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Results for “amplification”

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PubMed1983

[Amplification of portions of the genome in the somatic cells of mammals resistant to colchicine. V. The induction of gene amplification in the cells of the Djungarian hamster and the mouse].

The influence of some agents on gene amplification in Djungarian hamster and mouse cells was studied. The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), the epidermal growth factor (EGF), insulin, and 5-bromodeoxyuridine (BUdR) increase the incidence of colchicine-resistance, connected with amplification of the genes, which probably encode the polypeptide p22. The highest frequency of gene amplification was observed after the pretreatment of cells with TPA, which enhanced the number of colchicine-resistant colonies 44-200-fold. Mitostatic agents colchicine and colcemid increased the number of methotrexate-resistant cells, 2.0-6.5 times. These cells usually arise as the result of amplification of dihydrofolate reductase genes. Dexamethasone and ethidium bromide did not change the portion of cells resistant to colchicine. Ethylmethane sulfonate (EMS) decreased the number of colchicine-resistant cells. The cells of two Djungarian hamster colchicine-resistant clones obtained after treatment with TPA did not differ from those of spontaneously derived colchicine-resistant clones. Both have similar survival patterns in the medium with different colchicine concentrations, unstable inheritance of the drug resistance, the additional chromosome 4 and small chromatin bodies-the structures containing the amplified genes. Possible mechanisms of the induction of gene amplification by the agents used are discussed.

Animals↗

PubMed1998

Quantitative reverse transcription strand displacement amplification: quantitation of nucleic acids using an isothermal amplification technique.

Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens. The most prevalent testing is carried out for HIV viral load. Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties. SDA was adapted for quantitative RNA amplification (QRT-SDA) of an HIV gag sequence by including AMV reverse transcriptase, a quantitative control sequence, and 32P-labeled detector oligonucleotides for the HIV and the control sequences. We have also improved the amplification efficiency by including the single-strand binding protein from gene 32 of T4 bacteriophage (T4gp32) to enhance strand displacement replication. In a preliminary analytical demonstration of the technique, RT-SDA was quantitative to within twofold over a range of 500-500,000 transcripts that were generated from a plasmid bearing an HIV gag sequence. QRT-SDA potentially represents a convenient alternative for viral load testing in a clinical setting.

Base Sequence↗

PubMed2002

Linear amplification coupled with controlled extension as a means of probe amplification in a cDNA array and gene expression analysis during cold acclimation in alfalfa (Medicago sativa L.).

This study describes a rapid and simple way to amplify limited amounts of probes used for cDNA array hybridization while maintaining the original representation of transcripts in the samples. The approach is based on linear amplification of cDNA-coupled controlled extension of amplified products and yielded a 50-75-fold increases in hybridization signal intensity. Controlled extension of products is achieved either by adjusting the amplification conditions or by using a digested template. Linear amplification with controlled extension generates a population of fragments consisting mainly of 3'-end portions of original transcripts and ranging in length from 200 to 800 nucleotides. cDNA array analysis revealed that amplified and non-amplified probes generate expression profiles with correlations ranging from r=0.857 to 0.895. Up to 90% of cDNA clones, differentially expressed during cold acclimation in alfalfa, could be detected with both types of probes. This amplification method should increase the utility of cDNA arrays for identifying novel differentially expressed genes as well as expression profiling in specialized tissues or cells when the amount of analysed material is limited. The possibility of diminishing cross-hybridization of long genes sharing high sequence homology and improving the hybridization kinetics of complex probes after amplification is also discussed.

Acclimatization↗

PubMed1995

Colcemid resistance in murine SEWA cells: non-Pgy gene amplification at low levels of resistance and preferential Pgy2 gene amplification at high levels of resistance.

Mammalian cell lines often become multidrug-resistant to cytotoxic drugs by amplification and/or overexpression of the P-glycoprotein (Pgy) genes. However, several malignant cell lines seem to acquire low levels of drug resistance by non-P-glycoprotein mediated mechanisms. We report here on cytogenetical signs of non-Pgy gene amplification in murine SEWA cells during the early steps of selection in Colcemid (COL). In line TC13COL0.01, rare cells exhibited a homogeneously staining region (HSR) distally in chromosome 16. As the COL-concentration was raised the HSR-chromosome was retained and, in addition, the cells developed numerous double minutes (DMs). The DMs, but not the HSR, contained amplified Pgy genes. The HSR may correspond to amplified heat shock protein 70 (Hsp70) genes, detected by Southern analysis. A second low-level COL-resistant line, TC13D70.01, contained DMs but showed no amplification of Pgy, Hsp70, Hsp90, alpha- or beta-tubulin genes. In higher COL-concentration, P-glycoprotein mediated drug resistance was induced. In contrast to actinomycin D-resistant SEWA cells, in which higher amplification levels of Pgy1 than of Pgy2 are regularly present, the COL-resistant lines showed a preference for Pgy2 gene amplification. These results are in line with the suggestion that the murine Pgy1 and Pgy2 genes have overlapping but distinct drug specificities.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcription-mediated amplification: identification of urinary substances associated with inhibition and removal of inhibitory activity.

The presence of endogenous amplification inhibitors in urine may produce false-negative results for the detection of Chlamydia trachomatis nucleic acids by tests such as PCR, ligase chain reaction (LCR), and transcription-mediated amplification (TMA). Consecutive urine specimens from 101 pregnant women and 287 nonpregnant women submitted for urinalysis were processed for C. trachomatis detection. Aliquots were spiked with the equivalent of one C. trachomatis elementary body and were tested by three commercial assays: AMPLICOR CT/NG, Chlamydia LCX, and Chlamydia TMA. The prevalence of inhibitors resulting in complete inhibition of amplification was 4.9% for PCR, 2.6% for LCR, and 7.5% for TMA. In addition, all three assays were partially inhibited by additional urine specimens. Only PCR was more often inhibited by urine from pregnant women than by urine from nonpregnant women (9.9 versus 3.1%; P = 0.011). A complete urinalysis including dipstick and a microscopic examination was performed. Logistic regression analysis revealed that the following substances were associated with amplification inhibition: beta-human chorionic gonadotropin (odds ratio [OR], 3.3) and crystals (OR, 3.3) for PCR, nitrites for LCR (OR, 14.4), and hemoglobin (OR, 3.3), nitrites (OR, 3.3), and crystals (OR, 3.3) for TMA. Aliquots of each inhibitory urine specimen were stored at 4 and -70 degreesC overnight or were extracted with phenol-chloroform and then retested at dilutions of 1:1, 1:4, and 1:10. Most inhibition was removed by storage overnight at 4 or -70 degreesC and a dilution of 1:10 (84% for PCR, 100% for LCR, and 92% for TMA). Five urine specimens (three for PCR and two for TMA) required phenol-chloroform extraction to remove inhibitors. The results indicate that the prevalence of nucleic acid amplification inhibitors in female urine is different for each technology, that this prevalence may be predicted by the presence of urinary factors, and that storage and dilution remove most of the inhibitors.

Adolescent↗

PubMed2003

Direct comparison of microarray gene expression profiles between non-amplification and a modified cDNA amplification procedure applicable for needle biopsy tissues.

Global gene expression profiling by cDNA microarray analysis has been used to discover the biomarkers for early diagnosis of various cancers, subclassing cancer type, and prediction of patient's treatment outcome. The information provided by gene expression profiling may contribute to the design of molecular mechanism-based strategies for cancer prevention and/or treatment. However, the standard procedure for cDNA microarray analysis requires 5mug of good quality total RNA as starting material for each target preparation reaction. Thus, there is a limit for needle biopsy samples, laser capture microdissected tissues, or flow-sorted cells to successfully utilize the microarray technology. In order to profile the gene expression of needle biopsy tissue, we have modified the standard protocol by carrying out cDNA amplification after cDNA synthesis. We compared percentage present calls, absent calls, reproducibility, and concordance in needle biopsy samples processed by standard microarray protocol (cDNA non-amplification method) and our modified protocol (cDNA amplification method). The results showed that cDNA amplification method provided high reproducibility, representation, and concordance with the standard cDNA non-amplification method. We have successfully analyzed the gene expression profiles of needle biopsy tissues using the modified method without significantly changing the expression profiles. These results suggest that the global gene expression profiles of small biopsy samples can be achieved by our modified method to facilitate the analysis of gene expression profiles for clinical application.

Biopsy, Needle↗

PubMed2002

Comprehensive human genome amplification using multiple displacement amplification.

Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multiple displacement amplification (MDA), which provides a highly uniform representation across the genome. Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4-6 orders of magnitude for PCR-based WGA methods. Average product length was >10 kb. MDA is an isothermal, strand-displacing amplification yielding about 20-30 microg product from as few as 1-10 copies of human genomic DNA. Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells. MDA-amplified human DNA is useful for several common methods of genetic analysis, including genotyping of single nucleotide polymorphisms, chromosome painting, Southern blotting and restriction fragment length polymorphism analysis, subcloning, and DNA sequencing. MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies, forensics, diagnostics, and long-term sample storage.

Blotting, Southern↗

PubMedDate not supplied

[The characteristics of the enzymatic amplification of locus D17S5 alleles: the need to assess the stability of the amplification profile].

Amplification behavior of one chromosome locus (D17S5), widely used in identification studies, is investigated. The effect of preferable amplification of alleles, characteristic of this locus, often leads to an erroneous conclusion about homozygosity. Our studies showed, that along with the hazard of typing false homozygotes in analysis of locus D17S5, more intricate cases with genotype distortion can occur in expert practice, which may be characterized not only by partial loss of the true alleles, but even by amplification of nonspecific (nonallele) fragments simulating the false-heterozygotic allele profile. Analysis of the stability of amplification profile helps solve this problem.

Alleles↗

PubMed1993

PCR-amplification and detection of the human D1S80 VNTR locus. Amplification conditions, population genetics and application in forensic analysis.

A series of experiments has been performed to evaluate amplification and typing of the D1S80 VNTR locus. The validation study that has been carried out showed that correct D1S80 typing results can be obtained when a defined amplification protocol and a high-resolution polyacrylamide gel electrophoresis method are used. The use of the Chelex extraction protocol has substantially reduced the processing time. DNA-extraction, amplification and subsequent typing can be performed in one day. The discrimination power of this locus is 0.94 in a Dutch Caucasian population sample. The system is extremely sensitive: 0.1 ng of genomic DNA gave a correct typing result. The test could also detect the correct genotypes in mixed samples containing DNA from different individuals. Even if the major type was in a 20-fold excess, the minority type could still be amplified and typed correctly. We have found no deviation from Hardy-Weinberg equilibrium in a Dutch Caucasian population sample. Evidence for the somatic stability of this locus was obtained from a set of experiments where we compared DNA-profiles from corresponding blood, semen and saliva samples. The results of this study suggest that in the near future analysis of the D1S80 locus by DNA-amplification can be applied in actual forensic case work.

Alleles↗

PubMed2006

Molecular crowding increases the amplification success of multiple displacement amplification and short tandem repeat genotyping.

Multiple displacement amplification (MDA) can generate large quantities of genomic DNA product from small amounts of template. We investigated the ability of MDA to amplify samples containing very small amounts of target DNA (5 pg to 1 ng) in the presence of a second, larger DNA sample for downstream short tandem repeat (STR) multiplex genotyping. We observed that STR amplification success of the minor fraction was increased in these mixed samples when compared with standard PCR only or MDA containing only the single trace DNA sample. Increased numbers of alleles were detected, with less amplification bias between loci than in single source samples undergoing the same protocol. To improve the STR genotyping accuracy, animal DNA was substituted for the additional human DNA, maintaining the increase in the number and quality of human-specific STR loci amplified. Polyethylene glycol 400, a commonly used crowding agent, was used as a replacement for the added genomic DNA in the MDA reaction and produced very similar results. Therefore, we suggest that additional DNA is acting as a molecular crowding agent during MDA. Performing MDA on trace amounts of DNA under crowded conditions results in greater numbers of alleles being amplified and more balanced amplification occurring between alleles.

Alleles↗

PubMed2004

Amplification of MGC2177, PLAG1, PSMC6P, and LYN in a malignant mixed tumor of salivary gland detected by cDNA microarray with tyramide signal amplification.

Gene amplifications have been observed in many different tumor cells, and many of these changes are related to tumor pathogenesis. Comparative genomic hybridization (CGH) using metaphase chromosomes can detect changes in chromosome copy number with a resolution of 10-20 Mb. Current advances in CGH analysis in a microarray format allow us to refine such changes down to the gene level. We applied microarray technology to detect novel gene amplification in a malignant mixed tumor of salivary gland. Besides detecting previously known gene amplifications (MDM2 and MYC), we identified four other highly amplified genes located at 8q11.2 approximately q13: MGC2177, PLAG1, PSMC6P, and LYN. The amplification was further validated with real-time quantitative polymerase chain reaction.

Adenosine Triphosphatases↗

PubMed2004

MYCN-status in neuroblastoma: characteristics of tumours showing amplification, gain, and non-amplification.

While the role of MYCN-amplification (MNA) for risk assessment in neuroblastoma is undisputed, the phenomenon of gene copy excess below the amplification threshold is rarely described. To discuss biological characteristics and the clinical impact of the so-called MYCN-gain versus amplified or non-amplified cases, we investigated the MYCN status of 659 patients uniformly analysed by fluorescence in situ hybridisation. The number of MYCN-amplified tumours in our cohort was 18% (116/659); an additional 38 tumours (6%) displayed MYCN-gain. Both alterations were associated with an advanced stage disease, an increased patient age and further chromosomal alterations. Most of the amplified neuroblastomas displayed 1p aberrations, whereas MYCN-gain tumours correlated with 11q alterations. In contrast to the amplified cases, tumours with gain displayed no increased MYCN RNA levels. MNA versus non-amplification discriminated between good and poor outcomes, independent of stage, age and the degree of amplification. However, patients with amplified tumours showed a significantly better outcome when this was combined with non-stage 4 disease and age <1 year versus stage 4 and age < 1 year. Although MYCN-gain was associated with poor event-free-survival (EFS) in stages 1-3, 4S (P=0.005), this might be related to associated genetic aberrations and not to the MYCN-gain itself. A survival difference between neuroblastomas with gain and single copy MYCN could not be delineated. In conclusion, MNA predicts a poor outcome for neuroblastoma patients of all stages and age. MYCN-gain is also a characteristic feature of advanced stage tumours and older patients, but is not associated with higher MYCN expression and appears not to be discriminative in predicting patient outcome.

Blotting, Southern↗

PubMed2006

Whole-metagenome amplification of a microbial community associated with scleractinian coral by multiple displacement amplification using phi29 polymerase.

Limitations in obtaining sufficient specimens and difficulties in extracting high quality DNA from environmental samples have impeded understanding of the structure of microbial communities. In this study, multiple displacement amplification (MDA) using phi29 polymerase was applied to overcome these hindrances. Optimization of the reaction conditions for amplification of the bacterial genome and evaluation of the MDA product were performed using cyanobacterium Synechocystis sp. strain PCC6803. An 8-h MDA reaction yielded a sufficient quantity of DNA from an initial amount of 0.4 ng, which is equivalent to approximately 10(5) cells. Uniform amplification of genes randomly selected from the cyanobacterial genome was confirmed by real-time polymerase chain reaction. The metagenome from bacteria associated with scleractinian corals was used for whole-genome amplification using phi29 polymerase to analyse the microbial diversity. Unidentified bacteria with less than 93% identity to the closest 16S rDNA sequences deposited in DNA Data Bank of Japan were predominantly detected from the coral-associated bacterial community before and after the MDA procedures. Sequencing analysis indicated that alpha-Proteobacteria was the dominant group in Pocillopora damicornis. This study demonstrates that MDA techniques are efficient for genome wide investigation to understand the actual microbial diversity in limited bacterial samples.

Animals↗

PubMed1994

Hairpin structures are the primary amplification products: a novel mechanism for generation of inverted repeats during gene amplification.

Early events of DNA amplification which occur during perturbed replication were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (CO60) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contain molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated CO60 cells. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several experimental procedures such as sucrose gradients, "snap-back" assay, and two-dimensional gel electrophoresis, we show that the overreplicated DNA contains IRs which are synthesized de novo as hairpins or stem-loop structures which were detached from the template molecules. The fully replicated SV40 molecules synthesized by the HeLa extracts do not contain such IRs. We propose "U-turn replication" as a novel mechanism for gene amplification, accounting for the generation of extrachromosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases.

Animals↗

PubMed2004

Amplification targeting: a modified pretargeting approach with potential for signal amplification-proof of a concept.

UNLABELLED: Conventional nuclear medicine imaging with large radiolabeled molecules such as antitumor antibodies suffers from slow localization and clearance. Pretargeting is under active investigation as an alternative using either (strept)avidin/biotin, bispecific antibodies, or oligomers. However, only the use of oligomers such as phosphorodiamidate morpholinos (MORFs) in pretargeting offers the potential of signal amplification at the target. Amplification targeting is a multistep procedure with the potential to greatly improve target localization of radioactivity (and eventually drugs) through the intermediate use of polymers conjugated with multiple copies of oligomers. OBJECTIVE: This study was conducted to prove the concept in vivo in tumored mice of amplfication targeting. METHODS: Nude mice bearing LS174T tumors received in order: the anti-CEA antibody MN14 conjugated with MORF, a polymer conjugated with multiple copies of complementary MORFs (cMORFs), and, finally, (99m)Tc-MORF. RESULTS: In tumored animals, dual radiolabels ((99m)Tc and (111)In) were used to demonstrate that, after 18 h, about 25% of antibody MORFs in tumor were targeted with polymeric cMORFs and, after 3 h, about 12% of the polymeric cMORFs in tumor were targeted with (99m)Tc-MORF. Therefore, hybridization in tumor in both cases (i.e., polymeric cMORF to antibody MORF and radiolabeled MORF to polymeric cMORF) was surprisingly efficient given the barriers to targeting in vivo and the competition between targeting and clearance. Moles of radiolabeled MORF accumulating in tumor were more than tripled for study animals receiving all 3 injections compared with control animals not receiving the antibody or the polymer. Furthermore, MORF expression (on antibody) and cMORF expression (on polymer) were rapidly lost in normal organs such as liver, spleen, and kidneys but not in tumor, thus improving the target-to-nontarget ratios. CONCLUSION: Although signal amplification has not yet been convincingly demonstrated and amplification targeting will require further studies for optimization, the concept has now been shown to be feasible.

Animals↗

PubMed2006

[RNA amplification from low numbers of bacteria in human blood to solve low sensitivity problem of conventional PCR amplification].

Blood culture from a febrile patient is often unsuccessful when a number of bacteria is lower than 10 CFU/ml in circulation. Theoretically, PCR amplification of bacteria in this low bacterial level in human blood is beyond the sensitivity of PCR method. RNA based Amplification (NASBA) method is more sensitive than PCR base assay because living bacteria carries several hundred to thousand RNA molecules in a cell. Increasing evidences also revealed another problem that human blood stream is often contaminated with bacterial genome. This often cause false positive results and bring difficulty of the interpretation to determine causative agent. Direct DNA amplification should be limited to severe causative agent such as Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitides, and etc. and also applied only for slow growing and fastidious pathogens such as mycoplasmae, chlamydiae, bartonellae, mycobacteria, and etc. Most cases blood culture should be carried out first and genetic amplification should be selected as a next step to detect pathogens from a blood culture bottle, because in these cases, patients are not in life threatening stages. These attempts also decrease the chance to detect circulating bacterial DNA fragment in blood stream.

Bacterial Infections↗

PubMed1994

Int-2/FGF3 amplification is a better independent predictor of relapse than c-myc and c-erbB-2/neu amplifications in primary human breast cancer.

The prognostic significance of the three genes most frequently amplified in breast tumors was investigated by multivariate analysis in a retrospective study of 112 primary human breast cancers. These three genes, c-myc, int-2/FGF3 (a marker for the 11q13 amplicon), and c-erbB-2/neu, were amplified in 37%, 14%, and 10% of breast tumors studied, respectively. Amplification of the c-myc gene was not related to metastasis-free survival in the total population but was a discriminant prognostic indicator in premenopausal patients. Int-2/FGF3 gene amplications were good indicators of prognosis, especially in premenopausal patients, and also in lymph-node-positive and steroid-receptor-negative patients. Int-2/FGF3 amplification and progesterone receptor status together proved to be the only independent variable predictive of metastasis-free survival. The risk of relapse in the subgroup of progesterone-receptor-negative patients was 5 times greater for those with int-2/FGF3 amplification than for those without this alteration. Amplifications at the c-erbB-2/neu locus were not significantly associated with any standard prognostic indicator or with an increased risk of recurrence. These results suggest that the combined use of classical prognostic factors and molecular markers may improve prognostic value and be applicable to patients with specific tumor phenotypes.

Adult↗

PubMed2002

Amplification-mutagenesis: evidence that "directed" adaptive mutation and general hypermutability result from growth with a selected gene amplification.

When a particular lac mutant of Escherichia coli starves in the presence of lactose, nongrowing cells appear to direct mutations preferentially to sites that allow growth (adaptive mutation). This observation suggested that growth limitation stimulates mutability. Evidence is provided here that this behavior is actually caused by a standard Darwinian process in which natural selection acts in three sequential steps. First, growth limitation favors growth of a subpopulation with an amplification of the mutant lac gene; next, it favors cells with a lac(+) revertant allele within the amplified array. Finally, it favors loss of mutant copies until a stable haploid lac(+) revertant arises and overgrows the colony. By increasing the lac copy number, selection enhances the likelihood of reversion within each developing clone. This sequence of events appears to direct mutations to useful sites. General mutagenesis is a side-effect of growth with an amplification (SOS induction). The F' plasmid, which carries lac, contributes by stimulating gene duplication and amplification. Selective stress has no direct effect on mutation rate or target specificity, but acts to favor a succession of cell types with progressively improved growth on lactose. The sequence of events--amplification, mutation, segregation--may help to explain both the origins of some cancers and the evolution of new genes under selection.

Electrophoresis, Agar Gel↗