridization-triggered fluorescence. These fluorogenic probes, known commercially as MGB Eclipse probes, are characterized by having a conjugated minor groove binder (MGB) ligand at the 5'-end and a fluorophore at the 3'-end. Additionally, they have an efficient quencher moiety at the 5'-end that is useful with a wide variety of fluorescent dyes. Fluorescence of the single-stranded MGB Eclipse probe is efficiently quenched by the interaction of the terminal dye and quencher groups when not hybridized. Upon hybridization to a complementary target, the MGB molecule folds into duplex and hyper-stabilizes it, allowing the use of shorter, more specific probe sequences. The 5'-MGB-quencher group also prevents nuclease digestion by Taq DNA polymerase during PCR. Because of the hybridization-triggered fluorescence and the excellent specificity imparted by the MGB, these 5'-MGB Eclipse probes have great versatility for real-time PCR applications. The high sensitivity and specificity are illustrated using single nucleotide polymorphism detection, viral load determination, and gene expression analysis.

DNA↗
PubMed1998

Quantitation of host cell DNA contaminate in pharmaceutical-grade plasmid DNA using competitive polymerase chain reaction and enzyme-linked immunosorbent assay.

The rising interest in gene therapy for the treatment of numerous disorders necessitates the need for the large-scale production of therapeutic biopharmaceuticals that meet stringent purity standards. Residual host cell DNA in recombinant pharmaceuticals has been identified as a potential risk factor that must be quantitated carefully both during the manufacturing process and in the final product. We describe a PCR method to quantitate contaminating levels of host cell DNA in clinical plasmid DNA preparations intended for human gene therapy. The quantitation is based on the coamplification of two similar templates, the target DNA and a synthetic competitor, and the quantitation of the resulting PCR products. The competitor is identical to the target DNA PCR product except for a 29-bp internal replacement. As a result, the two PCR products can easily be distinguished from each other. The competitive nature of the assay allows the use of the ratio of the target DNA PCR product to the competitor DNA PCR product to determine the original amount of target DNA in a sample. The primers used in this assay anneal to a conserved region of the E. coli 23S rRNA gene. One of the primers is biotinylated, allowing the PCR products to be detected colorimetrically after their capture on microtiter plates. The capture is accomplished by differential hybridization to target and competitor-specific probes covalently attached to wells of microtiter plates. The entire assay is performed in less than 2 hr postamplification. This method represents an attractive alternative to Southern blot analysis, which is the currently established method for DNA quantitation.

Binding, Competitive↗
PubMed1981

Inverted terminal repetition in adeno-associated virus DNA: independence of the orientation at either end of the genome.

Complementary strands of adeno-associated virus DNA labeled with 32P at the 5' ends were separated and then self-annealed to form single-stranded circles stabilized by hydrogen bonds between the complementary sequences in the inverted terminal repetitions. We have previously shown that there are two distinct sequences in the terminal repetition which represent an inversion of the first 125 nucleotides (E. Lusby et al., J. Virol. 34:402-409, 1980; I. S. Spear et al., Virology 24:627-634, 1977). Base pairing between terminal sequences of the same orientation leads to a normal double helical structure. If sequences of the opposite orientation pair, an aberrant secondary structure is formed. HpaII digestion of the self-annealed, single-stranded circles led to labeled terminal fragments that corresponded both to those generated from termini of a normal double helical structure and those generated from an aberrant terminal secondary structure. Thus, the orientation of the terminal repetition at one end of the genome is not influenced by the orientation at the other end.

DNA Replication↗
PubMed1980

Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA.

The inverted terminal repetition in adeno-associated virus type 2 DNA has been sequenced. The terminal repetition contain 145 nucleotides of which the first 125 nucleotides can self-base pair to form a T-shaped hairpin structure. Both restriction endonuclease analysis with SmaI and BglI and direct sequence analysis of the SmaI fragments provide evidence for two sequences in the region of the terminal repetition between nucleotides 44 and 81. The two sequences represent an inversion of the first 125 nucleotides of the terminal repetition. Based on these data a model for adeno-associated virus DNA replication is presented which agrees in detail with a general model for eucaryotic DNA replication originally proposed by Cavalier-Smith (T. Cavalier-Smith, Nature [London] 18:672--684, 1976).

Base Sequence↗