Evaluation of arrayed primer extension for TP53 mutation detection in breast and ovarian carcinomas

Mutations in the tumor suppressor gene TP53 are associated with a wide range of different cancers and may have prognostic and therapeutic implications. Methods for rapid and sensitive detection of mutations in this gene are therefore required. In order to make screening more effective, a commercially available TP53 genotyping microarray from Asper Biotech has been constructed by arrayed primer extension (APEX). The present study is the first report that blindly evaluates the efficiency of the second generation APEX TP53 genotype chip outside the Asper laboratory and compares it to temporal temperature gradient electrophoresis (TTGE) and sequencing of TP53 for mutation detection in ovarian and breast cancer samples. All nucleotides in the TP53 gene from exon 2-9 are included on the chip by synthesis and application of sequence-specific oligonucleotides. The chip was validated by screening 48 breast and 11 ovarian cancer cases, all of which had previously been analyzed by TTGE and sequencing. APEX scored 17 of 20 sequence variants, missing one deletion, one insertion, and a missense mutation. Resequencing efficiency using APEX was 92% for both DNA strands and 99.5% for sense and/or antisense strand. We conclude that the APEX TP53 microarray is a robust, rapid, and comprehensive screening tool for sequence alterations in tumors.     

Associations between milk performance traits in Holstein cows and 16 candidate SNPs identified by arrayed primer extension (APEX) microarray

An oligonucleotide microarray-which allows for parallel genotyping of many SNPs in genes involved in cow milk protein biosynthesis-was used to identify which of the 16 candidate SNPs are associated with milk performance traits in Holstein cows. Four hundred cows were genotyped by the developed and validated microarray. Significant associations were found between four single SNPs, namely DGAT1 (acyloCoA:diacylglycerol acyltransferase), LTF (lactoferrin), CSN3 (kappa-casein), and GHR (growth hormone receptor) and with fat and protein yield and percentage. Many significant associations between combined genotypes (two SNPs) and milk performance traits were found. The associations between the combined genotypes DGAT1/LTF and DGAT1/LEPTIN analyzed traits are presented as examples. The microarray based on APEX (Arrayed Primer Extension) is a fast and reliable method for multiple SNP analysis of potential application in marker-assisted selection. After further development, the chip may prospectively be used for dairy cattle paternity analysis and evolutionary studies.     

Microsatellite genotyping by primer extension and MALDI-ToF mass spectrometry

A novel method for genotyping microsatellite alleles using primer extensions and mass spectrometry analysis has been developed. Following PCR amplification of the target region, a genotyping primer, with its 3′ end directly flanking the microsatellite repeats, was extended by a mixture of dNTPs complementary to the nucleotides composing the microsatellite. The length and molecular weight of extended primers vary with the number of repeats present in the allele(s) under examination. The weights of extension products were determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) and used to identify genotypes on the basis of differential primer extension. This is a platform that is not gel based and is amenable to multiplexing and automation. The technique enables identification of heterozygous progeny in which alleles differ by a single trinucleotide repeat. The method is illustrated by genotyping a polymorphic microsatellite identified in an intron of the barleyMlo gene.     

Fluorescence-based DHPLC for allelic quantification by single-nucleotide primer extension

We have investigated the possibility of determining quantitatively the alleles of binary DNA polymorphisms by single-nucleotide primer extension (SNuPE) and fluorescence-based DHPLC. Using a polymorphism of interest to our group, ROX-labeled dideoxy CTP (ROX-ddCTP) was incorporated at the 3' end of the primer annealed to the template adjacent to the polymorphic site. The primer extension product was then resolved from the unincorporated dye terminator by ion-pair reversed-phase liquid chromatography. The signal intensity of incorporated ROX-ddCTP correlated well over one order of magnitude with the relative amount of the C-allele present in the genomic DNA template. We conclude that SNuPE, when combined with fluorescence-based DHPLC, can accurately determine the relative molar proportion of one allele in total DNA.     

Template-directed primer extension catalyzed by the Tetrahymena ribozyme.

The Tetrahymena ribozyme has been shown to catalyze an RNA polymerase-like reaction in which an RNA primer is extended by the sequential addition of pN nucleotides derived from GpN dinucleotides, where N = A, C, or U. Here, we show that this reaction is influenced by the presence of a template; bases that can form Watson-Crick base pairs with a template add as much as 25-fold more efficiently than mismatched bases. A mutant enzyme with an altered guanosine binding site can catalyze template-directed primer extension with all four bases when supplied with dinucleotides of the form 2-aminopurine-pN.     

SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays

This study reports the development of a microarray-based allele-specific extension method for typing of single nucleotide polymorphisms (SNPs). The use of allele-specific primers has been employed previously to identify single base variations but it is acknowledged that certain mismatches are not refractory to extension. Here we have overcome this limitation by introducing apyrase, a nucleotide-degrading enzyme, to the extension reaction. We have shown previously that DNA polymerases exhibit slower reaction kinetics when extending a mismatched primer compared with a matched primer. This kinetic difference is exploited in the apyrase-mediated allele-specific extension (AMASE) assay, allowing incorporation of nucleotides when the reaction kinetics are fast but degrading the nucleotides before extension when the reaction kinetics are slow. Here we show that five homozygous variants (14% of the total number of variants) that were incorrectly scored in the absence of apyrase were correctly typed when apyrase was included in the extension reaction. AMASE was performed in situ on the oligonucleotide microarrays using fluorescent nucleotides to type 10 SNPs and two indels in 17 individuals generating approximately 200 genotypes. Cluster analysis of these data shows three distinct clusters with clear-cut boundaries. We conclude that SNP typing on oligonucleotide microarrays by AMASE is an efficient, rapid and accurate technique for large-scale genotyping.     

Identifying the targets of aminoacyl-tRNA synthetase inhibitors by primer extension inhibition

Aminoacyl-transfer RNA (tRNA) synthetases (RS) are essential components of the cellular translation machinery and can be exploited for antibiotic discovery. Because cells have many different RS, usually one for each amino acid, identification of the specific enzyme targeted by a new natural or synthetic inhibitor can be cumbersome. We describe the use of the primer extension technique in conjunction with specifically designed synthetic genes to identify the RS targeted by an inhibitor. Suppression of a synthetase activity reduces the amount of the cognate aminoacyl-tRNA in a cell-free translation system resulting in arrest of translation when the corresponding codon enters the decoding center of the ribosome. The utility of the technique is demonstrated by identifying a switch in target specificity of some synthetic inhibitors of threonyl-tRNA synthetase.     

Effects of single-stranded DNA binding proteins on primer extension by telomerase

We present a biochemical analysis of the effects of three single-stranded DNA binding proteins on extension of oligonucleotide primers by the Tetrahymena telomerase. One of them, a human protein designated translin, which was shown to specifically bind the G-rich Tetrahymena and human telomeric repeats, slightly stimulated the primer extension reactions at molar ratios of translin/primer of <1:2. At higher molar ratios, it inhibited the reactions by up to 80%. The inhibition was caused by binding of translin to the primers, rather than by a direct interaction of this protein with telomerase. A second protein, the general human single-stranded DNA binding protein Replication Protein A (RPA), similarly affected the primer extension by telomerase, even though its mode of binding to DNA differs from that of translin. A third protein, the E. coli single-stranded DNA binding protein (SSB), whose binding to DNA is highly cooperative, caused more substantial stimulation and inhibition at the lower and the higher molar ratios of SSB/primer, respectively. Both telomere-specific and general single-stranded DNA binding proteins are found in living cells in telomeric complexes. Based on our data, we propose that these proteins may exert either stimulatory or inhibitory effects on intracellular telomerases, depending on their local concentrations.     

A model for dynamics of primer extension by eukaryotic DNA primase

A mathematical model is proposed for processive primer extension by eukaryotic DNA primase. The model uses available experimental data to predict rate constants for the dynamic behavior of primase activity as a function of NTP concentration. The model also predicts some data such as the binding affinities of the primase for the DNA template and for the RNA primer.     

Genotyping single nucleotide polymorphisms by primer extension and high performance liquid chromatography

We have investigated the possibility of genotyping single nucleotide polymorphisms (SNPs) by primer extension and high performance liquid chromatography (HPLC). Using three polymorphisms of current interest to our group (an A/G polymorphism in the proneurotensin gene and A/G and T/C polymorphisms in the 5HT2a receptor gene), we show that robust signal is obtained using this simple analytic method which has the added advantages that sample loading and analysis are essentially automated, analytic time is brief, and no further purification step after primer extension is required. We also show that all stages of the HPLC-primer extension genotyping can be multiplexed which, together with automation, suggests that this system may be suitable for linkage studies based upon emerging SNP maps. Received: 27 April 1998 / Accepted: 26 November 1998     

Sinapinic acid can replace ascorbate in the biotin switch assay

Background

Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins.

Methods

The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO₂^- released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells ± S-nitrosocysteine (CysNO) exposure.

Results

We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges.

Conclusions

Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay.

General significance

The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay.     

ShcA tyrosine phosphorylation sites can replace ShcA binding in signalling by middle T-antigen.

ShcA and Grb2 are crucial components in signalling by most tyrosine kinase-associated receptors. How ever, it is not clear whether Grb2 bound directly to the receptor is equivalent to Grb2 associated via ShcA. We have used signalling stimulated by the middle T-antigen (MT) of polyoma virus to address this question. The two known Grb2-binding sites from murine ShcA, 313Y and 239/240YY, could functionally replace the MT ShcA-interacting region in transformation assays using Rat2 fibroblasts. This demonstrates that signal output from membrane-bound ShcA requires only these two sequences and the ShcA-binding site in MT does not recruit other signalling molecules. Two standard Grb2-interacting sequences, either from the EGF receptor or the ShcA 313Y region, could not replace the requirement for ShcA binding to MT, indicating an enhanced role for the ShcA 239/240YY motif. Sos1 and the docking protein Gab1 are brought into the MT complex through Grb2 association and this may be more effective using the 239/240YY sequence.     

Rapid genotyping of loci involved in antifolate drug resistance in Plasmodium falciparum by primer extension

Current methods used to genotype point mutations in Plasmodium falciparum genes involved in resistance to antifolate drugs include restriction digestion of PCR products, allele-specific amplification or sequencing. Here we demonstrate that known point mutations in dihydrofolate reductase and dihydropteroate synthase can be scored quickly and accurately by single-nucleotide primer extension and detection of florescent products on a capillary sequencer. We use this method to genotype parasites in natural infections from the Thai-Myanmar border. This approach could greatly simplify large-scale screening of resistance mutations of the type required for evaluating and updating antimalarial drug treatment policies. The method can be easily adapted to other P. falciparum genes and will greatly simplify scoring of point mutations in this and other parasitic organisms.     

Preparation of ferrocene-functionalized gold nanoparticles by primer extension reaction on the particle surface

DNA molecules possessing multiple ferrocene (Fc) molecules as a redox active probe were prepared by the primer extension (PEX) reaction using a 2′-deoxyuridine-5′-triphosphate derivative in which Fc was connected to the C5-position of the uridine by a diethylene glycol linker. Gold nanoparticles (AuNP) covered with DNA possessing the Fc molecules were prepared by the PEX reaction on the surface. The AuNP–FcDNA conjugates exhibit a detectable electrochemical signal due to the Fc molecules. Possible application of the PEX reaction on AuNP is demonstrated for the detection of a single nucleotide mutation in the target DNA.