A system for the quantitation of DNA using a microtiter plate-based hybridization and enzyme amplification technology

A quantitative hybridization technique for the detection of plasmid DNA by the action of a nuclease enzyme is described. The process utilizes the specific capture and detection of a sandwich hybridization, in a microtiter plate, that occurs in a single step. The detector probe is labeled with nuclease P1. The pH-dependent specificity of this enzyme for 3'-dinucleotides is used to generate a measurable signal by activating apo-glucose oxidase, which triggers an enzyme amplification cascade in the same microtiter plate. The sensitivity of the assay system is demonstrated in an assay of a mutated form of the human pancreatic ribonuclease gene inserted into the plasmid pUC 18. The system was able to detect 35 amol of target DNA in an assay composed of a 60-min hybridization and 20 min of signal generation. This use of nuclease P1 as the enzyme label and apo-glucose oxidase as the trigger for the amplification cascade results in an assay that is more sensitive than previously described enzyme amplification systems using colorimetric detection.     

Gene amplification in Chinese hamster DNA repair deficient mutants

In order to study the possible relationship between gene amplification and DNA repair we analyzed the amplification of the CAD gene in four mutants hypersensitive to UV light (CHO43RO, CHO7PV, UV5 and UV61) isolated in vitro from Chinese hamster cell lines (CHO-K1 and AA8). These mutants are characterized by different defects in the nucleotide excision repair mechanism and represent complementation groups 1, 9, 2, and 6 respectively. To evaluate the amplification ability of each cell line we measured the rate of appearance of PALA resistant clones with the Luria and Delbrück fluctuation test. Resistance to PALA is mainly due to amplification of the CAD gene. In the mutants CHO43RO, UV5 and CHO7PV we reproducibly found an amplification rate lower than in the parental cell lines (2-5 times), while in UV61 the amplification rate was about 4 times higher. This result indicates that each mutant is characterized by a specific amplification ability and that the unefficient removal of UV induced DNA damage can be associated with either a higher or a lower amplification rate. However, the analysis of randomly isolated CHO-K1 clones with normal UV sensitivity has shown variability in their amplification ability, making it difficult to relate the specific amplification ability of the mutants to the DNA repair defect and suggesting clonal heterogeneity of the parental population.     

Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: an analysis of resected gastric carcinoma

Although several factors affecting the sensitivity of polymerase chain reaction (PCR) amplification from formalin-fixed tissues have been investigated mostly by experiments, the feasibility of archival formalin-fixed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2-3 days and 14% (4/28) of the samples fixed for 4-6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified p53 products from archival tissues using a non-isotopic method, temperature gradient gel electrophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding just after 1 day or less fixation for subsequent use in PCR amplification.     

Optimization of a hexaplex DNA amplification from short tandem repeat and amelogenin loci

An automated DNA profiling system based on the multiplex amplification of highly polymorphic short tandem repeat (STR) markers and the amelogenin locus was developed. Five STR loci with nonoverlapping allele size ranges have been utilized in the multiplex amplifications, including HUMD1S103, HUMTH01, HUMD21S11, HUMD18S51, and HUMFIBRA. One primer for each locus was labeled with a fluorescent dye (fluorescein) which allows detection on the single wavelength ALF DNA Sequencer (Pharmacia Biotech). As part of the detailed evaluation of the suitability of the hexaplex system for routine forensic use, the effect of variation in amplification parameters on the efficiency of the system was examined. Polymerase chain reaction amplification conditions were optimized to provide specific, robust amplification of forensic samples.     

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1-1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)-this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.     

Simultaneous analysis of various mutations on the 21-hydroxylase gene by multi-allele specific amplification and capillary gel electrophoresis

A detailed study is presented on the detection of various known point mutations using polymerase chain reaction (PCR) based multi-allele specific amplification (MASA) in conjunction with capillary gel electrophoresis (CGE) separation. The resulting PCR products, corresponding to the individual mutations, are labeled with ethidium bromide during CGE separation, and detected by laser-induced fluorescence. MASA proved to be a novel, fast and cost-effective method for simultaneous analysis of multiple known mutation sites, employing more than one allele specific primers in a single PCR reaction. It results in coexisting amplification of numerous DNA fragments differing in size, which are subsequently separated by CGE. In the present study, several point mutations were analyzed simultaneously by MASA-CGE on the 21-hydroxylase gene of a patient with congenital adrenal hyperplasia.     

Benefits of linear amplification and multichannel compression for speech comprehension in backgrounds with spectral and temporal dips

People with cochlear hearing loss have markedly higher speech-receptions thresholds (SRTs) than normal for speech presented in background sounds with spectral and/or temporal dips. This article examines the extent to which SRTs can be improved by linear amplification with appropriate frequency-response shaping, and by fast-acting wide-dynamic-range compression amplification with one, two, four, or eight channels. Eighteen elderly subjects with moderate to severe hearing loss were tested. SRTs for sentences were measured for four background sounds, presented at a nominal level (prior to amplification) of 65 dB SPL: (1) A single female talker, digitally filtered so that the long-term average spectrum matched that of the target speech; (2) a noise with the same average spectrum as the target speech, but with the temporal envelope of the single talker; (3) a noise with the same overall spectral shape as the target speech, but filtered so as to have 4 equivalent-rectangular-bandwidth (ERB) wide spectral notches at several frequencies; (4) a noise with both spectral and temporal dips obtained by applying the temporal envelope of a single talker to speech-shaped noise [as in (2)] and then filtering that noise [as in (3)]. Mean SRTs were 5-6 dB lower (better) in all of the conditions with amplification than for unaided listening. SRTs were significantly lower for the systems with one-, four-, and eight-channel compression than for linear amplification, although the benefit, averaged across subjects, was typically only 0.5 to 0.9 dB. The lowest mean SRT (-9.9 dB, expressed as a speech-to-background ratio) was obtained for noise (4) and the system with eight-channel compression. This is about 6 dB worse than for elderly subjects with near-normal hearing, when tested without amplification. It is concluded that amplification, and especially fast-acting compression amplification, can improve the ability to understand speech in background sounds with spectral and temporal dips, but it does not restore performance to normal.     

Consonant perception with linear and compression amplification

The consonant perception of 15 subjects with mild to moderate sensorineural hearing loss was evaluated using linear amplification and two different types of compression amplification. A specially modified hearing aid was used which allowed for variation of the amplifier input/output function in three steps, such that the compression ratio could be set to 1 (linear), 1.3 or 1.8. The Nonsense Syllable Test (NST) was recorded through the aid in quiet and in two different noise conditions (four-talker babble and a background noise with sharp intermittent sounds), and replayed to the listeners through headphones. No differences in consonant perception were found between the different types of amplification in the quiet condition. In the babble condition, consonant perception was significantly better with linear amplification than with either form of compression. In the sharp noise condition, there was no difference in performance between linear amplification and compression amplification with the ratio of 1.8. Consonant perception was adversely affected, however, by the compression and amplification with the ratio of 1.3 in this condition. Overall NST results and results for particular classes of consonants are discussed.     

A wide-range survey of cross-species microsatellite amplification in birds

The possibility to perform cross-species microsatellite amplification in birds was surveyed by analysing sets of primers developed from the swallow and the pied flycatcher genomes on a panel of 48 different bird species. In total, 162 cases (species/marker combinations) of heterologous amplification were recorded. Ten amplification products were sequenced and all were found to be true homologues of the original loci. There was a significant and negative relationship between microsatellite performance and evolutionary distance between the original species and the tested species. As a rough indicator of expected cross-species microsatellite performance we estimate that 50% of markers will reveal polymorphism in a species with a DNA-DNA hybridization delta T(m)H value of 5 separating it from the original species. This corresponds to a divergence time of = 11 million years before present for passerine birds. The established relationship between performance and evolutionary distance agrees very well with data obtained from some mammalian species. The proportion of polymorphic loci among those markers that amplified decreased with increasing genetic distance, suggesting that few long repeats are preserved during evolution. One of the swallow markers, HrU2, amplified a specific product in all species analyzed and will thus allow access of nuclear sequence data over a broad range of species. The only predictor of cross-species performance was the amount of non-specific amplification seen in the original species. An analysis of 10 species from within the family Hirundinidae with the swallow primers consistently revealed extensive polymorphism with average probabilities of identical genotypes ranging from 6 x 10(-4) to 6 x 10(-7). There were distinct allele frequency differences between the Hirundinidae species and we envisage that microsatellite cross-species amplification will be a useful tool in phylogeny construction and in species identification.     

Heterogeneity of MYCN amplification in a child with stroma-rich neuroblastoma (ganglioneuroblastoma)

Amplification of MYCN portends rapid tumor progression and poor prognosis in neuroblastoma. MYCN copy number has been described as homogeneous within a tumor and congruent in primary tumor and metastasis. We report a child with stage III favorable histology stroma-rich neuroblastoma (ganglioneuroblastoma) and a poor outcome with an apparent change in MYCN gene amplification by Southern blot. Initial biopsy revealed a ganglioneuroblastoma with predominance of differentiating cells designated as neuroblastoma, stroma-rich, intermixed (Shimada). Southern blot failed to demonstrate MYCN gene amplification. After front-line chemotherapy failed, a total resection was performed. In this specimen, Southern blot demonstrated MYCN amplification (15-20 copies) in the undifferentiated component and no amplification in the differentiated. Fluorescence in situ hybridization (FISH) analysis performed retrospectively on both tumor biopsies demonstrated MYCN amplification in the undifferentiated sections of both tumor specimens but not in the differentiated ones. This is the first well-documented case report of heterogeneous MYCN amplification in a child with neuroblastoma. Because key therapeutic decisions are based on the presence of MYCN amplification, physicians diagnosing and treating children with neuroblastoma need to be aware of the possibility that MYCN amplification may be heterogeneous within a tumor and may be missed using techniques based on pooled DNA such as Southern blotting. FISH may be a preferable method for determining MYCN amplification.     

Combined determination of N-myc oncogene amplification and DNA ploidy in neuroblastoma. Complementary prognostic indicators

BACKGROUND: N-myc gene amplification is a well-established prognostic indicator in neuroblastoma. Flow cytometric analysis of nuclear DNA content has shown that an abnormal nuclear DNA content in neuroblastoma is associated with a better prognosis. Because some patients with N-myc unamplified tumors have a poor prognosis, factors other than N-myc amplification may play a role in determining the clinical behavior of neuroblastoma. In the current study, the authors correlated N-myc gene amplification and flow cytometric nuclear DNA content with respect to prognosis. METHODS: Forty-one patients with neuroblastoma, including 15 screened patients, served as subjects. The copy number of the N-myc gene was determined by Southern blot analysis. DNA ploidy analysis was done on nuclei isolated from formalin-fixed, paraffin-embedded blocks. RESULTS: Of 40 specimens of neuroblastoma, 7 involved tumors containing amplification of the N-myc gene and 33 did not; 13 specimens showed DNA diploidy, and 27 showed DNA aneuploidy (including 4 with DNA tetraploidy). The Kaplan-Meier survival analysis indicated a significantly better prognosis in patients with unamplified N-myc tumors compared with those with N-myc amplified tumors (87.3% versus 28.6%, P < 0.05) and in patients with DNA aneuploid tumors compared with those with DNA diploid tumors (96.3% versus 43.0%, P < 0.001). The difference in the survival of the two extreme combinations, (e.g., 25 with N-myc unamplified and DNA aneuploidy [4 tetraploidy] versus 5 with N-myc amplified and DNA diploidy) was more significant (96.0% versus 20.0%, P < 0.001) than any other combination. CONCLUSION: Evaluations of N-myc gene amplification and DNA ploidy are complementary, and the combined determination of these two factors may be one of the most powerful prognostic indicators in neuroblastoma.     

DNA extraction and amplification from Giemsa-stained blood smears

DNA extraction was attempted from Giemsa-stained blood smears on glass slides that had been stored for several years. High molecular weight DNA bands were clearly visible after electrophoresis when DNA was extracted from specimens stored up to 2 years. Specimens more than 4 years old demonstrated a smeared pattern, suggesting degeneration of the DNA, but they could be rescued by PCR amplification using primers of HLA-DQA1 genes. The recovery could be pursued even in 11-year-old specimens.     

Cell cycle control of chorion gene amplification

Over-replication of two clusters of chorion genes in Drosophila ovarian follicle cells is essential for rapid eggshell biosynthesis. The relationship of this amplification to the follicle cell cycles has remained unclear. To investigate the regulation of amplification, we developed a technique to detect amplifying chorion genes in individual follicle cells using BrdU incorporation and FISH. Amplification occurs in two developmental phases. One of the gene clusters begins to amplify periodically during S phases of follicle cell endocycles. Subsequently, after endocycles have ceased, both clusters amplify continuously during the remainder of oogenesis. In contrast to the early phase, late amplification commences synchronously among follicle cells. The pattern of Cyclin E expression mirrors these two phases. We present evidence that Cyclin E is required positively for amplification. We suggest that Cyclin E also acts negatively to inhibit refiring of most origins within a cycle, and that specific factors at chorion origins allow them to escape this negative rereplication control. Our findings suggest that chorion amplification is a model for understanding metazoan replicons and the controls that restrict replication to once per cell cycle.     

Prognostic value of amplification of c-erb-B2 in bladder carcinoma

Amplification of c-erb-B2 is examined in patients with transitional cell bladder carcinoma. In a pilot study we found that the amplification correlated with high tumor grade. Tumor grade is a known prognostic factor. Therefore, we next examined the prognostic value of c-erb-B2 amplification in patients with >16 years of clinical follow-up. The gene copy number was determined, using semiquantitative PCR, in archival formalin-fixed tissues. Twenty-three percent (37/163 patients) showed the amplification. The amplification correlated with grade and stage. Moreover, we found that tumor grade (P < 0.001) and c-erb-B2 amplification (P < 0.001) showed prognostic information for survival. Patients with grade 3 tumors and concomitant c-erb-B2 amplification showed the worst prognosis. Multivariate analysis indicates that grade and c-erb-B2 amplification are independent prognostic factors.     

Bone conduction implants for amplification: comparison of results

We compared the results from the North American patient database on the Xomed Audiant Bone Conductor to those reported on the NobleBiocare (previously Noblepharma) HC200 bone-anchored hearing aid (BAHA) implant, using the literature and specific results provided by one of the authors. It has been proposed that the percutaneous coupling of the NobleBiocare implant transduces energy more powerfully than the Audiant transcutaneous coupling. If true, percutaneous coupling could provide greater amplification, helping patients experiencing both conductive and sensorineural hearing loss. Aided sound-field thresholds corresponding to bone-conduction thresholds were compared retrospectively through the speech frequencies. Both the BAHA and the Audiant devices amplified in the sound field to approximate preoperative bone-conduction thresholds. No statistically significant differences existed between the amplification of warble tones through the speech frequencies for either device. We conclude that amplification with the Audiant device offers as much gain as the HC200 device through the speech frequencies. While both devices can supply effective amplification for select patients suffering from conductive hearing loss, neither provides gain superior to preoperative bone-conduction thresholds to address the needs of select patients with a substantial sensorineural component.     

Regulation and mechanisms of gene amplification

Amplification in rodent cells usually involves bridge-breakage-fusion (BBF) cycles initiated either by end-to-end fusion of sister chromatids, or by chromosome breakage. In contrast, in human cells, resistance to the antimetabolite N-(phosphonacetyl)-L-aspartate (PALA) can be mediated by several different mechanisms that lead to overexpression of the target enzyme carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase (CAD). Mechanisms involving BBF cycles account for only a minority of CAD amplification events in the human fibrosarcoma cell line HT 1080. Here, formation of a 2p isochromosome and overexpression of CAD by other types of amplification events (and even without amplification) are much more prevalent. Broken DNA is recognized by mammalian cells with intact damage-recognition pathways, as a signal to arrest or to die. Loss of these pathways by, for example, loss of p53 or pRb tumour suppressor function, or by increased expression of ras and myc oncogenes, causes non-permissive rat and human cells to become permissive both for amplification and for other manifestations of DNA damage. In cells that are already permissive, amplification can be stimulated by overexpressing oncogenes such as c-myc or ras, or by damaging DNA in a variety of ways. To supplement genetic analysis of amplification in mammalian cells, an amplification selection has been established in Schizosaccharomyces pombe. Selection with LiCl yields cells with amplified sod2 genes in structures related to those observed in mammalian cells. The effect on amplification in S. pombe can now be tested for any mutation in a gene involved in repair of damaged DNA or in normal cellular responses to DNA damage.     

5'-degenerate 3'-dideoxy-terminated competitors of PCR primers increase specificity of amplification

Amplification of a product in PCR with specific primers may be viewed as an artificial Darwinian-type "selection of the fittest". In other selective systems, such as general evolution, immune system and probably brain cortex, the stringency of selection is not absolute but rather degenerate, with selection of many highly fit units, not limited, however, to only the fittest. In PCR also, annealing of the primers is not absolutely specific. The subsequent amplification frequently leads to amplification of not only the desired product but also to less-specific sequences. Using theoretical analysis of the degenerate mode of selection, we predict theoretically and prove experimentally that 5'-degenerate, 3'-dideoxy-terminated competitors of PCR primers can be used to dramatically improve the specificity of PCR amplification without affecting the quantitation of the final specific product.     

In vitro evolution of molecular cooperation in CATCH, a cooperatively coupled amplification system

BACKGROUND: One of the key issues in the investigation of evolution is how complex systems evolved from simple chemical replicators. Theoretical work proposed several models in which complex replicating systems are kinetically stabilized. The development of powerful isothermal amplification technique allows complex nucleic acid based evolving in vitro systems to be set up, which may then serve to verify experimentally current theories of evolution. Recently such a system based on the 3SR (self-sustained sequence replication) reaction has been established to investigate the evolution of cooperation: the trans-cooperatively coupled CATCH (cooperative amplification by cross hybridization). RESULTS: Over four rounds of serial transfer, the cooperatively coupled two species CATCH system evolved into a more complex cooperative four species system, which then was overgrown by CATCH-derived RNA-Z-like hairpin species. In contrast to the classical RNA-Z species, these molecules have complementary loop sequences and self-amplify using a dual mechanism that includes concentration-dependent phases of noncooperative and cooperative amplification. CONCLUSIONS: The evolution of a cooperative system, under conditions that were alternately unfavorable and favorable for cooperative amplification, led to a system showing facultative cooperation. This principle of facultative cooperation preserves the complexity of the system investigated and could have general implications for the evolution and stabilization of cooperation under oscillating reaction conditions.