Loss of heterozygosity assay for molecular detection of cancer using energy-transfer primers and capillary array electrophoresis

Microsatellite DNA loci are useful markers for the detection of loss of heterozygosity (LOH) and microsatellite instability (MI) associated with primary cancers. To carry out large-scale studies of LOH and MI in cancer progression, high-throughput instrumentation and assays with high accuracy and sensitivity need to be validated. DNA was extracted from 26 renal tumor and paired lymphocyte samples and amplified with two-color energy-transfer (ET) fluorescent primers specific for loci associated with cancer-induced chromosomal changes. PCR amplicons were separated on the MegaBACE-1000 96 capillary array electrophoresis (CAE) instrument and analyzed with MegaBACE Genetic Profiler v.1.0 software. Ninety-six separations were achieved in parallel in 75 minutes. Loss of heterozygosity was easily detected in tumor samples as was the gain/loss of microsatellite core repeats. Allelic ratios were determined with a precision of +/- 10% or better. Prior analysis of these samples with slab gel electrophoresis and radioisotope labeling had not detected these changes with as much sensitivity or precision. This study establishes the validity of this assay and the MegaBACE instrument for large-scale, high-throughput studies of the molecular genetic changes associated with cancer.     

Fluorescence melting curve analysis for the detection of the bcl-1/JH translocation in mantle cell lymphoma

PCR amplification and product analysis for the detection of chromosomal translocations such as bcl-1/JH have traditionally been performed as a two-step process with separate amplification and product detection. PCR product detection has generally entailed gel electrophoresis, hybridization, or sequencing for confirmation of assay specificity. By using a microvolume fluorimeter integrated with a thermal cycler and the PCR compatible double-stranded DNA (dsDNA) binding dye SYBR Green I, we simultaneously amplified and detected bcl-1/JH translocation products by using rapid cycle PCR and fluorescence melting curve analysis. We analyzed DNA from 25 cases of lymphoproliferative disorders comprising 12 previously documented bcl-1/JH-positive mantle cell lymphomas, and 13 reactive lymphadenopathies. The samples were coded and analyzed in a blind manner for the presence of bcl-1/JH translocations by fluorescence melting curve analysis. The results of fluorescence analysis were compared with those of conventional PCR and gel electrophoresis. All of the 12 cases (100%) previously determined to be bcl-1/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at about 86 degrees C by melting curve analysis. For easier visualization of melting temperatures (Tm), fluorescence melting peaks were obtained by plotting the negative derivative of fluorescence over temperature (-dF/dT) versus temperature (T). Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 40-fold. Our results indicate that nucleic acid amplification integrated with fluorescence melting curve analysis is a simple, reliable, sensitive, and rapid method for the detection of bcl-1/JH translocations. The feasibility of specific PCR product detection without electrophoresis or expensive fluorescently labeled probes makes this methodology attractive for studies in molecular pathology.     

Detection of Aspergillus fumigatus PCR products by a microtitre plate based DNA hybridisation assay.

AIMS: To develop a DNA based plate hybridisation assay for the detection of polymerase chain reaction (PCR) products amplified from Aspergillus fumigatus DNA; and to determine the sensitivity of this technique and compare it with Southern blotting. METHODS: A half-log dilution series of DNA extracted from A fumigatus was amplified with specific primers, one of which was 5' end labelled with biotin. PCR products were subsequently detected by agarose gel electrophoresis, Southern blotting, and binding of the products to a streptavidin coated microtitre well, followed by non-radioactive colorimetric detection. Amplification was carried out 10 times for each DNA dilution and a plot of initial DNA concentration against signal intensity was made. RESULTS: A DNA concentration of 1.5 pg could be detected by agarose gel electrophoresis and Southern blotting with a non-radioactively labelled aspergillus specific probe; 1.5 pg was detectable by streptavidin binding of the PCR products to a microtitre plate. The signal from the microtitre plate detection was proportional to the amount of DNA in the PCR reaction on a log-log scale between 100 and 1 pg of DNA. CONCLUSIONS: A DNA based plate hybridisation assay for the detection of A fumigatus PCR products is as sensitive as Southern blotting. However, results are obtained in three hours rather than the three days required for agarose gel electrophoresis, blotting, hybridisation, and detection.     

Detection of adenovirus using PCR and molecular beacon.

The polymerase chain reaction (PCR) and a molecular beacon probe were used for the detection of Adenovirus. A 307 bp DNA fragment from a conserved region of the hexon gene was amplified. The specific molecular beacon was characterized with respect to its efficiency of quenching, and signal to noise ratio by spectrofluorometric analysis of its hybridization with virus specific complementary single stranded oligonucleotide target. Amplification was carried out in the presence of the molecular beacon probe, and the amplified target was detected by measurement of fluorescence signal in the post PCR sample. Separately, a 32P-labeled linear probe (having the same sequence as that of molecular beacon probe) was liquid-phase hybridized with the product of PCR performed in the absence of the molecular beacon. The virus specific target was then detected by electrophoresis of the hybridized product in a nondenaturing polyacrylamide gel and subsequent autoradiographic analysis. The detection limit of adenovirus by PCR in the presence of the molecular beacon probe was found to be similar to that obtained by labeled linear probe hybridization following PCR.     

Separation and detection of DNA polynucleotides using capillary electrophoresis. Application to detection of polymerase chain reaction-amplified human immunodeficiency virus and HLA DNA.

The polymerase chain reaction (PCR) is a technique for rapid amplification of target DNA sequences. During the past several years, a large number of research applications of PCR have appeared, many of which may prove to be useful clinically. We report the use of capillary electrophoresis, a fully automated technique, as an alternative to polyacrylamide gel electrophoresis for the detection of PCR-amplified viral and cellular DNA. We describe conditions for rapid separation, detection, and discrimination of PCR products from the human immunodeficiency virus type 1 gag gene and the HLA-DQ-alpha gene amplified from the human immunodeficiency virus provirus-containing U1.1 cell line. The sensitivity achieved with the use of capillary electrophoresis analysis was roughly equivalent to that of ethidium bromide staining of polyacrylamide gel electrophoresis gels. Further refinement of capillary electrophoresis for automated detection and quantitation of PCR-amplified products should expedite more widespread application of PCR analysis in the clinical laboratory.     

Investigation of a pseudo-outbreak of Nocardia asteroides infection by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA PCR.

Molecular strain typing by pulsed-field gel electrophoresis and by randomly amplified polymorphic DNA analysis was used to investigate a cluster of four Nocardia asteroides isolates associated with the BACTEC 460 TB system. An instrument motor drive misalignment resulted in inadequate needle sterilization and cross-contamination of BACTEC vials. This pseudo-outbreak illustrates the importance of proper BACTEC 460 needle sterilization and maintenance and confirms the usefulness of molecular typing methods for epidemiologic investigations.     

Detection of Epstein-Barr virus DNA sequences in nasopharyngeal carcinoma cells by enzymatic DNA amplification.

The presence of Epstein-Barr virus (EBV) DNA sequences was examined by the polymerase chain reaction in 50 nasopharyngeal carcinoma (NPC) biopsy specimens and in two primary epithelial tumor cell cultures derived from patients with NPC. The detection limit was a single EBV genome equivalent by agarose gel electrophoresis followed by Southern blot analysis of the amplified products. EBV DNA sequences were detected in all 41 undifferentiated NPC cell specimens, in 2 of 4 moderately differentiated NPC cell specimens, and in 3 of 5 keratinized NPC cell specimens. Undifferentiated NPC cells were also found to contain higher copy numbers of EBV than cells of the other two types of NPC. Our data suggest that EBV replication may be closely associated with the differentiation of NPC tumor cells. The results also demonstrated a sensitive and specific method for the detection of EBV DNA sequences in NPC tumor cells.     

An integrated microfluidic platform for rapid tumor cell isolation, counting and molecular diagnosis

Ovarian cancer is the second most common of the gynecological cancers in Taiwan. It is challenging to diagnose at an early stage when proper treatment is the most effective. It is well recognized that the detection of tumor cells (TCs) is critical for determining cancer growth stages and may provide important information for accurate diagnosis and even prognosis. In this study, a new microfluidic platform integrated with a moving-wall micro-incubator, a micro flow cytometer and a molecular diagnosis module performed automated identification of ovarian cancer cells. By efficiently mixing the cells and immunomagnetic beads coated with specific antibodies, the target TCs were successfully isolated from the clinical samples. Then counting of the target cells was achieved by a combination of the micro flow cytometer and an optical detection module and showed a counting accuracy as high as 92.5 %. Finally, cancer-associated genes were amplified and detected by the downstream molecular diagnosis module. The fluorescence intensity of specific genes (CD24 and HE4) associated with ovarian cancer was amplified by the molecular diagnosis module and the results were comparable to traditional slab-gel electrophoresis analysis, with a limit of detection around 10 TCs. This integrated microfluidic platform realized the concept of a “lab-on-a-chip” and had advantages which included automation, disposability, lower cost and rapid diagnosis and, therefore, may provide a promising approach for the fast and accurate detection of cancer cells.     

Mutation detection in the alpha-1 antitrypsin gene (PI) using denaturing gradient gel electrophoresis

A method for mutation detection in the alpha-1 antitrypsin gene (protease inhibitor 1; PI) has been developed using denaturing gradient gel electrophoresis of PCR amplified gene fragments. Using this experimental approach, all common phenotypes and mutations could be detected. Denaturing gradient gel electrophoresis (DGGE) was compared with standard isoelectric focusing (IEF) in 20 potential alpha1-antitrypsin deficient patients and their relatives. The genotype determined by DGGE was found to be more reliable in some cases than IEF, which is essential for a proper diagnosis of alpha-1 antitrypsin malfunctioning.     

Molecular epidemiology provides evidence of genotypic heterogeneity of multidrug-resistant Pseudomonas aeruginosa serotype O:12 outbreak isolates from a pediatric hospital.

Ribotyping randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis were used for the epidemiologic evaluation of eight Pseudomonas aeruginosa O:12 isolates obtained from eight children and two P. aeruginosa O:12 environmental isolates from a hematology ward. Randomly amplified polymorphic DNA analysis and pulsed-field gel electrophoresis were able to discriminate isolates that were indistinguishable by biochemical typing, O serotyping or ribotyping.     

Detection of Shiga toxin-producing Shigella dysenteriae type 1 and Escherichia coli by using polymerase chain reaction with incorporation of digoxigenin-11-dUTP.

A technique has been developed for the detection of Shiga toxin- and Shiga-like toxin type I (ShT/SLT-I)-producing Shigella dysenteriae type 1 and Escherichia coli by using the polymerase chain reaction with the incorporation of digoxigenin-11-dUTP. Target DNA liberated from whole cells was amplified, using primer pairs homologous to the A-subunit genes of ShT/SLT-I. The TTP analog digoxigenin-11-dUTP was incorporated into the reaction mixture, permitting nonradioactive labeling of the amplified DNA. The labeled polymerase chain reaction products were hybridized to specific gene sequences immobilized on a nitrocellulose membrane and detected by using an alkaline phosphatase-conjugated antibody to digoxigenin and the enzyme substrates. Toxin-producing strains of E. coli and S. dysenteriae type 1 were identified as colored spots on the membrane. Because this technique does not require DNA purification, gel electrophoresis, or radioactive DNA probes, it is suitable for the clinical detection of ShT/SLT-I-producing strains of S. dysenteriae type 1 and E. coli.     

Y染色体特异短串联重复序列初步研究

目的了解人类Ｙ染色体特异短串联重复序列等位片段结构特征,揭示成都汉族群体５个Ｙ染色体特异ＳＴＲ基因座的遗传多态性。方法无血缘关系样本采自成都地区汉族群体。ＰＣＲ扩增５个Ｙ染色体特异ＳＴＲ基因座。ＰＣＲ产物由非变性聚丙烯酰胺凝胶不连续缓冲系统水平电泳分型和自动激光荧光测序仪分析。结果观察到人类Ｙ染色体特异ＳＴＲ等位片段具有复杂的串联重复结构。制备了５个Ｙ染色体特异ＳＴＲ基因座分型标准物用于分型。观察到我国人群５个Ｙ染色体特异ＳＴＲ基因座均具有遗传多态性。获得了成都汉族群体５个Ｙ染色体特异ＳＴＲ基因座的等位基因频率。结论本研究为群体遗传亲缘关系分析提供了高效遗传标记,为研究我国人群Ｙ染色体特异ＳＴＲ的群体遗传提供了具有可比性的资料,为研究人类近期进化事件提供了新的可能性     

Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid technique, which can be applied for disease diagnosis in aquaculture. Using the LAMP method, a highly specific and sensitive diagnostic system for infectious hypodermal and hematopoietic necrosis virus (IHHNV) detection was designed. A set of four primers was designed by targeting the IHHNV genome DNA. By the detection system, target DNA was amplified and visualized on agarose gel within 60min under isothermal condition at 64 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by the white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. The assay had a detection limit of 5-500 copies of DNA template with gel electrophoresis, SYBR Green I and white turbidity with naked-eye inspection. The detection sensitivity of LAMP was 100-fold higher than the PCR. A diagnostic procedure which is rapid and highly sensitive was developed for IHHNV detection.     

Electrophoretic isolation of extrachromosomal DNA from tumor cells

Gene amplification allows transformed cells to overexpress specific genes and gain a survival advantage. For this reason, cloning and characterization of amplified genes can improve our understanding of the biology of transformed cells. The techniques of in-gel renaturation and chromosome microdissection can enrich for amplified DNA sequences, but both are labor intensive and have other drawbacks. We have developed an alternative strategy of enriching for amplified DNA sequences that involves two-directional agarose gel electrophoresis of extrachromosomal circular DNA. Extrachromosomal circles can be detected with repetitive DNA probes and can be used to produce DNA probes suitable for fluorescence in situ hybridization for location of genomic origin. The ability to enrich for amplified DNA without specialized equipment or transformed cell metaphases should prove useful in the search for new genes which are important in tumor cell progression.     

Detection of Mycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods.

Detection of Mycobacterium tuberculosis in clinical specimens by the polymerase chain reaction (PCR) was compared with detection by culture. A 317-bp segment within the M. tuberculosis-specific insertion sequence IS6110 was amplified. The detection limit of the PCR assay for cultured mycobacteria was 50 cells per reaction by ethidium bromide-stained agarose gel electrophoresis and 5 cells per reaction by hybridization with an oligonucleotide probe conjugated with either digoxigenin or alkaline phosphatase (AP). This sensitivity was reduced fivefold in sputum specimens seeded with M. tuberculosis. Seventy-six clinical specimens were amplified and examined by the three detection methods. Both the digoxigenin and AP procedures were found to be more sensitive than agarose gel electrophoresis, but they were occasionally associated with a high background. An additional 308 specimens were examined only by agarose gel electrophoresis and the AP procedure. Of 71 specimens found to contain M. tuberculosis, amplified products were detected from 56 (79%) samples by agarose gel electrophoresis and/or the AP procedure. Of the additional 313 specimens that were culture negative for M. tuberculosis, 19 (6%) had amplified products detectable by agarose gel electrophoresis and/or the AP procedure. Compared with culture, PCR showed sensitivities and specificities of 55 and 98%, respectively, for agarose gel electrophoresis and 74 and 95%, respectively, for the AP procedure. Despite this low sensitivity, a rapid positive PCR result was accurate and clinically useful.     

An HLA Class-II allele frequent in Eskimos and Amerindians is found in the Tyrolean Ice Man

DNA was extracted from specimens derived from the calcaneus of the Tyrolean Ice Man under sterile conditions in a laboratory, where no DNA extractions and PCR experiments had been performed before. Agarose gel electrophoresis and ethidium bromide staining did not reveal any evidence of genomic DNA in the preparation obtained, indicating a high degree of DNA degradation. Nevertheless, we performed PCR amplifications with this sample using primer pairs specific for HLA class II alleles. HLA-DRB and DQB1 alleles were amplified in a nested PCR approach. In one of the reactions, we observed a distinct amplification product, which we directly sequenced. By comparing the obtained nucleotide sequence with a database of HLA alleles we assigned the HLA- DRB1 *1402 type to the amplified sample. None of the investigators involved possesses this allele, indicating that no contamination with modern DNA had occured. The HLA- DRB1 *1402 allele is extremely rare in Europe, but is common in Inuits and South American Indians and has previously only once been identified in the laboratory.     

Simple two-color array-based approach for mutation detection

The ability to analyze multiple polymorphic/mutation sites rapidly and accurately is pivotal in all areas of genetic analysis. We have applied single nucleotide primer extension (SNE) for detection of multiple point mutations in a micro-array format using two-color, fluorescent dye-tagged dideoxynucleoside triphosphate terminators (ddNTPs). The oligonucleotide primer ending one nucleotide short of the mutation site being probed is bound to the slide and single-base extended in place with two different Cy5/Cy3 dye-tagged terminators using solution-phase, locus-specific, single-stranded complementary templates generated by PCR from genomic DNA. The composite fluorescence produced contains peaks of distinct wave lengths corresponding to each Cy dye-tagged terminator incorporated, resulting in a fluorescent 'fingerprint' for each DNA target. DNA polymerase-catalyzed incorporation of Cy dye-tagged dideoxynucleoside triphosphates was dependent on the particular dyes, the specific ddNTP, the DNA target concentration, sequence of the template, on-slide temperature cycling and washing conditions. Results from analysis of mutations in the human hemochromatosis and connexin 26 genes show that this approach has several advantages over existing methods and is simple, rapid, robust, cost effective and accurate with potential applications in many areas of genetic analysis.     

Comparative evaluation of three commercial software packages for analysis of DNA polymorphism patterns

Objective   In the present study we have compared three commercial software packages, GelCompar, Molecular Analyst Fingerprinting, and BioImage, to determine if the results generated by the programs were comparable and correlated adequately with visual interpretation of electrophoretic gels, in the analysis of several well characterized incidents of infections.
Methods   Infections caused by Pseudomonas aeruginosa , Candida dubliniensis , C. albicans , and serotypes of Salmonella were characterized by restriction endonuclease analysis, macrorestriction analysis of genomic DNA with pulsed-field gel electrophoresis, and random amplified polymorphic DNA. The genotypes were visually detected based on band presence or absence in the different gels. The similarity values of DNA profiles were computed using Dice coefficient and were presented in dendrograms by upgma . The concordance or agreement between the number of genotypes obtained and their clustering, using the computerized programs, was determined.
Results   In general, agreement in number of genotypes obtained visually and by using the commercial DNA analysis software was achieved, but discrepancies were also denoted between the systems. The concordance between the visual and the computerized analysis ranged from 72% to 100%.
Conclusion   In our experience, although the programs evaluated in the present study performed acceptably well, such programs may be used as an aid in the analysis of complex banding patterns, and they do not provide an indisputably correct analysis in genotype definition.     

Rapid genotyping of Achromobacter xylosoxidans, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia isolates using melting curve analysis of RAPD-generated DNA fragments (McRAPD)

Typing of bacteria is important for monitoring newly emerging pathogens and for examining local outbreaks. We evaluated the randomly amplified polymorphic DNA technique in combination with melting curve analysis (McRAPD) of the amplified DNA fragments to genotype isolates from five Gram-negative species, i.e. Achromobacter xylosoxidans, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia.By determining the melting temperature peaks of the amplified DNA fragments, we were able to distinguish the different genotypes of isolates, as they had been assessed by other genotyping techniques, i.e. agarose gel electrophoresis of RAPD fragments, multilocus sequence typing and/or AFLP™.According to our results, McRAPD may offer the possibility of genotyping a limited number of bacterial isolates, e.g. in case of suspicion of hospital outbreak, via a less costly, more rapid, less laborious and more user-friendly technique than RAPD followed by electrophoresis.