Multiplex fluorescence-based primer extension method for quantitative mutation analysis of mitochondrial DNA and its diagnostic application for Alzheimer's disease

A sensitive and highly reproducible multiplexed primer extension assay is described for quantitative mutation analysis of heterogeneous DNA populations. Wild-type and mutant target DNA are simultaneously probed in competitive primer extension reactions using fluorophor-labeled primers and high fidelity, thermostable DNA polymerases in the presence of defined mixtures of deoxy- and dideoxynucleotides. Primers are differentially extended and the resulting products are distinguished by size and dye label. Wild-type:mutant DNA ratios are determined from the fluorescence intensities associated with electrophoretically resolved reaction products. Multiple nucleotide sites can be simultaneously interrogated with uniquely labeled primers of different lengths. The application of this quantitative technique is shown in the analysis of heteroplasmic point mutations in mitochondrial DNA that are associated with Alzheimer's disease.     

Molecular diagnostic tests for ascertainment of genotype at the mucopolysaccharidosis type VII locus in dogs

OBJECTIVE: To develop a molecular diagnostic test to ascertain genotype of the mucopolysaccharidosis type VII (MPS VII) locus. SAMPLE POPULATION: Blood samples from 45 mixed-breed (German Shepherd Dog X Beagle) dogs that were phenotypically normal or affected with MPSVII. PROCEDURE: The canine beta-glucuronidase gene (exon 3) was amplified by polymerase chain reaction (PCR), using 2 pairs of primers to determine the genotype of the MPSVII locus by 2 independent methods. For the first method, PCR products were used for heteroduplex analysis, using conformation-sensitive gel electrophoresis. In the second method, an allele-specific restriction site was created by use of a mismatch primer in PCR. The amplified DNA fragment was digested with a restriction enzyme (Eag I) to enable identification of the wild-type and mutant alleles. RESULTS: Conformation-sensitive gel electrophoresis resulted in a single DNA band representing homoduplex when the sample contained a wild-type or MPS VII allele, but 2 bands representing hetero- and homoduplexes when both alleles were in the sample. Restriction digestion of the DNA fragment obtained by use of a mismatch primer was cleaved only when the template was a wild-type allele. Thus, samples from phenotypically normal carrier dogs that contained wild-type and MPS VII alleles were partially digested by the enzyme. CONCLUSIONS: The diagnostic test used 2 strategies for independently ascertaining the wild-type or mutant MPS VII alleles in dogs. Thus, test results can distinguish phenotypically normal MPS VII-carrier dogs from homozygous normal dogs.     

Detection of basepair substitution mutation at a frequency of 1 x 10(-7) by combining two genotypic selection methods, MutEx enrichment and allele-specific competitive blocker PCR

The detection of rare mutations has many important applications, including risk assessment of drugs and chemicals, measuring environmental exposures to genotoxins, and cancer cell detection. A sensitive genotypic selection method has been developed that combines two different mutant allele selection techniques, MutEx enrichment and allele-specific competitive blocker PCR (ACB-PCR). This method was developed and evaluated for the detection of a CAA --> AAA mutation at codon 61 of the mouse H-ras gene. The MutEx enrichment is based on MutS binding to a mismatched basepair in heteroduplex DNA. The bound MutS protects the mutant allele from degradation during subsequent exonuclease treatment. ACB-PCR preferentially amplifies a mutant allele in a PCR reaction using a primer that has more mismatches to the wild-type allele than the mutant allele. By combining these two approaches, the codon 61 mutation was detected at mutant fractions as low as 1 in 10(7). This sensitivity was achieved with the thermostable Thermus aquaticus MutS protein but not the Escherichia coli MutS protein. Using the combined approach, the average Pfu DNA polymerase error rate +/- the standard error of the mean for this particular basepair was estimated to be 8 +/- 3 x 10(-7) errors per duplication. The results indicate that MutEx/ACB-PCR is among the most sensitive genotypic selection methods for the detection of mutation.     

Tacrolimus (FK506): validation of a sensitive enzyme-linked immunosorbent assay kit for and application to a clinical pharmacokinetic study

OBJECTIVES: Hepatitis B virus (HBV) with a stop mutation at precore codon 28 (TGG-->TAG, tryptophan-->stop) was investigated to clarify if such a mutant virus might play a role in hepatocarcinogenesis. METHODS: A total of 73 patients with HBV-related hepatocellular carcinoma were included in this study. Polymerase chain reaction (PCR) was performed in DNA samples extracted from 73 sera to amplify a HBV-DNA segment involving the precore and proximal core regions, and sequences of PCR products were analyzed to see the presence of the mutations at precore codon 28 by a direct sequencing method. RESULTS: HBV-DNA was detectable in 64 (88%) patients by PCR. The stop mutation at precore codon 28 was identified in 50 of 58 PCR products (86%), in which direct sequencing was performed. Among patients with this mutant HBV, 21/50 (42%) patients were co-infected with wild-type HBV. The mutant virus was found in 23/28 (82%) patients with hepatitis B e antigen (HBeAg) and 27/30 (90%) patients without HBeAg. The mutant HBV alone was found in 10/28 (36%) patients with HBeAg and 19/30 (63%) without HBeAg. Among those patients on whom laparoscopy was performed, 22/24 (92%) with the precore codon 28 stop mutant alone had cirrhosis, compared to 12/19 (63%) co-infected by both the mutant and the wild-type (p < 0.05). The association of this mutant virus with both the presence and absence of HBeAg, and its association with cirrhosis when there is no co-infection with wild-type HBV, suggests an evolving pattern of liver pathology. CONCLUSION: The high prevalence of a stop mutation at precore codon 28 in these patients with hepatocellular carcinoma suggests that HBV with this mutation may contribute to the development of hepatocellular carcinoma.     

Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays

We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.     

STR DNA typing: increased sensitivity and efficient sample consumption using reduced PCR reaction volumes

Improvements in detection limits/sensitivity and lower sample consumption are potential benefits of reducing PCR reaction volumes used in forensic DNA typing of crime scene samples. This premise was studied first with experimental mixtures and a nine-loci megaplex, which demonstrated stochiometric amplification and accurate detection. Next, adjudicated casework samples were subjected to amplification under 15 different template DNA to PCR reaction volume ratios. Reduction of PCR reaction volume and DNA down to 10 microL and 0.500 ng, respectively, produced identical profiles with the same signal intensity and heterozygous allele peak height ratio (HR). Reduction to 5 microL and 0.063 ng yielded HR values that were slightly affected in one to three STR loci. PCR reaction volume reduction can enhance detection and sensitivity while reducing the consumption of irreplaceable crime scene samples.     

Rapid diagnostic test for hereditary nonpolyposis colon cancer kindred using polymerase chain reaction

PURPOSE: We have identified a mutation in the hMLH1 gene from the proband of a hereditary nonpolyposis colorectal cancer kindred. We wished to develop a rapid test for this specific mutation to facilitate screening of other family members. METHOD: An allele-specific polymerase chain reaction strategy was used to detect a T insertion at the + 3 splice site post exon 9 in the hMLH1 gene. The test was evaluated on DNA in which the mutation status was known. RESULTS: A 130-base pair fragment was reliably amplified using the allele-specific polymerase chain reaction. The test is able to identify the mutant allele and to distinguish between normal, carriers (heterozygous), and tumor DNA samples. The mutant allele is not present in an unrelated hereditary nonpolyposis colorectal cancer cell line or in a sample of the normal population (n=49). CONCLUSIONS: This is a simple, rapid test that can determine carrier status in the members of a kindred at risk for this mutation. This mutation is unlikely to be a polymorphism. This test may now be evaluated in a clinical setting.     

Model multiple antigenic and homopolymeric peptides from non-repetitive sequences of malaria merozoite proteins elicit biologically irrelevant antibodies

We describe here a new type of mitochondrial mutation (dum24; for dark uniparental minus inheritance) of the unicellular photosynthetic alga Chlamydomonas reinhardtii. The mutant fails to grow under heterotrophic conditions and displays reduced growth under both photoautotrophic and mixotrophic conditions. In reciprocal crosses between mutant and wild-type cells, the meiotic progeny only inherit the phenotype of the mating-type minus parent, indicating that the dum24 mutation exclusively affects the mitochondrial genome. Digestion with various restriction enzymes followed by DNA gel blot hybridizations with specific probes demonstrated that dum24 cells contain four types of altered mitochondrial genomes: deleted monomers lacking cob, nd4, and the 3' end of the nd5 gene; deleted monomers deprived of cob, nd4, nd5, and the 5' end of the cox1 coding sequence; and two types of dimers produced by end-to-end fusions between monomers similarly or differently deleted. Due to these mitochondrial DNA alterations, complex I activity, the cytochrome pathway of respiration, and presumably, the three phosphorylation sites associated with these enzyme activities are lacking in the mutant. The low respiratory rate of the dum24 cells results from the activities of rotenone-resistant NADH dehydrogenase, complex II, and alternative oxidase, with none of these enzymes being coupled to ATP production. To our knowledge, this type of mitochondrial mutation has never been described for photosynthetic organisms or more generally for obligate aerobes.     

A mutation in a novel yeast proteasomal gene, RPN11/MPR1, produces a cell cycle arrest, overreplication of nuclear and mitochondrial DNA, and an altered mitochondrial morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24 degreesC growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36 degreesC on either carbon source. Microscopic observation of cells growing on glucose at 24 degreesC shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho degrees] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.     

Single-fiber polymerase chain reaction for detection of mutant mitochondrial DNA]

Single-fiber PCR amplifies mitochondrial DNA (mtDNA) in single muscle fiber isolated from cross frozen section. The PCR products are digested with a restriction enzyme to distinguish mutant mtDNA from wild-type mtDNA. The proportion of mutant mtDNA is higher in ragged-red fiber (RRF) than in non-RRF in mitochondrial encephalomyopathies with mutations of mtDNA. This method may be applied to evaluate amount of mtDNA and mRNA in single muscle fiber, and become a powerful tool to elucidate the pathogenetic mechanism in mitochondrial encephalomyopathies.     

Inhibition of PCR amplification by a point mutation downstream of a primer

A T-->C point mutation is shown to specifically inhibit PCR amplification when compared to wild-type controls in exon H of the factor IX gene. Multiple primers of different lengths and locations were designed to examine this phenomenon. The experiments suggest that poor annealing and/or extension from the downstream primer are responsible for the observed inhibition and that the mutation can exert an inhibitory effect upon PCR amplification at a distance of at least 84 bp. The inhibition was not alleviated when amplification conditions such as annealing temperature, time of extension, type of DNA polymerase or concentration of DNA template, primer or DNA polymerase were varied. The inhibitory factor(s) are likely to be contained within the amplified segment itself because neither the use of a previously amplified PCR product as template for nested PCRs nor the restriction enzyme digestion of that previously amplified product relieved the inhibition of PCR amplification in the mutant sample. Computer analyses with the FOLDRNA and FOLDDNA programs did not reveal the mechanism of inhibition. Although dramatic inhibition, as shown here, may be uncommon, more subtle inhibition may be frequent. Documentation of differential amplification caused by a single-base substitution in template sequence has implications for certain commonly used PCR-based methods such as quantitative PCR, differential display and DNA fingerprinting. In addition, heterozygous single-base pair mutations down-stream of a primer may be missed if the PCR is inhibited; alternatively; the mutation may appear to be homozygous if amplification of the mutated allele is selectively enhanced.     

The RFC2 gene, encoding the third-largest subunit of the replication factor C complex, is required for an S-phase checkpoint in Saccharomyces cerevisiae

Replication factor C (RF-C), an auxiliary factor for DNA polymerases delta and epsilon, is a multiprotein complex consisting of five different polypeptides. It recognizes a primer on a template DNA, binds to a primer terminus, and helps load proliferating cell nuclear antigen onto the DNA template. The RFC2 gene encodes the third-largest subunit of the RF-C complex. To elucidate the role of this subunit in DNA metabolism, we isolated a thermosensitive mutation (rfc2-1) in the RFC2 gene. It was shown that mutant cells having the rfc2-1 mutation exhibit (i) temperature-sensitive cell growth; (ii) defects in the integrity of chromosomal DNA at restrictive temperatures; (iii) progression through cell cycle without definitive terminal morphology and rapid loss of cell viability at restrictive temperatures; (iv) sensitivity to hydroxyurea, methyl methanesulfonate, and UV light; and (v) increased rate of spontaneous mitotic recombination and chromosome loss. These phenotypes of the mutant suggest that the RFC2 gene product is required not only for chromosomal DNA replication but also for a cell cycle checkpoint. It was also shown that the rfc2-1 mutation is synthetically lethal with either the cdc44-1 or rfc5-1 mutation and that the restrictive temperature of rfc2-1 mutant cells can be lowered by combining either with the cdc2-2 or pol2-11 mutation. Finally, it was shown that the temperature-sensitive cell growth phenotype and checkpoint defect of the rfc2-1 mutation can be suppressed by a multicopy plasmid containing the RFC5 gene. These results suggest that the RFC2 gene product interacts with the CDC44/RFC1 and RFC5 gene products in the RF-C complex and with both DNA polymerases delta and epsilon during chromosomal DNA replication.     

Analysis of localization of mutated tissue-nonspecific alkaline phosphatase proteins associated with neonatal hypophosphatasia using green fluorescent protein chimeras

Hypophosphatasia is associated with a defect of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The onset and clinical severity are usually correlated in hypophosphatasia; patients with perinatal hypophosphatasia die approximately at the time of birth. In contrast, we describe a male neonatal patient with hypophosphatasia who had no respiratory problems and survived. He was compound heterozygous for the conversion of Phe to Leu at codon 310 (F310L) and the deletion of a nucleotide T at 1735 (delT1735), causing the frame shift with the result of the addition of 80 amino acids at the C-terminal of the protein. Because the C-terminal portion of TNSALP is known to be important for TNSALP to bind to the plasma membrane, the localization of wild-type and mutated TNSALP proteins was analyzed using green fluorescent protein chimeras. The expression vectors containing the complementary DNA of fusion proteins consisting of signal peptide, green fluorescent protein, and wild-type or mutated TNSALP, caused by delT1735 or F310L mutation, were introduced transiently or stably in Saos-2 cells. The delT1735 mutant failed to localize at the cell surface membrane, whereas the wild-type and the F310L mutants were located in the plasma membrane and cytoplasm. The assay for enzymatic activity of TNSALP revealed that the delT1735 mutant lost the activity and that the F310L mutant exhibited an enzymatic activity level that was 72% of the normal level. The F310L mutation was also detected in another neonatal patient with relatively mild (nonlethal) hypophosphatasia (reported in J Clin Endocrinol Metab, 81:4458-4461, 1996), suggesting that residual ALP activity of the F310L mutant contributes to the less severe phenotype. The patient is unique, with respect to a discrepancy between onset and clinical severity in hypophosphatasia.     

CCR: a rapid and simple approach for mutation detection

We describe a simple approach for detecting known mutations in genomic DNA. The strategy entails a DNA amplification reaction that combines the use of thermostable DNA polymerase and ligase, and that has been designated the Combined Chain Reaction (CCR). CCR consists of four phases: denaturation, annealing, elongation and ligation. Unlike most PCR-based mutation detection systems it relies on mismatch between primer and template at the primer 5'ends. It is rapid and simple, and requires neither the use of radioactivity, nor polyacrylamide gel electrophoresis, nor autoradiography for mutation detection at the single base-pair level.     

Nested PCR assay for detection of granulocytic ehrlichiae

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.     

Influence of the formamidine pesticide amitraz and its metabolites on porcine myometrial contractility: involvement of alpha 2-adrenoceptors and Ca2+ channels

We report an application of multiprimed polymerase chain reaction (PCR) which allows a rapid, nonradioactive detection of deletions in mitochondrial DNA using EDTA-blood and muscle samples. The use of two primer sets consisting of three forward and five reverse primers, respectively, allows a competitive PCR resulting in significant amplification products only in the presence of deletion-harbouring DNA species. Under the conditions described, deletions causing Kearns-Sayre syndrome (KSS) and progressive external ophthalmoplegia (PEO) have been successfully detected. The location of the primers on mitochondrial DNA used in this study should allow identification and localization of most of the large-scale deletions (i.e. more than 1 kb) of mitochondrial DNA reported so far.     

Direct detection of multiple point mutations in mitochondrial DNA

Mitochondrial defects can be caused by mutations in nuclear or mitochondrial DNA. Large deletion/duplication and point mutations are the two major types of mitochondrial DNA (mtDNA) mutations. Comprehensive molecular diagnosis requires the analysis of multiple point mutations. We developed an effective multiplex PCR/allele-specific oligonucleotide (ASO) method to simultaneously screen multiple point mutations in mtDNA. The system involved three pairs of primers to amplify mutation "hot spots" at tRNA(leu(UUR)), tRNA(lys)/ATPase, and ND4 regions, followed by detection of point mutations with ASO probes. Over 2000 specimens were analyzed and the results were compared with those from previous studies with the PCR/restriction fragment length polymorphism method. Our data demonstrate that the multiplex PCR/ASO method is much more sensitive in the detection of low mutant heteroplasmy. It is simple and cost effective, especially if a large number of samples are to be screened for multiple point mutations.