Fluorescent PCR Amplification Research Articles

Rapid screening test of most frequent BRCA1/BRCA2 pathogenic variants in the NGS era.

The average risk of breast cancer in general Slovak population of women is 4-5% and the risk of ovarian cancer is 2%. Probability of breast/ovarian cancer development is higher in individuals carrying a causative germline DNA variant in BRCA1 or BRCA2 gene responsible for hereditary breast/ovarian cancer (HBOC). Although a major proportion of inherited breast/ovarian cancers are due to the mentioned causal mutations, a number of new genes have emerged. Here we describe a rapid, multiplex and comprehensive approach for the detection of pathogenic variants in BRCA1 and BRCA2 genes which most frequently occur in Slovak HBOC population. Analysis comprises the combination of mutation specific methods. Fluorescent PCR amplification followed by fragment analysis for detection of insertions/deletions in exon 11 of BRCA1 gene. Second method is SNaPshot analysis for detection of the most frequent missense and ins/del variants in exons 2, 5, 13, 20 of BRCA1 and exons 11, 23 and 25 of BRCA2 gene. Altogether, we have analyzed 687 samples, 86 (12.5%) in group 1, which fulfilled indication criteria based on the positive family/personal history. Group 2 involved 601 (87.5%) cases, who did not meet the indication criteria and only the screening test was recommended. Using the combined approach, we have identified 47 mutated samples (6.8%), 40 in group 1 (46.5%) and 7 in group 2 (1.1%). However, the presented screening test would not provide complex results of BRCA1/2 gene analysis, it offers testing accessible to a broader spectrum of individuals under the threshold of indication for whole gene analysis. This approach may provide valuable information even in the NGS analysis era.

The Use of Methylation-Sensitive Multiplex Ligation-Dependent Probe Amplification for Quantification of Imprinted Methylation.

Imprinting disorders are a group of congenital diseases that can result from multiple mechanisms affecting imprinted gene dosage including cytogenetic aberration and epigenetic anomalies. Quantification of CpG methylation and correct copy-number calling is required for molecular diagnosis. Methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) is a multiplex method that accurately measures both parameters in a single assay. This technique relies upon the ligation of MLPA probe oligonucleotides and digestion of the genomic DNA-probe hybrid complexes with the Hha1 methylation-sensitive restriction endonuclease prior to fluorescent PCR amplification with a single primer pair. Since each targeted probe contains stuffer sequence of varying length, each interrogated position is visualized as an amplicon of different size upon capillary electrophoresis.

Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay.

Various detection methods have been developed to date for identification of animal species. New techniques based on PCR approach have raised the hope of developing better identification methods, which can overcome the limitations of the existing methods. PCR-based methods used the mitochondrial DNA (mtDNA) as well as nuclear DNA sequences. In this study, by targeting nuclear DNA, multiplex PCR and real-time PCR methods were developed to assist with qualitative and quantitative analysis. The multiplex PCR was found to simultaneously and effectively distinguish four species (fox, dog, mink, and rabbit) ingredients by the different sizes of electrophoretic bands: 480, 317, 220, and 209bp. Real-time fluorescent PCR's amplification profiles and standard curves showed good quantitative measurement responses and linearity, as indicated by good repeatability and coefficient of determination R 2>0.99. The quantitative results of quaternary DNA mixtures including mink, fox, dog, and rabbit DNA are in line with our expectations: R.D. (relative deviation) varied between 1.98 and 12.23% and R.S.D. (relative standard deviation) varied between 3.06 and 11.51%, both of which are well within the acceptance criterion of ≤25%. Combining the two methods is suitable for the rapid identification and accurate quantification of fox-, dog-, mink-, and rabbit-derived ingredients in the animal products.

Clinical, histological, and molecular risk factors for cancer recurrence in patients with stage II colon cancer.

The assessment of risk factors of cancer recurrence in patients with stage II colon cancer (CC) is crucial. Our aim was to study the clinical, histological, and molecular features associated with 3-year disease-free survival in a series of consecutive patients with stage II CC treated in three regional digestive oncology centers. Clinical and histological data of all patients after curative surgery for stage II CC, treated from 2001 until 2009, were collected retrospectively. Histological samples were obtained and tested prospectively for microsatellite instability using fluorescent PCR amplification. Cox proportional hazards regression models were used to calculate P values, hazard ratios (HRs), and 95% confidence intervals (CIs). Among 195 patients studied, 22 (11%) had disease recurrence during the 3-year period following diagnosis. On multivariate analysis, only low number of lymph nodes (HR=3.81, 95% CI: 1.19-12.19, P=0.02) and T4 status (HR=5.49, 95% CI: 1.06-28.43, P=0.04) were associated significantly with an increased risk of relapse. In this series of stage II CC patients, only T4 status and low number of lymph nodes were independent risk factors for poor 3-year disease-free survival, suggesting that patients with these features should be considered for adjuvant chemotherapy.

Circulating microRNAs: Understanding the Limits for Quantitative Measurement by Real‐Time PCR

Ever since the discovery of small, non-coding RNAs in circulation, microRNAs have gained significant attention as biomarkers or, in some instances, regulators of disease and/or associated complications. Until now, most articles published use real-time quantitative PCR (qPCR) for assessment of

Is whole genome amplification really offering advantages to traditional nested fluorescent PCR amplification in single cells as promised?

Is whole genome amplification really offering advantages to traditional nested fluorescent PCR amplification in single cells as promised?

Application of an erythroblast scoring system and DNA polymorphism sequence analysis for the gender-independent assessment of fetal cells in maternal blood

SEQUENCE ANALYSIS FOR THE GENDER-INDEPENDENT ASSESSMENT OF FETAL CELLS IN MATERNAL BLOOD DONGHYUN CHA, KIARASH KHOSROTEHRANI, DIANA BIANCHI, KIRBY JOHNSON, Pochon University, Obstetrics and Gynecology, Seoul, South Korea, Tufts University, Pediatrics, Boston, Massachusetts, Tufts University, Pediatrics, Obstetrics & Gynecology, Boston, Massachusetts OBJECTIVE: The aim of this study was to determine whether fetal nucleated red blood cells (fNRBCs) could be distinguished from maternal cells in peripheral blood using an erythroblast scoring system based on the unique morphological and hemoglobin staining characteristics of this cell type. Presumptive fNRBCs were further analyzed for the presence of paternally inherited DNA polymorphisms to prove fetal origin. STUDY DESIGN: fNRBCs were isolated by density gradient separation, CD15/ 45 depletion, and gamma hemoglobin positive selection from peripheral blood of nine women following termination of pregnancy for trisomy 21 (4 cases), 18 (1 case), 13 (2 cases), and other genetic abnormalities (2 cases). Candidate fetal NRBCs, based on four discrete morphological and hemoglobin staining criteria, were then subjected to fluorescent PCR amplification of chromosome 21 (D21S1411, D21S11) and chromosome 18 (D18S535) short tandem repeat (STR) DNA polymorphisms. RESULTS: In all cases candidate fetal NRBCs were accurately identified based on morphologic and hemoglobin staining characteristics and confirmed to be fetal in origin based on the presence of shared and non-shared polymorphic DNA alleles when compared to DNA isolated from maternal cells. CONCLUSION: Using the erythroblast scoring system and subsequent analysis of inherited DNA polymorphisms, we were able to distinguish fetal NRBCs from maternal cells and prove fetal origin independent of gender. These results suggest that this novel combined approach to fetal cell isolation and genetic analysis is a promising method for noninvasive prenatal diagnostic applications. Women and Infants’ Hospital, Maternal Fetal Medicine, Providence, Rhode Island, University of North Carolina, Department of Obstetrics and Gynecology, Chapel Hill, North Carolina OBJECTIVE: Vaginal bleeding in the first trimester may result in a disruption in the maternal-fetal interface with subsequent transfer of hormones into the maternal circulation. Elevated maternal serum concentrations of AFP have been observed in the first and second trimester in women with a history of first trimester vaginal bleeding. Our goal was to examine the effect of first trimester vaginal bleeding on first trimester serum levels of PAPP-A, free b-hCG, and nuchal translucency (NT) using a prospectively-collected large database. STUDY DESIGN: Women enrolled in the FASTER trial had a NT measurement and PAPP-A and free b-hCG levels drawn at 10 3/7-13 6/7 weeks. Patients with fetal anomalies, a diagnosis of diabetes prior to becoming pregnant or who conceived with invitro fertilization were excluded from this analysis. Patients were divided into three groups: (1) no bleeding, (2) light bleeding, or (3) heavy bleeding. The log transformed multiples of the medians (MoMs) of NT, PAPPA, and free b-hCG for the three groups were compared using analysis of variance for crude effects and analysis of covariance adjusting for possible confounding variables. For presentation, the results were back transformed to medians in MoMs. RESULTS: The study included 34,548 patients: 29,639 patients without bleeding, 4341 patients with light bleeding, and 547 patients with heavy bleeding. Adjusted medians of the MoMs of the markers are listed in the table. CONCLUSION: First trimester vaginal bleeding does not appear to affect PAPP-A or free b-hCG levels nor does bleeding impact NT measurement in this large study.

Rapid identification of pickle yeasts by fluorescent PCR and microtemperature-gradient gel electrophoresis

Monitoring the yeast populations within pickle soaking fluid is imperative for ensuring optimum taste, but these analyses have proven time-consuming and expensive, limiting their industrial application. Here, yeasts were identified in the soaking fluid from Japanese radish pickles using fluorescent PCR amplification of the variable D1/D2 region of the 26S rDNA, followed by analysis with microtemperature-gradient gel electrophoresis (μ-TGGE). This smaller version of the normal TGGE apparatus is capable of analyzing samples 10- to 20-fold faster without sacrificing data quality. Each primer set was labeled with a different fluorescent dye, allowing easy isolation of the various PCR products and identification of the bands corresponding to the various yeasts. The results indicate that fluorescent PCR and μ-TGGE may be a useful new method for rapid, easy monitoring of yeast flora in various food industries. This new method can be used on a daily basis to provide overviews of yeast flora during pickle production, allowing producers to quickly grasp pickle readiness at a single glance.

Rapid identification of pickle yeasts by fluorescent PCR and microtemperature-gradient gel electrophoresis

Monitoring the yeast populations within pickle soaking fluid is imperative for ensuring optimum taste, but these analyses have proven time-consuming and expensive, limiting their industrial application. Here, yeasts were identified in the soaking fluid from Japanese radish pickles using fluorescent PCR amplification of the variable D1/D2 region of the 26S rDNA, followed by analysis with microtemperature-gradient gel electrophoresis (μ-TGGE). This smaller version of the normal TGGE apparatus is capable of analyzing samples 10- to 20-fold faster without sacrificing data quality. Each primer set was labeled with a different fluorescent dye, allowing easy isolation of the various PCR products and identification of the bands corresponding to the various yeasts. The results indicate that fluorescent PCR and μ-TGGE may be a useful new method for rapid, easy monitoring of yeast flora in various food industries. This new method can be used on a daily basis to provide overviews of yeast flora during pickle production, allowing producers to quickly grasp pickle readiness at a single glance.

Software-based assessment of fluorescent PCR amplification pattern for a highly integrative laboratory management

Software-based assessment of fluorescent PCR amplification pattern for a highly integrative laboratory management

Diagnosis of Trisomy 21 in Fetal Nucleated Erythrocytes from Maternal Blood by Use of Short Tandem Repeat Sequences

The purpose of this study was to determine whether aneuploid fetal nucleated erythrocytes (NRBCs) could be detected in maternal blood through the use of fluorescent PCR amplification with polymorphic short tandem repeat (STR) markers as an alternative or complementary method to analysis by fluorescent in situ hybridization (FISH). Peripheral blood samples were obtained from women who had just undergone termination of pregnancy because of fetal trisomy 21 (three cases, 47,XY,+21; four cases, 47,XX,+21). Candidate fetal cells were isolated by flow-sorting by antibodies to the gamma chain of fetal hemoglobin and Hoechst 33342. FISH analysis was performed by the use of chromosome-specific probes for X, Y, and 21. Fetal NRBCs, as defined by the presence of gamma staining, characteristic morphology, and three chromosome 21 signals, along with maternal leukocytes, defined as gamma negative and two chromosome 21 signals, were micromanipulated separately and subjected to fluorescent PCR amplification of chromosome 21 STR markers (D21S11, D21S1411, and/or D21S1412). In five of seven cases analyzed, fetal NRBCs were aneuploid, as determined by the presence of triallelic or diallelic peaks of chromosome 21 sequences when compared with sequences from the maternal leukocytes. Fluorescent PCR amplification of STRs can detect fetal aneuploidy and may be useful in the setting of poor hybridization efficiency with FISH analysis. These results suggest that combined fetal aneuploidy and single-gene diagnoses by the use of DNA microarrays may be feasible in the near future.

Detection of maternal DNA in umbilical cord plasma by fluorescent PCR amplification of short tandem repeat sequences.

Recently, maternal DNA was detected in umbilical cord blood using PCR amplification of minisatellite sequences. The presence of maternal DNA was demonstrated in 1% to 100% of umbilical cord blood samples. The objective of this study was to determine the frequency of cord blood contamination by maternal genetic material. We used fluorescent PCR amplification of highly polymorphic short tandem repeat (STR) markers to detect maternal DNA in umbilical cord plasma.

Microsatellite marker analysis of allelic imbalance in a rat fibrosarcoma model

Fibrosarcomas were induced by subcutaneous injections of DMBA (7,12-dimethylbenz[a]-anthracene) in progeny from a cross between the inbred rat strains BN/Han and LE/Mol. Twenty-one of 34 treated animals (62%) developed fibrosarcomas at the injection site. The tumor DNA was analyzed for allelic imbalance that would be indicative of amplification or deletion of genetic material. In order to cover the entire length of the rat linkage map, including all 20 autosomes and the X chromosome, 215 polymorphic microsatellite markers were selected. The markers were grouped into panels of 6–12 markers, which could be analyzed simultaneously on polyacrylamide gels after fluorescent PCR amplification. Numerous cases of allelic imbalance were revealed using the panels on the fibrosarcoma material. Most of them involved markers situated on rat chromosome 1 and 2, suggesting that genes located on these chromosomes are important in sarcomagenesis. The development of microsatellite marker panels such as those described will greatly facilitate future analysis of rat tumors by genome-wide scanning.

Rapid trisomy diagnosis (21, 18, and 13) using fluorescent PCR and short tandem repeats: applications for prenatal diagnosis and preimplantation genetic diagnosis.

Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis from amniotic fluid. This requires lengthy laboratory procedures and high costs and is unsuitable for large-scale screening of pregnant women. An alternative method, which is rapid and inexpensive and may potentially be suitable for diagnosing trisomies even from single fetal cells, is the fluorescent polymerase chain reaction (F-PCR) using polymorphic small tandem repeats (STRs). In this paper we present data demonstrating that fluorescent PCR amplification of STRs can be used for rapid diagnosis of trisomy 21, trisomy 18, and trisomy 13 and can be successfully applied to both prenatal diagnosis and diagnosis of single cells. This study also reports significant numbers of prenatal diagnoses using quantitative fluorescent PCR. We believe that further studies of greater numbers of samples will determine the absolute reliability of this technique. These results also provide a model for trisomy diagnosis from single cells using multiple STR markers for either preimplantation genetic diagnosis or, potentially, diagnosis from fetal cells isolated from maternal blood.