Round Of Polymerase Chain Reaction Research Articles

DNA Fountain enables a robust and efficient storage architecture.

DNA is an attractive medium to store digital information. Here we report a storage strategy, called DNA Fountain, that is highly robust and approaches the information capacity per nucleotide. Using our approach, we stored a full computer operating system, movie, and other files with a total of 2.14 × 106 bytes in DNA oligonucleotides and perfectly retrieved the information from a sequencing coverage equivalent to a single tile of Illumina sequencing. We also tested a process that can allow 2.18 × 1015 retrievals using the original DNA sample and were able to perfectly decode the data. Finally, we explored the limit of our architecture in terms of bytes per molecule and obtained a perfect retrieval from a density of 215 petabytes per gram of DNA, orders of magnitude higher than previous reports.

Facilitation of Polymerase Chain Reaction with Poly(ethylene glycol)-Engrafted Graphene Oxide Analogous to a Single-Stranded-DNA Binding Protein.

Polymerase chain reaction (PCR), a versatile DNA amplification method, is a fundamental technology in modern life sciences and molecular diagnostics. After multiple rounds of PCR, however, nonspecific DNA fragments are often produced and the amplification efficiency and fidelity decrease. Here, we demonstrated that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly improve the PCR specificity and efficiency. PEG-nGO allows the specificity to be maintained even after multiple rounds of PCR, allowing reliable amplification at low annealing temperatures. PEG-nGO decreases the nonspecific annealing of single-stranded DNA (ssDNA), such as primer dimerization and false priming, by adsorbing excess primers. Moreover, PEG-nGO interrupts the reannealing of denatured template DNA by preferentially binding to ssDNA. Thus, PEG-nGO enhances the PCR specificity by preferentially binding to ssDNA without inhibiting DNA polymerase, which is analogous to the role of ssDNA binding proteins.

Multiple infections of Anaplasma platys variants in Philippine dogs.

Aim:Anaplasma platys, the causative agent of infectious canine cyclic thrombocytopenia, is a tick-borne pathogen that also has been implicated as potentially zoonotic. To provide molecular evidence on the multiple infections of A. platys variants in Philippine dogs.Materials and Methods:DNA fragments of A. platys from infected dogs in the Philippines were molecularly characterized. For screening, 25 dogs suspected to have canine anaplasmosis were tested using a 16S rRNA-based nested polymerase chain reaction (PCR). Infection was confirmed by sequencing of positive amplicons. Second round PCR targeting a longer 16S rRNA fragment was subsequently performed on the first round PCR amplicons of the positive samples. Further characterization using the heat-shock operon (groEL) gene was also performed on the A. platys-positive samples.Results:10 16S rRNA sequences were obtained and found 99.6-100% identical to each other and 99.6-99.7% identical to the closest registered A. platys sequences. On the other hand, 36 groEL clone sequences were obtained and found to be 85.1-99.8% identical with each other and 85.0-88.9% identical to the closest previously registered A. platys sequences. Four dogs were found coinfected with 2-3 groEL variant sequences. Phylogenetic analyses suggest that the detected A. platys in the Philippines may represent unique variants.Conclusion:A. platys variants were detected in Philippine dogs. Coinfection of different A. platys variants in dogs was also demonstrated. The present study may indicate the potential genetic diversity of A. platys in the country.

Occurrence of Blastocystis sp. in water catchments at Malay villages and Aboriginal settlement during wet and dry seasons in Peninsular Malaysia.

In the tropics, there are too few studies on isolation of Blastocystis sp. subtypes from water sources; in addition, there is also an absence of reported studies on the occurrence of Blastocystis sp. subtypes in water during different seasons. Therefore, this study was aimed to determine the occurrence of Blastocystis sp. subtypes in river water and other water sources that drained aboriginal vicinity of highly endemic intestinal parasitic infections during wet and dry seasons. Water samples were collected from six sampling points of Sungai Krau (K1–K6) and a point at Sungai Lompat (K7) and other water sources around the aboriginal villages. The water samples were collected during both seasons, wet and dry seasons. Filtration of the water samples were carried out using a flatbed membrane filtration system. The extracted DNA from concentrated water sediment was subjected to single round polymerase chain reaction and positive PCR products were subjected to sequencing. All samples were also subjected to filtration and cultured on membrane lactose glucuronide agar for the detection of faecal coliforms. During wet season, Blastocystis sp. ST1, ST2 and ST3 were detected in river water samples. Blastocystis sp. ST3 occurrence was sustained in the river water samples during dry season. However Blastocystis sp. ST1 and ST2 were absent during dry season. Water samples collected from various water sources showed contaminations of Blastocystis sp. ST1, ST2, ST3 and ST4, during wet season and Blastocystis sp. ST1, ST3, ST8 and ST10 during dry season. Water collected from all river sampling points during both seasons showed growth of Escherichia coli and Enterobacter aerogenes, indicating faecal contamination. In this study, Blastocystis sp. ST3 is suggested as the most robust and resistant subtype able to survive in any adverse environmental condition. Restriction and control of human and animal faecal contaminations to the river and other water sources shall prevent the transmission of Blastocystis sp. to humans and animals in this aboriginal community.

Effects of breed, age and season on quality of frozen-thawed semen in stallions

The aim of this investigation was to develop a real-time polymerase chain reaction (PCR) assay for detecting circulating cell-free fetal DNA in Arabian pregnant mare plasma and predicting fetal sex through identifying SRY gene. Peripheral blood samples of 28 pregnant mares were collected into tubes containing EDTA, at a single time point, between 8 and 20 weeks of gestation. Circulating cell-free fetal DNA was extracted from 3 mL of maternal plasma. Using outer and inner primers, real-time PCR was performed for the SRY gene and for the glyceraldehyde-3-phosphate dehydrogenase gene as the internal control. Amplicons were labeled as positive, negative, or inconclusive according to threshold cycle and melting curve analysis. Of the total 28 Arabian pregnant mares, 16 were carrying male and 12 were carrying female fetuses. On first round of real-time PCR, results were inconclusive for SRY gene; however, on the second round, conclusive results were obtained. In total, the accuracy of test was 88% (22/25) (confidence interval [CI]: 65.5, 95.6); it had a sensitivity of 85.7% (CI: 65.6, 92.5), whereas its specificity was 90.9% (CI: 65.3, 99.5). We had a positive predictive value of 92.3% (CI: 70.7, 99.6) and a negative predictive value of 83.3% (CI: 59.9, 91.2). To conclude, SRY gene can permit detection of equine fetal sex with a good accuracy by analyzing cell-free fetal DNA in maternal plasma just after 8 weeks of gestation.

Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.

Molecular identification of phytoplasmas associated with some weeds in West Azarbaijan province, Iran

During field surveys in 2013 and 2014, about 14 weed plants showing phytoplasma diseases symptoms including yellowing and witches’broom were collected and tested by polymerase chain reaction (PCR) using universal primers for 16SrRNA starting by primer pairs P1/P7 in first round PCR followed by primer pair R16F2n/R16R2 in nested PCR. The detected phytoplasmas were characterized and differentiated through sequence analysis of PCR-amplified rDNA and virtual restriction fragment length polymorphism (RFLP). The phytoplasmas detected in symptomatic horseweed (Erigeron canadensis L.), common madder (Rubia tinctorum L.), Johnson grass (Sorghum halepense [L.] Pers.) and Sophora root (Sophora alopecuroides L.) were identified as members of the clover proliferation group (16SrVI group) by construction of phylogenetic trees. Further analysis by virtual RFLP classified the phytoplasmas of Erigeron canadensis L. and Sorghum halepense L. in subgroup 16SrVI-A and phytoplasmas of Rubia tinctorum L. and Sophora alpecuriodes L. in subgroup 16SrVI-D. This is the first report on the occurrence of phytoplasma diseases of weeds in west Azarbaijan, Iran.

Developing a Nested Real-Time PCR Assay for Determining Equine Fetal Sex Prenatally

The aim of this investigation was to develop a real-time polymerase chain reaction (PCR) assay for detecting circulating cell-free fetal DNA in Arabian pregnant mare plasma and predicting fetal sex through identifying SRY gene. Peripheral blood samples of 28 pregnant mares were collected into tubes containing EDTA, at a single time point, between 8 and 20 weeks of gestation. Circulating cell-free fetal DNA was extracted from 3 mL of maternal plasma. Using outer and inner primers, real-time PCR was performed for the SRY gene and for the glyceraldehyde-3-phosphate dehydrogenase gene as the internal control. Amplicons were labeled as positive, negative, or inconclusive according to threshold cycle and melting curve analysis. Of the total 28 Arabian pregnant mares, 16 were carrying male and 12 were carrying female fetuses. On first round of real-time PCR, results were inconclusive for SRY gene; however, on the second round, conclusive results were obtained. In total, the accuracy of test was 88% (22/25) (confidence interval [CI]: 65.5, 95.6); it had a sensitivity of 85.7% (CI: 65.6, 92.5), whereas its specificity was 90.9% (CI: 65.3, 99.5). We had a positive predictive value of 92.3% (CI: 70.7, 99.6) and a negative predictive value of 83.3% (CI: 59.9, 91.2). To conclude, SRY gene can permit detection of equine fetal sex with a good accuracy by analyzing cell-free fetal DNA in maternal plasma just after 8 weeks of gestation.

Pooled Nucleic Acid Testing to Detect Antiretroviral Treatment Failure in HIV-Infected Patients in Mozambique.

In resource-limited settings, viral load monitoring of HIV-infected patients receiving antiretroviral therapy (ART) is not readily available because of high costs. Here, we compared the accuracy and costs of quantitative and qualitative pooled methods with standard viral load testing. Blood was collected prospectively from 461 patients receiving first-line ART in Mozambique who had not been evaluated previously with viral load testing. Screening for virologic failure of ART was performed quantitatively (ie, standard viral loads) and qualitatively [one and 2 rounds of polymerase chain reaction (PCR)]. Individual samples and minipools of 5 samples were then analyzed using both methods. The relative efficiency, accuracy, and costs of each method were calculated based on viral load thresholds for ART failure. Standard viral load testing of individual samples revealed a high rate of ART failure (19%-23%) across all virologic failure thresholds, and the majority of the patients (93%) with viral loads >1500 copies per milliliter had genotypic resistance to drugs in their ART regimen. Pooled quantitative screening and deconvolution testing had positive and negative predictive values exceeding 95% with cost savings of $11,250 compared with quantitative testing of each sample individually. Pooled qualitative screening and deconvolution testing had a higher cost savings of $30,147 for 1 PCR round and $25,535 for 2 PCR rounds compared with quantitative testing each sample individually. Both pooled qualitative PCR methods had positive and negative predictive values ≥90%, but the pooled 1-round PCR method had a sensitivity of 64%. Given the high rate of undiagnosed ART failure and drug resistance in this cohort, it is clear that virologic monitoring is urgently needed in this population. Here, we compared alternative methods of virologic monitoring with standard viral load testing of individual samples and found these methods to be cost saving and accurate. The test characteristics of each method will likely need to be considered for each local population before it is adopted.

A new nested allele-specific multiplex polymerase chain reaction method for haplotyping of VKORC1 gene to predict warfarin sensitivity.

The vitamin K epoxide reductase complex 1 gene (VKORC1) is commonly assessed to predict warfarin sensitivity. In this study, a new nested allele-specific multiplex polymerase chain reaction (PCR) method that can simultaneously identify single nucleotide polymorphisms (SNPs) at VKORC1 381, 861, 5808, and 9041 for haplotype analysis was developed and validated. Extracted DNA was amplified in the first PCR DNA, which was optimized by investigating the effects of varying the primer concentrations, annealing temperature, magnesium chloride concentration, enzyme concentration, and the amount of DNA template. The amplification products produced from the first round of PCR were used as templates for a second PCR amplification in which both mutant and wild-type primers were added in separate PCR tubes, followed by optimization in a similar manner. The final PCR products were resolved by agarose gel electrophoresis and further analysed by using a VKORC1 genealogic tree to infer patient haplotypes. Fifty patients were identified to have H1H1, one had H1H2, one had H1H7, 31 had either H1H7 or H1H9, one had H1H9, eight had H7H7, and one had H8H9 haplotypes. This is the first method that is able to infer VKORC1 haplotypes using only conventional PCR methods.

First report of the molecular identification of a phytoplasma associated with dieback disease of papaya in Sri Lanka

Papaya (Carica papaya) is a very popular fruit crop in Sri Lanka, grown as a home garden and commercial crop for local and export markets. Papaya is commercially cultivated in Sri Lanka in about 8,000 ha with production of about 32,000 tonnes/year. As elsewhere, phytoplasma diseases have become a threat to the papaya crop in Sri Lanka. During November 2012, dieback-like symptoms were observed in papaya fields at Monaragala, Uva Province, in Sri Lanka. Symptoms in the field initially included a bright yellowing of the upper young leaves (Fig. 1a) that progressively evolved to tip necrosis in the tender leaves (Fig. 1b), followed by drying of the upper leaves and eventual death of the whole plant (Fig. 1c). Moreover, diseased plants exhibited discolouration of the vascular tissues, abnormal growth and shapes of fruits and premature fruit fall. Total genomic DNA was extracted from the midribs of fresh leaf samples (1 g) from four plants with symptoms and two symptomless plants using the DNeasy Plant Mini Kit (QIAGEN, UK) according to the manufacturer's protocol. Phytoplasma rDNA was amplified with the universal phytoplasma primer pairs P1/P7 in the first round of polymerase chain reaction (PCR), and R16F2n/R16R2 in a nested PCR (Dickinson et al., 3). Amplifications of the 16S rDNA were performed in 25 ül reactions using Ready-To-Go PCR beadsTM (Amersham Pharmacia Biotech, UK) in a TECHNE TC-3000 thermocycler, and resulted in PCR bands of approximately 1.2 kb from symptom-bearing plants but not from the symptomless papaya plants (Fig. 2). Three representative nested PCR products were purified (PCR Cleanup kit, Sigma, UK) and directly sequenced. A phylogenetic tree (Fig. 3) was constructed based on the 16S rDNA consensus sequence using the neighbour-joining method with default values and 1000 replicates for bootstrap analysis (MEGA version 3.1). The partial 16S rDNA sequences of the three representative phytoplasma isolates shared 100% sequence identity with each other. The BLAST analysis of the consensus sequence of the Monaragala papaya dieback phytoplasma (GenBank Accession No. KJ027722) revealed 99% sequence identity with that of phytoplasmas of group 16SrI (‘Candidatus Phytoplasma asteris’). Acosta et al. (1) previously reported a phytoplasma from group 16SrI associated bunchy top-like symptoms of papaya in Cuba. Moreover, phytoplasmas belonging to different 16Sr groups have been identified in papaya, particularly in the Asian continent including group 16SrII (‘Candidatus Phytoplasma aurantifolia’) in India associated with similar dieback symptoms (Rao et al., 5) and axillary shoot proliferation (Verma et al., 6); and group 16SrXII ‘Stolbur’ associated with a dieback-like disease of papaya in Israel (Gera et al., 4), and yellows disease in Taiwan (Bau et al., 2). To our knowledge, this is the first record of the identification of a 16SrI ‘Ca. Phytoplasma asteris’ isolate associated with a dieback-like disease of papaya in Sri Lanka. This finding demonstrates significant phytosanitary impact for the papaya industry in Sri Lanka, and the region, considering that 16SrI is the group that possesses the widest plant host range and the most complex epidemiology worldwide. The authors acknowledge the research grant (Pro/DVC/0185) received through the TURIS project from University of Ruhuna, Sri Lanka.

Generation of Helper Plasmids Encoding Mutant Adeno-associated Virus Type 2 Capsid Proteins with Increased Resistance against Proteasomal Degradation.

Adeno-associated virus type 2 (AAV2) vectors are widely used for both experimental and clinical gene therapy. A recent research has shown that the performance of these vectors can be greatly improved by substitution of specific surface-exposed tyrosine residues with phenylalanines. In this study, a fast and simple method is presented to generate AAV2 vector helper plasmids encoding capsid proteins with single, double or triple Y→F mutations. A one-step, high-fidelity polymerase chain reaction (PCR) cloning procedure involving the use of two partially overlapping primers to amplify a circular DNA template was applied to produce AAV2 cap genes encoding VP1 mutants with Y→F substitutions in residues 444, 500 or 730. The resulting constructs were used to make the different double and triple mutant by another round of PCR (Y444500F mutant), subcloning (Y444730F and Y500730F mutants) or a combination of both techniques (Y444500730F mutant). Nucleotide sequence analysis revealed successful introduction of the desired mutations in the AAV2 cap gene and showed the absence of any unintended mutations in the DNA fragments used to assemble the final set of AAV2 vector helper plasmids. The correctness of these plasmids was further confirmed by restriction mapping. PCR-based, single-step site-directed mutagenesis of circular DNA templates is a highly efficient and cost-effective method to generate AAV2 vector helper plasmids encoding mutant Cap proteins for the production of vector particles with increased gene transfer efficiency.

Comparative Evaluation of Uniplex, Nested, Semi-nested, Multiplex and Nested Multiplex PCR Methods in the Identification of Microbial Etiology of Clinically Suspected Infectious Endophthalmitis

Purpose: This study is aimed to determine the utility of various polymerase chain reaction (PCR) methods in vitreous fluids (VFs) for detecting the infectious genomes in the diagnosis of infectious endophthalmitis in terms of sensitivity and specificity.Methods: This prospective and consecutive analysis included a total of 66 VFs that were submitted for the microbiological evaluation, which were obtained from 66 clinically diagnosed endophthalmitis patients presented between November 2010 and October 2011 at the tertiary eye care referral centre in South India. Part of the collected VFs were subjected to cultures and smears, and the remaining parts were utilized for five PCR methods: uniplex, nested, semi-nested, multiplex and nested multiplex after extracting DNA, using universal eubacterial and Propionibacterium acnes species-specific primer sets targeting 16S rRNA gene in all bacteria and P. acnes, and panfungal primers, targeting 28S rRNA gene in all fungi.Results: Of the 66 VFs, five (7.5%) showed positive results in smears, 16 (24%) in cultures and 43 (65%) showed positive results in PCRs. Among the 43 positively amplified VFs, 10 (15%) were positive for P. acnes genome, one for panfungal genome and 42 (62%) for eubacterial genome (including 10 P. acnes positives). Among 42 eubacterial-positive VFs, 36 were positive by both uniplex (first round) and multiplex (first round) PCRs, while nested (second round) and nested multiplex (second round) PCRs produced positive results in 42 and 41 VFs, respectively. Of the 43 PCR-positive specimens, 16 (37%) had positive growth (15 bacterial and one fungal) in culture. Of 50 culture-negative specimens, 27 (54%) were showed positive amplification, of which 10 were amplified for both P. acnes and eubacterial genomes and the remaining 17 were for eubacterial genome alone.Conclusions: Nested PCRs are superior than uniplex and multiplex PCR. PCRs proved to be a powerful tool in the diagnosis of endophthalmitis, especially for detecting uncultured microbes.

Detecting DNA Methylation of the BCL2, CDKN2A and NID2 Genes in Urine Using a Nested Methylation Specific Polymerase Chain Reaction Assay to Predict Bladder Cancer

Detection of methylated DNA has been shown to be a good biomarker for bladder cancer. Bladder cancer has the highest recurrence rate of any cancer and, as such, patients are regularly monitored using invasive diagnostic techniques. As urine is easily attainable, bladder cancer is an optimal cancer to detect using DNA methylation. DNA methylation is highly specific in cancer detection. However, it is difficult to detect because of the limited amount of DNA present in the urine of patients with bladder cancer. Therefore, an improved, sensitive and noninvasive diagnostic test is needed. We developed a highly specific and sensitive nested methylation specific polymerase chain reaction assay to detect the presence of bladder cancer in small volumes of patient urine. The genes assayed for DNA methylation are BCL2, CDKN2A and NID2. The regions surrounding the DNA methylation sites were amplified in a methylation independent first round polymerase chain reaction and the amplification product from the first polymerase chain reaction was used in a real-time methylation specific polymerase chain reaction. Urine samples were collected from patients receiving treatment at Wolfson Medical Center in Holon, Israel. In a pilot clinical study using patient urine samples we were able to differentiate bladder cancer from other urogenital malignancies and nonmalignant conditions with a sensitivity of 80.9% and a specificity of 86.4%. We developed a novel methylation specific polymerase chain reaction assay for the detection and monitoring of bladder cancer using DNA extracted from patient urine. The assay may also be combined with other diagnostic tests to improve accuracy.

Single step detection of HIV-1 proviral DNA and housekeeping β-actin gene from dried blood spots by a monoplex polymerase chain reaction

There is a growing need for developing a simple, rapid, reliable and cost effective method for detection of HIV-1 for early diagnosis of the infection especially in developing countries and in resource limited settings. A method for simultaneous detection of the HIV-1 p17 gene and the house keeping human β-actin gene from dried blood spots (DBS) by a monoplex polymerase chain reaction (PCR) is described. Genomic DNA was extracted from 40 HIV-1 positive and 40 HIV-1 negative DBS and used as templates to amplify both the HIV-1 p17 and β-actin genes simultaneously under the same cycling condition by a single round PCR. This method of detection of HIV-1 may provide a simple, rapid and cost effective alternative in resource limited settings; however, it would require testing a larger number of samples before widespread use.

Graphene Enhances the Specificity of the Polymerase Chain Reaction

Graphene can inhibit non-specific DNA fragments, and the specificity of the polymerase chain reaction (PCR) can be retained even after eight rounds of repeated amplification in the presence of graphene in the form of reduced graphene oxide (RGO). In the figure, the numbers at the top give the number of rounds of PCR; lanes marked with C correspond to controls (no RGO), and the concentration of RGO in the other samples is 12 μg mL(-1) .

Detection and molecular characterization of phytoplasma associated with chickpea phyllody disease in south India

Chickpea (Cicer arietinum L.) plants showing typical symptoms of infection by a phytoplasma that causes phyllody disease have been commonly observed in recent years in parts of south India. The symptoms included pale green leaves, bushy appearance due to excessive stunting of shoots, reduced internodal length and excessive axillary proliferation. The causal agent of the phyllody disease was identified based on symptoms, amplification of 16S rDNA of the phytoplasma by polymerase chain reaction (PCR) from infected samples, as well as by sequencing and phylogenetic analysis. First round PCR and nested-PCR protocols were standardized for improved efficiency and reliability of the diagnostic protocols. Using the primers P1/P7 and R16F2n/R16R2, 1,800 bp and 1,200 bp size products were amplified in first round PCR and nested-PCR protocols, respectively. The PCR product was cloned and sequenced and compared with the reference phytoplasma sequences from the database (NCBI). The Indian chickpea phyllody phytoplasma 16S rDNA sequences shared the highest nucleotide identity (>98%) with the 16S rII group phytoplasma candidates, also infecting chickpea from Australia and Pakistan. This is the first report of a phytoplasma of the 16SrII-group infecting chickpea from India. The genetic similarities and the potential threat of this new disease to chickpea cultivation in India are discussed.

Single-cell polymerase chain reaction-based pre-implantation genetic diagnosis using fragment analysis for β-thalassemia in an Indian couple with β-globin gene mutations

Despite advances in diagnostic techniques, approximately 10,000 babies with β-thalassemia major are born annually in India. Pre-implantation genetic diagnosis (PGD), an alternative to prenatal diagnosis, helps in negative selection of affected embryos prior to implantation. Hereby, we report the first successful β-thalassemia PGD pregnancy in an Indian carrier couple. β-Thalassemia mutation analysis by Amplification-Refractory Mutation Sequence (ARMS)-polymerase chain reaction (PCR) in the parents, followed by PGD for β-thalassemia mutation in embryos in two consequent in vitro fertilization (IVF) cycles, with transfer for three β-thalassemia minor embryos, resulted in singleton successful pregnancy, the results of which were confirmed on prenatal diagnosis. With advances in assisted reproductive techniques and molecular diagnosis, PGD for monogenic diseases is feasible in high-risk couples. The methodology in the current study included two rounds of PCR using fluorescently labeled primers, fragment analysis using the ABI 3100 nucleotide sequencer and the GeneMapper software, purification, and concentration of PCR product, which enabled distinct clear peaks making the analysis and interpretation non-ambiguous.

Rapid detection of viable bacteria by nested polymerase chain reaction via long DNA amplification after ethidium monoazide treatment

In assays to determine whether viable cells of Enterobacteriaceae are present in pasteurized milk, the typical ethidium monoazide (EMA) polymerase chain reaction (PCR) targets a short stretch of DNA. This process often triggers false-positive results owing to the high level of dead cells of Enterobacteriaceae that had initially contaminated the sample. We have developed a novel, direct, real-time PCR that does not require DNA isolation (DQ-PCR) to detect low levels of cells of Enterobacteriaceae regardless of live and dead cells first. We confirmed that the DQ-PCR targeting a long DNA (the 16S ribosomal RNA [rRNA] gene, amplified length of 1514 bp) following EMA treatment is a promising tool to detect live bacteria of all genera owing to the complete suppression of background signal from high levels of dead bacteria in pasteurized milk. However, when identifying viable bacteria in pasteurized milk, commercial PCR primers designed for detecting long stretches of DNA are generally not available. Thus, we treated samples with EMA and then carried out an initial round of PCR of a long stretch of DNA (16S gene, 1514 bp). We then performed another round of PCR, a novel nested PCR to generate short products using commercial primers. This procedure resulted in the rapid detection of low levels of viable cells of Enterobacteriaceae.

Laser Capture Microdissection: Understanding the Techniques and Implications for Molecular Biology in Nursing Research Through Analysis of Breast Cancer Tumor Samples

The purpose of this paper is to review the techniques and implications of laser capture microdissection (LCM) to isolate tissue and DNA of interest using breast biopsy tissue as an example. Tissues are a heterogeneous mix of different cell types, and molecular alterations are often specific to a single cell type. An accurate correlation of molecular and morphologic pathologies requires the ability to procure pure populations of morphologically similar cells for molecular analysis. LCM is a technique for isolating highly pure cell populations of morphologically similar cells from a heterogeneous tissue section. Nine invasive, paraffin-embedded breast biopsy specimens were obtained and analyzed. Depending on the size of the lesion, 500-1,000 shots using the 7.5- or 15-µm infrared laser beam were utilized to obtain an average of 2,000 cells. DNA was isolated from normal tissue and carcinomas and polymerase chain reaction (PCR) amplification was examined by agarose gel electrophoresis. The HER2/neu gene was amplified by standard PCR. A second round of PCR using nested primers to re-amplify the HER2/neu fragment was performed. Amplification of the HER2/neu gene with DNA isolated from pure cell populations by LCM was performed. The results indicated that 22% of the cases studied were positive for HER2/neu amplifications, which corresponds to the literature regarding HER2/neu amplification/overexpression. HER2/neu amplification could be detected as early as the ductal carcinoma in situ (DCIS) stage. LCM is an accurate and reliable method to acquire nucleic acid and protein profiles from a specific cell population in heterogeneous tissue.