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P M Lizardi

Publications and source records attributed to P M Lizardi.

At least 19 recordsLinked to original sources
PubMed2000

Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection.

We describe an adaptation of the rolling circle amplification (RCA) reporter system for the detection of protein Ags, termed "immunoRCA. " In immunoRCA, an oligonucleotide primer is covalently attached to an Ab; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, amplification results in a long DNA molecule containing hundreds of copies of the circular DNA sequence that remain attached to the Ab and that can be detected in a variety of ways. Using immunoRCA, analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays in ELISA and microparticle formats. The signal amplification afforded by immunoRCA also enabled immunoassays to be carried out in microspot and microarray formats with exquisite sensitivity. When Ags are present at concentrations down to fM levels, specifically bound Abs can be scored by counting discrete fluorescent signals arising from individual Ag-Ab complexes. Multiplex immunoRCA also was demonstrated by accurately quantifying Ags mixed in different ratios in a two-color, single-molecule-counting assay on a glass slide. ImmunoRCA thus combines high sensitivity and a very wide dynamic range with an unprecedented capability for single molecule detection. This Ag-detection method is of general applicability and is extendable to multiplexed immunoassays that employ a battery of different Abs, each labeled with a unique oligonucleotide primer, that can be discriminated by a color-coded visualization system. ImmunoRCA-profiling based on the simultaneous quantitation of multiple Ags should expand the power of immunoassays by exploiting the increased information content of ratio-based expression analysis.

Animals↗
PubMed1998

PNA molecular beacons for rapid detection of PCR amplicons.

The authors have developed a method for rapid detection of polymerase chain reaction (PCR) amplicons based on surface immobilized PNA-DNA hybrid probes ('molecular beacons') that undergo a fluorescent-linked conformational change in the presence of a complementary DNA target. Amplicons can be detected by simply adding a PCR reaction to a microtitre-well containing the previously immobilized probe, and reading the generated fluorescence. No further transfers or washing steps are involved. The authors demonstrate the specificity of the method for the detection of ribosomal DNA from Entamoeba histolytica.

Animals↗
PubMed1998

Mutation detection and single-molecule counting using isothermal rolling-circle amplification.

Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the - strand of DNA, a complex pattern of DNA strand displacement ensues that generates 10(9) or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.

Alleles↗
PubMed1997

Entamoeba histolytica contains a gene encoding a homologue to the 54 kDa subunit of the signal recognition particle.

We have determined the nucleotide sequence and predicted amino acid sequence of the 54 kDa subunit of the signal recognition particle (SRP54) from the amitochondrial protist Entamoeba histolytica. The SRP54 gene was isolated from a genomic library using a polymerase chain reaction (PCR) probe. Nucleotide sequence analysis of a 2.3 kb fragment, derived from a 7 kb genomic clone, revealed an open reading frame encoding a protein of 487 amino acids (MW 53.8 kDa). The identities of the predicted amino acid sequence with its homologues from other species were between 24 and 47%. Functional domains previously defined for the SRP54-type proteins were present in the entamoebal sequence, such as the amino-terminal GTP binding domain (G domain) and the carboxy-terminal methionine rich domain (M domain). SRP54 mRNA contains an extra G residue at the 5' end, suggesting that capping of poly-A(+) transcripts is present in E. histolytica. Evolutionary analysis of the SRP54 based on phylogenetic inference placed the E. histolytica sequence as an early divergence of the eukaryotic tree. Although the function of the entamoebal homologue remains to be elucidated, the identification of the SRP54 gene constitutes the first evidence for SRP related proteins in protozoans.

Amino Acid Sequence↗
PubMed1996

Identification and analysis of the u6 small nuclear RNA gene from Entamoeba histolytica.

Among the small nuclear RNAs (snRNAs) involved in the spliceosomal processing of pre-mRNA, U6 is the most conserved. As a first evidence for the presence of the splicing machinery in the amitochondrial protozoan Entamoeba histolytica (Eh), we have cloned the u6 snRNA gene. We find that in this organism u6 is a single copy gene that is transcribed as a poly(A)- RNA molecule of approximately 105 nucleotides. We have mapped the 5' end of the U6 snRNA transcript, and identified typical elements of a putative polymerase III promoter. This is the first snRNA gene reported in Eh. Sequence analysis indicates that this gene contains all the conserved nucleotides known to be important for U6 snRNA function. These results, in conjunction with the earlier finding of genes that contain pre-mRNA introns, suggest that Eh has a functional spliceosomal complex.

Animals↗
PubMed1996

Extremely sensitive, background-free gene detection using binary probes and beta replicase.

We have developed a specific and sensitive nucleic acid amplification assay that is suitable for routine gene detection. The assay is based on a novel molecular genetic strategy in which two different RNA probes are hybridized to adjacent positions on a target nucleic acid and then ligated to form an amplifiable reporter RNA. The reporter RNA is then replicated up to a hundred billion-fold in a 30-min isothermal reaction that signals the presence of the target. The assay can detect fewer than 100 nucleic acid molecules; it provides quantitative results over a wide range of target concentrations and it employs a universal format that can detect any infectious agent.

Base Sequence↗
PubMed1996

Sequence-specific detection of PCR-amplified DNA by restriction enzyme release of hybrids.

We have developed a method to detect PCR amplicons that contain a restriction enzyme site. The method is called Polymerase Chain Reaction-Hybridization and Enzymatic Release Assay (PCR-HERA). The PCR product is hybridized to a synthetic oligonucleotide that is immobilized on a microtitre plate. Specific cleavage of the resulting hybrid by a restriction endonuclease liberates a fluorescein labeled arm of the immobilized DNA. We demonstrate the use of PCR-HERA to detect species-specific sequences of extra-chromosomal, ribosomal DNA, of Entamoeba histolytica. This highly specific assay does not require any washing steps and can be easily automated.

Animals↗
PubMed1995

Identification and analysis of the start site of ribosomal RNA transcription of Entamoeba histolytica.

In this article we report the identification of the start site of ribosomal RNA transcription unit of the enteric parasite E. histolytica. We cloned the upstream region of the ribosomal RNA and we defined the 5' boundary of the transcription unit with nuclear run-on assays. We report that ribosomal transcription starts 2447 bp upstream the SSU ribosomal gene, at an adenosine residue. This data was supported both by S1 mapping and by primer extension analysis; that the mapped site was indeed the transcription start point was demonstrated by RNAse protection of the in vitro capped RNA. Our sequence data around the transcription start point shows two different tandem repeat clusters immediately downstream from the transcription start point.

Animals↗
PubMed1995

An allosteric hammerhead ribozyme.

We have constructed an RNA molecule containing a hammerhead ribozyme that is under allosteric control. In the inactive state, the RNA enzyme is unable to cleave a suitable substrate. The formation of the active state of the ribozyme is triggered by a specific interaction with a DNA oligonucleotide effector that is complementary to a single-stranded loop in the RNA enzyme molecule. Other DNA or RNA molecules containing unrelated nucleotide sequences do not function as allosteric effectors. This work demonstrates the feasibility of designing RNA enzymes that are specifically activated in response to an artificially designed molecular recognition event. Such enzymes may have practical applications.

Allosteric Regulation↗