Amplification Of DNA Sequences Research Articles

Effects of Six Different Tissue Fixatives on DNA Quality as Determined by Polymerase Chain Reaction Amplification of Genes Ranging from 100 to 574 Base Pairs

As smaller samples (e.g. fine-needle aspiration) are being obtained for pathologic diagnosis, the need to incorporate ancillary studies including molecular genetic tests is increasing.For some diseases, such as lymphoproliferative disorders, polymerase chain reaction (PCR) amplification can be used as a powerful diagnostic tool. The poor quality of DNA extracted from some fixatives, however, can preclude amplification of DNA sequences of interest. In this study, we analyzed the quality of DNA extracted from hypesplastic tonsil and lymphomatous spleen fixed in six different fixatives by using PCR to amplify DNA target sequences ranging from 100 bp to 574 base pairs. We have shown that PCR can successfully amplify genes in this size range using tissues fixed in five fixatives tested, including three formalin-based fixatives, Hollande's, and Bouin's fixatives. In contrast, the poor quality of genomic DNA extracted from B5 fixed tissue precluded PCR amplification of all target genes tested in this study. We conclude that B5 fixation is a significant limiting factor for PCR analysis. In cases in which PCR analysis is likely to be needed, B5 fixative should be avoided. (The J Histotechnol 26:111, 2003)Submitted: July 5, 2002; Accepted with revisions: April 7, 2003

PROGRESSIVE OUTER RETINAL NECROSIS CAUSED BY VARICELLA-ZOSTER VIRUS IN CHILDREN WITH ACQUIRED IMMUNODEFICIENCY SYNDROME

We report three HIV-infected children with bilateral progressive outer retinal necrosis caused by varicella-zoster virus. All three lost vision in both eyes. Aggressive treatment strategies with combination intravenous antiviral therapy in addition to intravitreal antiviral therapy and appropriate surgical management may prevent blindness. Rapid recognition of this distinct clinical syndrome is essential to improve visual outcome.

Applicazioni della PCR e PCR in situ nella diagnosi di infezioni batteriche e virali da biopsie fissate in formalina e incluse in paraffina

In situ PCR, amplification of target DNA sequences in fixed cells, is a very useful molecular biology tecnique with potential to combine the high sensitivity of tube PCR with the precise anatomical localization of the targeted bsequences. It allows the study of low copy viral or bacterial DNA. In this study we document the utility of directin situ PCR with single primer pair by applying it to 3 infectious agents in different model systems: Borrelia burgdorferi in 5 Eritema migrans lesions and 55 primitive cutaneous B cell lymphomas, Chlamydia pneumoniae in 200 autoptic atheromasic lesions, and Papilloma virus in 20 CIN 1 (mild cervical dysplasia). In situ PCR seems to be a very promising tecnique; however, the prerequisite for the success of in situ PCR is conditioned by optimal standardization of the key variables which, on the other hand, are influenced by tissue composition.

Broad-range ribosomal RNA real-time PCR after removal of DNA from reagents: melting profiles for clinically important bacteria.

With the ability to exponentially amplify regions of DNA, two different PCR-based strategies have been developed for nonculture diagnosis of bacteremia. The first approach targets the species-specific genes for amplification (1), whereas the second approach involves universal PCR amplification of conserved bacterial DNA sequences, such as the 16S rRNA, the 23S rRNA, and the 16S-23S rRNA interspace regions (2)(3)(4). Although universal PCR is not able to distinguish bacteria to the species level, numerous studies have shown that this method provides valuable information complementary to the results of time-consuming and subjective phenotypic tests used in detection of bacterial infection (5) and can be used to differentiate bacterial from viral or other infections (6)(7). In clinical applications, real-time PCR for broad-range amplification of bacterial DNA sequences could offer additional benefits: it is less labor-intensive, less time-consuming, and reduces the risk of PCR carryover contamination. A major obstacle for broad-range PCR amplification is the presence of bacterial DNA in the Taq DNA polymerase and real-time PCR master mixture (8)(9)(10). The contaminating DNA is effectively amplified, giving rise to false-positive results. Efforts have been taken to eliminate contaminating DNA, including the use of Sau 3AI restriction endonuclease (11), DNase I (12)(13), and ultraviolet irradiation (7)(8). Although these strategies may be effective for conventional PCR, the study by Corless et al. (14) indicated that the contamination issue could not be avoided without affecting sensitivity when TaqMan probe-based real-time PCR is performed. Therefore, an appropriate method for removal of contaminating bacterial DNA and subsequent real-time amplification is required. In this study, we address this issue and report optimized conditions for broad-range amplification of the bacterial 16S rRNA gene by real-time PCR using SYBR Green I dye (15) as the fluorescent signal. Bacterial …

Positional mapping for amplified DNA sequences on 1q21–q22 in hepatocellular carcinoma indicates candidate genes over-expression

Background/Aims : Comparative genomic hybridization analysis on hepatocellular carcinoma (HCC) indicated frequent gains of 1q and an amplicon at 1q21–q22. Current cytogenetic evidences confer much importance on 1q21–q22, where a role in drug resistance, tumor metastasis and shorter patient survival had been implicated. Methods : Using positional mapping by interphase cytogenetics, we investigated the amplicon 1q21–q22 in five HCC cases. Three amplification maxima represented by yeast artificial chromosomes (YACs) 955E11, 876B11 and 945D5 that mapped to regions 1q21.1, 1q21.2 and 1q22, respectively, were indicated. We further investigated candidate genes expression in the mapped YACs by quantitative reverse-transcription-polymerase chain reaction. A panel of genes encoding protein transcripts involved in apoptosis, cell cycle progression, calcium binding and jumping translocation was studied. Results : Among ten HCC cases with the amplicon 1q21–q22 examined, we found a significant gene expression level of JTB , SHC1 , CCT3 and COPA in the tumors than the paired adjacent non-malignant liver tissues ( P ≤0.04). Conclusions : Our interphase findings on 1q21–q22 pinpointed three affected loci between D1S305 and D1S2369. Up-regulation of candidate genes identified within these over-represented regions may represent targets in the progression of HCC and may carry prognostic significance.

Polymerase chain reaction in cutaneous tuberculosis: is it a reliable diagnostic method in paraffin-embedded tissues?

Most cutaneous tuberculosis lesions contain few bacilli, so identification of Mycobacterium tuberculosis in conventional laboratory tests is difficult. In vitro amplification of specific DNA sequences using polymerase chain reaction (PCR) has become a valuable tool in the rapid detection of slow-growing organisms like M. tuberculosis. To investigate the presence of M. tuberculosis DNA in cutaneous tuberculosis. Twenty-two archival biopsy specimens diagnosed as cutaneous tuberculosis were investigated for the presence of M. tuberculosis DNA by PCR. Normal skin samples of 29 healthy patients were used as a control. Amplification of the M. tuberculosis DNA was observed in one of the cutaneous tuberculosis specimens and in a healthy control. Although PCR is a rapid diagnostic method, fixation procedures may decrease its sensitivity.

Cytochrome p450 genotyping by multiplexed real-time dna sequencing with pyrosequencing technology.

Individual differences in xenobiotic metabolism influence the therapeutic value of many drugs and are of major concern during the development of new drug candidates. A number of polymorphic cytochrome p450 enzymes account for a significant part of this variation. A better understanding of these genetic factors would be of value for drug development, as well as clinical practice. To fulfill the goal of a personalized medicine, methods for simple and accurate assessment of cytochrome p450 genes are required. We report on the development of multiplex assays for genotyping of the cytochrome p450 drug-metabolizing enzymes CYP2D6, CYP2C9, and CYP2C19 with Pyrosequencing technology. Eleven variable positions, representing 12 of the most frequent alleles, were scored: CYP2D6 alleles *2, *3, *4, *6, *7, *8, and *14, CYP2C19 alleles *2, *3, and *4, and CYP2C9 alleles *2 and *3. Four multiplex Pyrosequencing reactions per patient sample were performed to cover these positions, using either simplex or multiplex PCR for amplification of target DNA sequences. Unequivocal genotypes were obtained for all patient samples, and the results were validated by comparing with results obtained using PCR-RFLP. For positions addressed with both methods, the results were in complete agreement. Pyrosequencing technology offers a highly automated, rapid, and accurate method for identification of cytochrome p450 alleles, which is suitable for pharmacogenomic research, as well as for routine assessment of patient genotypes.

Origin of an apparent B chromosome by mutation, chromosome fragmentation and specific DNA sequence amplification.

The present study documents the de novo origin of an apparent B chromosome in Plantago lagopus. The origin was associated with mutation (aneuploidy), chromosome fragmentation, specific DNA sequence amplification, addition of telomeric repeats, and centromeric misdivision. It originated in the progeny of trisome 2, from the excision of 5S rDNA and 18S, 5.8S, 25S rDNA sequences located on chromosome 2, and within a few generations acquired many characteristics of an apparent B chromosome. The B chromosome has preferential transmission through the male (41%, P<0.025) and female gametes (42%, P<0.01) but does not affect plant phenotype. The B chromosome is completely heterochromatic, has a functional centromere and does not pair at meiosis with any A chromosomes of the standard complement. Fluorescence in situ hybridization analysis showed that it arose from massive amplification of 5S rDNA sequences, has 18S, 5.8S, 25S rDNA sequences at the ends of both arms and telomeric repeats at both termini. Ag-NOR-banding and determination of the maximum number of nucleoli in interphase cells indicate that the nucleolar organizer regions at the ends of both arms of the B chromosome are active in organizing nucleoli. RNA blot analysis showed that the 5S rDNA sequences are not transcribed. To our knowledge, this is the first report that fully documents one of the mechanisms by which B chromosomes may arise in nature.

Amplification of Trypanosoma cruzi-specific DNA sequences in formalin-fixed raccoon tissues using polymerase chain reaction.

This investigation applied polymerase chain reaction (PCR) using 3 sets of Trypanosoma cruzi-specific primers to amplify DNA from 31 archived formalin-fixed and fresh-frozen raccoon hearts. PCR successfully amplified T. cruzi-specific sequences, with at least 1 primer set, from multiple sites within the myocardium of formalin-fixed and fresh-frozen raccoon hearts that had previously tested positive using enzyme-linked immunosorbent assay and indirect immunofluorescent antibody titer in the absence of positive hemoculture results. Trypanosoma cruzi DNA was most frequently amplified from the interventricular septum, right ventricle, and left atrium. In addition, T. cruzi DNA was amplified with all 3 primers in at least I raccoon that was hemoculture positive and 2 animals that were borderline negative for the T. cruzi antibody and hemoculture negative. The amplification of T. cruzi-specific DNA sequences in the presence of an elevated antibody titer and negative culture results suggests good sensitivity of this method for detecting the presence of the parasite in archival tissues.

Combined eco-epidemiological and molecular biology approaches to assess Echinococcus granulosus transmission to humans in Mauritania: occurrence of the ‘camel’ strain and human cystic echinococcosis

Mauritania lies between West-Central Africa where human cystic echinococcosis (CE) is considered extremely rare and West Maghreb where CE accounts for a real public health problem. Until 1992, Mauritania was considered as human CE-free even though CE seemed well known in livestock. In 1992, the introduction of ultrasonography led to the diagnosis of the first human CE cases. In 1997, a veterinary study revealed that dogs living around Nouakchott were commonly infected by Echinococcus granulosus. To assess E. granulosus transmission and to identify the most relevant animal reservoir responsible for human CE emerging in Mauritania, a simultaneous eco-epidemiological and molecular biology approach was performed. The fieldwork included sample collection and investigation of relationships between intermediate hosts, definitive hosts and humans. Typing of E. granulosus strains was performed using comparison of polymerase chain reaction (PCR)-amplified DNA sequences with one nuclear (BG 1 3 ) and 2 mitochondrial (COI, NDI) targets. Results show that the ‘camel’ strain is actually infectious to humans and circulates between intermediate hosts including camels and cattle. It is suggested that preventive measures at slaughtering places could reduce human contamination.

Human hematopoietic progenitors engraft in fetal canine recipients and expand with neonatal injection of fibroblasts expressing human hematopoietic cytokines

The development of large-animal models for human hematopoiesis will facilitate the study of human hematopoietic stem cells and their progenitors in vivo. In previous studies, human hematopoietic progenitors engrafted in fetal dogs and contributed to hematopoiesis for one year. Despite initially high levels of human cells, the proportion declined to less than 0.1% at 6 months, possibly due to inability of the canine hematopoietic microenvironment to support ongoing human hematopoiesis. In the current experiments we examined the potential of co-transplanting fibroblasts expressing human hematopoietic cytokines with the hematopoietic graft to increase the contribution of human progenitors to chimeric hematopoiesis. Mid-gestation canine fetuses were injected with 1-3 x 10(7) human cord blood cells and 1 x 10(7) murine fibroblasts engineered to express human cytokines. Neonatal pups were boosted with additional injections of cytokine-expressing fibroblasts. Human cell engraftment was monitored by PCR amplification of human-specific DNA sequences from recipient hematopoietic tissues. Human hematopoietic cells were detected in 13/15 fetal recipients for at least 7 months. At time points up to 30 weeks of age, human DNA was detected in stimulated lymphocyte cultures, approximately 0.1% of blood leukocytes and 1.5% (85/5757) of myeloid colonies. Eight months postinfusion, 1.7% of colony-forming units (CFUs) were of human origin. By one year 0.5% or less of myeloid colonies and less than 0.01% of blood leukocytes carried human DNA. Following an infusion of cytokine-expressing fibroblasts at one year, the proportion of human myeloid progenitors rose to 11.5% and remained detectable for 8 months. These studies confirm that human hematopoietic progenitors can engraft in fetal pups and contribute to multilineage hematopoiesis. Infusion of cells expressing human cytokines is one approach to stimulate human hematopoietic progenitors in vivo and thus increase their contributions to chimeric hematopoiesis.

Polymorphism of the eosinophil cationic protein-gene is related to the expression of allergic symptoms.

We have found a polymorphism in the ECP (eosinophil cationic protein)-gene at position 434 according to GenBank accession number NM 002935. This polymorphism would cause the change of the amino acid arginine (base at position 434 is G) at position 97 to threonine (base at position 434 is C). To investigate the prevalence of the ECP-polymorphism and to screen for disease associations. DNA of 209 medical students and 76 asthmatic subjects was analysed. The 434 genotype in the ECP-gene was detected by cleavage of the amplified DNA sequence with the restriction enzyme PstI and analysis of the cleaved product by agarose gel electrophoresis. The prevalences of the polymorphism in the student population were 53%, 39% and 8% for the 434GG, the 434GC and the 434CC genotype, respectively, with allele frequencies of 72% (434G) and 28% (434C). Subjects reporting allergy had a higher prevalence of the 434G allele than non-allergic subjects (P = 0.0056). Of the students who were Phadiatop-positive and had allergic symptoms, 79% had the 434GG genotype, whereas the 434GC and 434CC genotypes were present in 82% of those who did not express allergic symptoms (P < 0.001). Among the 76 patients with asthma, patients with allergic asthma had a significantly higher proportion of 434GG compared with patients with non-allergic asthma (P = 0.04). None of the 18 subjects of the two groups with the 434CC genotype had allergy. The 434(G > C) polymorphism in the ECP-gene is related to the development of allergic symptoms, suggesting a central role for the ECP molecule in the process.

A Simple Method for Storing Mosquito Bloodmeals for Human DNA Profiling

A simple method for storing mosquito bloodmeal samples, which permits extraction and detection of human DNA after polymerase chain reaction (PCR) amplification of target DNA sequences, was tested. Abdomens of bloodfed field-collected Anopheles gambiae s.l. and An. funestus mosquitoes (Diptera: Culicidae) were directly expressed onto filter paper, air-dried and stored at room temperature. DNA was extracted and amplified at human hypervariable loci TC11, VWA and D1S80. The amplified products were separated using polyacrylamide gel electrophoresis, visualised by silver staining, and results compared with those from mosquitoes that had been preserved in liquid nitrogen. DNA from blooded abdomens stored on dried filter papers could be amplified with greater than 95 % success for any locus, storage temperature, mosquito species or storage duration. Collection and drying of mosquito bloodmeals directly onto filter paper appears to be a more convenient method for sample transportation and storage than the conventional method involving cryopreservation.

Protein detection using proximity-dependent DNA ligation assays.

The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 x 10(-21) mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.

Establishment and characterization of human metastatic hepatocellular carcinoma cell line

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a poor prognosis. Recently, we established a HCC cell line from a metastatic HCC tumor. GTG banding analysis was performed and the karyotype showed that this metastatic HCC cell line is a hypertriploid (71–78 chromosomes) with a large marker chromosome containing a long homogeneously staining region (hsr). Comparative genomic hybridization was applied to characterize the chromosomal alterations in this metastatic HCC cell line. The results showed that the hsr was composed of amplified DNA sequences from 11q13. Further characterization of the hsr may lead to the isolation of the putative amplified oncogene at 11q13.

Genetic diversity of Paenibacillus polymyxa populations isolated from the rhizosphere of four cultivars of maize ( Zea mays) planted in Cerrado soil

A tropical Brazilian soil (Cerrado) was planted with four cultivars of maize (CMS04, CMS11, CMS22 and CMS36) and the genetic diversity of the Paenibacillus polymyxa populations present in their rhizospheres was determined after 90 days of sowing. For that, a total of 67 isolates were identified as P. polymyxa by classical biochemical tests and were analyzed for DNA polymorphism with the randomly amplified polymorphic DNA (RAPD) and amplification of repetitive DNA sequences (rep) methods. The amplification patterns obtained using three arbitrary primers and the primer BOXA1R were used separately to construct dendrograms based on the unweighted pair groups method with arithmetic means (UPGMA). Fifty-four genotypic groups were formed when data from different PCR amplifications were combined, showing a high level of genetic polymorphism among P. polymyxa strains. A dendrogram based also on combined PCR data, followed by cluster analysis with minimum-variance criteria (Ward) and Euclidean distance, showed that P. polymyxa strains could be divided into two main clusters. One cluster was formed predominantly by strains from maize cultivars CMS04 and CMS36, while the other cluster was formed predominantly by strains of maize cultivars CMS11 and CMS22. Multivariate analysis of variance (MANOVA) allowed the correlation between the genetic structure of P. polymyxa populations and the different cultivars of maize to be studied. The results showed that the strains isolated from the rhizospheres of the different maize cultivars were significantly different.

Integrated microfluidic electrophoresis system for analysis of genetic materials using signal amplification methods.

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), was performed within a microfluidic chip. The presence of DNA from methicillin-resistant Staphylococcus aureus was determined by signal amplification of a specific DNA sequence. The microfluidic device consisted of four channels intersecting to mix the sample and reagents within 55 s, as they were directed toward the reactor coil by electrokinetic pumping. The 160-nL CPT reactor occupied approximately 220 mm2. Gel-free capillary electrophoresis separation of the biotin- and fluorescein-labeled probe from the probe fragments was performed on-chip following the on-chip reaction. An off-chip CPT reaction, with on-chip separation gave a detection limit of 2 fM (0.03 amol) target DNA and an amplification factor of 85,000. Calibration curves, linear at <5% probe fragmentation, obeyed a power law relationship with an argument of 0.5 [target] at higher target DNA concentrations for both on-chip and off-chip CPT reaction and analysis. An amplification factor of 42,000 at 250 fM target (25,000 target molecules) was observed on-chip, but the reaction was approximately 4 times less sensitive than off-chip under the conditions used. Relative SD values for on-chip CPT were 0.8% for the peak migration times, 9% for the area of intact probe peak, and 8% for the fragment/probe peak area ratio.

Monomers of a satellite sequence of chaffinch (Fringilla coelebs L., Aves: Passeriformes) genome contains short clusters of the TTTAGGG repeat

A novel repeated sequence of chaffinch (Fringilla coelebs) designated as GS was isolated from genomic DNA after in vitro amplification of satellite DNA sequences using GSP-PCR technique. The proportion of this repeat in the chaffinch genome constitutes about 2%. Monomers are 176 to 199 bp in size and contain a short cluster of the TTAGGG telomeric tandem repeat. The oligomer of the telomeric hexanucleotide is flanked by the sequences that are significantly different in different monomers. The GS sequences are organized as tandemly repeated units and located in a number of chromomycin-positive blocks on the long arms of macrochromosomes 1, 2, 3, 5, and 6, as well as on several microchromosomes. The sequences homologous to the GS satellite of chaffinch were not found in the genomes of redwing (Turdus iliacus) and house sparrow (Passer domesticus).

Rapid amplification of genomic DNA sequences tagged by insertional mutagenesis.

Current and recent efforts to determine the genomic DNA sequence for numerous organisms (e.g., Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Arabidopsis thaliana, Zea mays, Caenorhabditis elegans, Mus musculus, Homo sapiens, Schizosaccharomyces pombe, Danio rerio, Drosophila melanogaster, Oryza sativa, and various archaea, and eubacteria) have revealed novel genes with unknown functions, and transposon mutagenesis provides a powerful method for assigning functions to these genes (e.g., ,-). In addition, restriction enzyme-mediated integration (REMI) has been used widely for mutagenesis (e.g., ,, reviewed in ). For both of these approaches, a unique DNA sequence is inserted by nonhomologous integration into genomic DNA to generate a disruption mutation that is physically tagged. Genetic analysis of the resulting mutants yields those with defects in the function of interest. Because each mutation is physically marked by a unique DNA sequence, it is possible to use this tag to identify each disrupted gene.

Multicolor spectral karyotyping of serous ovarian adenocarcinoma.

We applied multicolor spectral karyotyping (SKY) to decipher the chromosomal complexity of a panel of seven cell lines and four primary tumors derived from patients with high-grade serous adenocarcinoma of the ovary. By this method we identified a total of 188 unbalanced translocations, nine reciprocal translocations [t(2;15)(q13;q23), t(7;17) (q32;q21), t(8;22)(p11;q11), t(8;22) (q24;q13), t(10;19) (q24;q13.2), t(11;19) (q13;p11), t(12;21)(q13;q22),t(18;20) (q?11;q?11), t(18;22)(q?11;q?13)], 6 isochromosomes [i(1q), i(7q), i(8q), i(9p), i(17q), i(21q)], and 23 deletions. By detailed mapping of rearrangement breakpoints, it was possible to identify several recurring breakpoint clusters at chromosomal bands 1p36, 2p11, 2p23, 3p21, 3q21, 4p11, 6q11, 8p11, 9q34, 10p11, 11p11, 11q13, 12p13, 12q13, 17q21, 18p11, 18q11, 20q11, and 21q22. Recurrent interstitial deletion of chromosomal bands 8p11, 11p11, and 12q13 and a recurrent unbalanced translocation--der(6)t(6;8)(q11;q11)-were also identified. In addition, a homogeneously staining region localized in one cell line to 11q13 was found using SKY to be derived from genetic material originating from chromosome 12. Subsequent comparative genomic hybridization (CGH) studies on this tumor revealed the amplification of DNA sequences derived from the short arm of chromosome 12 at the 12p11.2 region. These studies demonstrate the power of SKY, CGH, and G-banding to resolve the full spectrum of chromosomal rearrangements in serous ovarian adenocarcinoma.