Detection Of Polymerase Chain Reaction Products Research Articles

Antibiofilm Activity of 4-(Adamantyl-1)-1-(1-Aminobutyl) Benzol against Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is a widespread opportunistic pathogen, causing community-acquired and nosocomial infections with both acute and chronic recurrent courses. The process of chronicity of the disease is provided by biofilms. Features of the structure and functioning of biofilms, in particular the presence of matrix, quorum sensing systems, persistent cells, and efflux pumps, provide microbial communities with resistance to antimicrobial drugs under their action in therapeutic concentrations. The insufficient eff ectiveness of modern antimicrobial chemotherapy against biofi lm microorganisms indicates the urgency of the problem to search for compounds with antibiofilm activity that can affect various stages of the biofilm formation and the formed biofilm. The aim of the study is to establish the antibiofilm activity of 4-(adamantyl-1)-1-(1-aminobutyl) benzol against methicillin-resistant S. aureus (MRSA) and to determine the mechanism of its action. Methods. The ability of adamantane-containing compound 4-(adamantyl-1)-1-(1-aminobutyl) benzol (AM-166) to prevent biofilm formation and destroy the formed biofilm of S. aureus was investigated on polystyrene plates by the sorption of gentian violet on its structures followed with desorption of the dye into the organic solvent. The viability of S. aureus cells at the first stage of biofilm formation and in the composition of mature biofilms was evaluated using specific dyes for living (acridine orange) and non-viable (propidium iodide) cells. Detection of genes responsible for antibiotic resistance and biofi lm formation was performed by the polymerase chain reaction (PCR) with detection of PCR products in agarose gel. Evaluation of the effect of AM-166 on the expression of genes regulating the biofilm formation (ica, agrA, sarA, and sigB) was investigated by the real-time PCR and semi-quantitative PCR. Results. It was found that the compound AM-166 shows activity against S. aureus biofilm formation. The most pronounced effect was registered at a concentration of 5.0 minimum inhibitory concentration (MIC) (92.3%.) Under the action of AM-166 on the formed 2-day biofilms, their destruction was marked: the biomass decreases by 30.9% at 5.0 MIC. According to the results of fluorescence microscopy, the adamantane derivative at 5.0 MIC helps to reduce the number of viable cells at different stages of formation of the S. aureus biofilm. The results of molecular genetic studies indicate that the ica gene expression is significantly inhibited by the action of subinhibitory concentrations of the compound AM-116. No significant changes in the expression of sarA, agrA, and sigB genes were registered. Conclusions. Experiments on the effect of adamantane derivative on S. aureus biofilms showed that the most pronounced activity of AM-116 was observed at the stage of biofilm formation, as evidenced by the inhibition of transcriptional activity of the ica gene responsible for early stages of the biofilm formation, in particular the adhesion of planktonic cells to the substrate.

ACE gene polymorphism and features of kidney disorders in patients with type 2 diabetes mellitus

Objective — to determine the prevalence of the Alu Ins/Del polymorphism of the angiotensin‑converting enzyme (ACE) gene in patients with type 2 diabetes mellitus (DM 2) with nephropathy and to identify possible associative relationship between the course of the disease and the genetic profile of the examined subjects.
 Materials and methods. Examinations involved 73 patients with diabetic nephropathy (DN), treated in the hospital of L. T. Mala Therapy National Institute. The control group consisted of 19 healthy individuals. After the initial examination and depending on the polymorphic variant of the ACE gene, patients were divided into three groups: group I — carriers of D/D polymorphism of the ACE gene (n = 23); group II — patients with I/D polymorphism of the ACE gene (n = 32); Group III — patients with I/I polymorphism of the ACE gene (n = 18). DNA amplification and genotyping were carried out by real‑time polymerase chain reaction (PCR) using the SNP‑Express‑Shot reagent kit (Litech, RF) and the CFX96 touch real‑time PCR product detection system (BioRad).
 Results. The distribution of the genotypes of the Alu I/D polymorphism of the ACE gene corresponded to the Hardy‑Weinberg equilibrium in all studied groups and did not differ significantly from European populations. Comparison of patients with DN, carriers of different Ins/Del polymorphic variants of the ACE gene, did not show significant differences in the stages of chronic kidney disease, creatinine level, and glomerular filtration rate (GFR). Patients with type 2 DM, homozygous for the I/I allele of the ACE gene, had significantly lower levels of urinary albumin (16.80 [12.61 — 34.20] mk/ mL), compared with I/D heterozygotes (26.07 [20.91 — 44.27] mkg/mL), p < 0.05, which demonstrates the negative effects of D allele on the kidney damage progression in this category of patients, even under the same GFR.
 Conclusions. Patients with DN who are homozygous for the ACE gene allele I, have significantly lower albumin levels than I/D heterozygotes, demonstrating an association between the D allele and the progression of renal disease in type 2 DM. Determining the ACE gene polymorphism in patients with type 2 DM allows to identify risk groups for the DN development and will help to make individualized adjustments to the scheme of pharmacological therapy.

Naked-eye detection of specific DNA sequences amplified by the polymerase chain reaction with nanocomposite beads

We developed a novel nanocomposite bead system for detection by the naked eye of specific DNA sequences amplified by the polymerase chain reaction (PCR). The DNA probes, which were complementary to the target DNA, are conjugated with the nanocomposite beads. If the amplified products contained sequences complementary to the probes, the beads aggregated through sandwich hybridization. The aggregation was detectable as precipitation of the nanocomposite beads. The results were determined visually and did not require instrumental detection. The assay was sensitive enough to detect PCR products with a detection limit of 10 copies/tube for DNA templates. This technique is that all needed components are included within the initial cap, so that the risk of carryover contamination is very low. The nanocomposite bead system has broad application prospects for the detection of specific DNA sequences in biological and biomedical research.

PCR Multiplexing Based on a Single Fluorescent Channel Using Dynamic Melting Curve Analysis.

Since its invention in 1986, the polymerase chain reaction (PCR), has become a well-established method for the detection and amplification of deoxyribonucleic acid (DNA) with a specific sequence. Incorporating fluorescent probes, known as TaqMan probes, or DNA intercalating dyes, such as SYBR Green, into the PCR mixture allows real-time monitoring of the reaction progress and extraction of quantitative information. Previously reported real-time PCR product detection using intercalating dyes required melting curve analysis (MCA) to be performed following thermal cycling. Here, we propose a technique to perform dynamic MCA during each thermal cycle, based on a continuous fluorescence monitoring method, providing qualitative and quantitative sample information. We applied the proposed method in multiplexing detection of hepatitis B virus DNA and complementary DNA of human immunodeficiency virus as well as glyceraldehyde 3-phosphate dehydrogenase in different concentration ratios. We extracted the DNA melting curve and its derivative from each PCR cycle during the transition from the elongation to the denaturation temperature with a set heating rate of 0.8 K·s–1and then used the data to construct individual PCR amplification curves for each gene to determine the initial concentration of DNA in the sample. Our proposed method allows researchers to look inside the PCR in each thermal cycle, determining the PCR product specificity in real time instead of waiting until the end of the PCR. Additionally, the slow transition rate from elongation to denaturation provides a dynamic multiplexing assay, allowing the detection of at least three genes in real time.

DESAIN PRIMER UNTUK AMPLIFIKASI REGIO PROMOTER GEN inhA ISOLAT P016 MULTIDRUG RESISTANCE Mycobacterium tuberculosis DENGAN METODE POLYMERASE CHAIN REACTION

Mutations in the inhA promoter region are responsible for isoniazid resistance and cross-resistance to ethionamide. To identify mutations with polymerase chain reaction (PCR), a pair of primers that can amplify the target region. This aim of this study was to obtain the best primer pair to amplify the inhA promoter region using the Clone Manager Suite 6 program. The inhA promoter sequence (Genbank: U66801) obtained from the www.ncbi.nlm.nih.gov was used as a template. The design results obtained the best primer pair tested in vitro using Polymerase Chain Reaction (PCR) method. The PCR amplification process was performed for 40 cycles with the following conditions: predenaturase (95oC for 15 minutes), denaturation (94 oC for 1 minute), annealing (56 oC for 1 minute 30 seconds), elongation (72 oC for 2 minutes), and final elongation (72 oC for 10 minutes). Detection of PCR products was performed in agarose gel electrophoresis 1.3% w / v and visualized by UV transluminator tool. The results obtained were forward primers of 5'-GGTCGAAGTGTGCTGAGTC-3 'and reverse primer 5'-TGCTCTTCTACCGCCGTGA-3' which met the good primary criterion based on Clone Manager Suite 6. The pair of primers has been able to amplify the inhA promoter region by the length of product produced at 373 bp.

Integrated Graphene Oxide Purification-Lateral Flow Test Strips (iGOP-LFTS) for Direct Detection of PCR Products with Enhanced Sensitivity and Specificity.

An integrated graphene oxide purification-lateral flow test strip (iGOP-LFTS) was developed for on-strip purifying and visually detecting polymerase chain reaction (PCR) products with an improved sensitivity as well as a more stringent specificity. PCR products amplified with a pair of biotin- and digoxin-labeled primers were directly pipetted onto GO pads, on which graphene oxide selectively adsorbed residual primers and primer-dimers with the aid of a running buffer containing MgCl2 and Tween 20. By stacking up three GO pads to increase the surface area for adsorption, 83.4% of double-stranded DNA with a length of 30 bp and 98.6% of 20-nt primers could be removed from a 10-μL DNA mixture. Since no primers interfered with detection, the increase of the sample loading volume from 5 to 20 μL could improve the signal-to-noise ratio of the test line 1.6 fold using the iGOP-LFTS while no changes were observed using the conventional LFTS. The limit of detection of the iGOP-LFTS was determined to be 30 copies of bacteriophage λ-DNA with naked eyes and this limit could be further decreased to 3 copies by loading 20 μL of the sample, which corresponded to a 1000-fold improvement compared to that of the LFTS detected by naked eyes. When the ImageJ analysis was employed, a 100-fold decrease of the detection limit can be obtained. In addition, due to the removal of the primer-dimers, the dim test line observed in the negative control of the LFTS was eliminated using the iGOP-LFTS. A mock clinical specimen spiked with defective HIV-1 (human immunodeficiency virus) viruses was successfully analyzed using a two-step reverse transcription-PCR with 30 amplification cycles followed by the iGOP-LFTS detection. These significant improvements were achieved without introducing any additional hands-on operations and instrumentations.

Glass-polytetrafluoroethylene-glass based sandwich microdevice for continuous-flow polymerase chain reaction and its application for fast identification of foodborne pathogens

A uniquely fabricated microdevice with a glass-polytetrafluoroethylene (PTFE)-glass (GPG) sandwich configuration was developed for the fast identification of foodborne pathogens by using continuous-flow polymerase chain reaction (PCR) followed by on-site fluorescence detection of PCR products. A PTFE tube was simply folded into a serpentine configuration and sandwiched between two glass slides to maintain a 2D planar structure. By using a PTFE tube, the microchannel has the advantage of seamlessness without the need for photolithography methods, and is less expensive and less time-consuming to fabricate than alternative devices. The non-porous and gas-impermeable PTFE tube ensured stable sample flow inside the microchannel. PCR conditions were optimized and evaluated by examining the effects of number of cycles and initial concentration of bacteria introduced. On-site detection of PCR products was performed using nucleic acid dye SYBR Green I. Using the GPG microdevice, Escherichia coli O157:H7 and Salmonella spp., two well-known foodborne pathogens, were amplified within 20min. Besides fast reaction, the simply fabricated microdevice demonstrated its potential as a portable device applicable for public healthcare diagnostics.

Smartphone-Enabled Detection Strategies for Portable PCR-Based Diagnostics.

Incredible progress continues to be made toward development of low-cost nucleic acid-based diagnostic solutions suitable for deployment in resource-limited settings. Detection components play a vitally important role in these systems, but have proven challenging to adapt for operation in a portable format. Here we describe efforts aimed at leveraging the capabilities of consumer-class smartphones as a convenient platform to enable detection of nucleic acid products associated with DNA amplification via the polymerase chain reaction (PCR). First, we show how fluorescence-based detection can be incorporated into a portable convective thermocycling system controlled by a smartphone app. Raw images captured by the phone's camera are processed to yield real-time amplification data comparable to benchtop instruments. Next, we leverage smartphone imaging to achieve label-free detection of PCR products by monitoring changes in electrochemical reactivity of embedded metal electrodes as the target DNA concentration increases during replication. These advancements make it possible to construct rugged inexpensive nucleic acid detection components that can be readily embedded in a variety of portable bioanalysis instruments.

A simple modification of PCR thermal profile applied to evade persisting contamination.

The polymerase chain reaction (PCR), one of the most commonly applied methods of diagnostics and molecular biology has a frustrating downside known as the false positive signal or contamination. Several solutions to avoid and to eliminate PCR contaminations have been worked out to date but the implementation of these solutions to laboratory practice may be laborious and time consuming. A simple approach to circumvent the problem of persisting PCR contamination is reported. The principle of this approach lies in shortening the steps of denaturation, annealing, and elongation in the PCR thermal cycle. The modification leads to the radical decline of false positive signals obtained for the no-template controls without affecting the detection of target PCR products. In the model experiments presented here, the signal of negative control was shifted by about ten cycles up above those for the examined samples so that it could be neglected. We do not recommend this solution in PCR diagnostics, where the sensitivity of detection is of the highest priority. However, the approach could be useful to pass by the problem of persisting contamination in quantitative PCR, where the range of quantitation is usually much above the limits of detection.Electronic supplementary materialThe online version of this article (doi:10.1007/s13353-015-0336-z) contains supplementary material, which is available to authorized users.

Real-time PCR applied to study on plant pathogens: potential applications in diagnosis - a review

Mirmajlessi S.M., Loit E., Mand M., Mansouripour S.M. (2015): Real-time PCR applied to study on plant pathogens: potential applications in diagnosis – a review. Plant Protect Sci., 51: 177–190. Quantitative real-time PCR (qPCR) technique incorporates traditional polymerase chain reaction (PCR) efficiency with the production of a specific fluorescent signal, measuring the kinetics of the reaction in the early PCR phases and providing quantification of specific targets in various environmental samples. There are an increasing number of chemistries to detect PCR products, which are widely used in plant pathology as they cluster into the amplicon sequence non-specific and sequence-specific techniques. In this review, we illustrate a general description of major chemistries and discuss some considerations for assay development as it applies for a wide range of applications in epidemiological studies. The technique has become the gold standard for early detection of pathogens and a funda mental tool in the research laboratory.

Biofunctionalization of Polyoxometalates with DNA Primers, Their Use in the Polymerase Chain Reaction (PCR) and Electrochemical Detection of PCR Products.

The bioconjugation of polyoxometalates (POMs), which are inorganic metal oxido clusters, to DNA strands to obtain functional labeled DNA primers and their potential use in electrochemical detection have been investigated. Activated monooxoacylated polyoxotungstates [SiW11 O39 {Sn(CH2 )2 CO}](8-) and [P2 W17 O61 {Sn(CH2 )2 CO}](6-) have been used to link to a 5'-NH2 terminated 21-mer DNA forward primer through amide coupling. The functionalized primer was characterized by using a battery of techniques, including electrophoresis, mass spectrometry, as well as IR and Raman spectroscopy. The functionality of the POM-labeled primers was demonstrated through hybridization with a surface-immobilized probe. Finally, the labeled primers were successfully used in the polymerase chain reaction (PCR) and the PCR products were characterized by using electrophoresis.

Optimization of Annealing Temperature for Amplification of 507 bp fragment of rpoB Gene of Clinical Multidrug-Resistant Mycobacterium tuberculosis Isolate 86

The gene of rpoB is the primary gene that is well known as a surrogate marker in MDR-TB (Multidrug-Resistant Tuberculosis) detection. The mutation of rpoB was studied around the world in its core region called RRDR (Rifampicin Resistance Determining Region). To learn the mutation in this fragment, PCR (Polymerase Chain Reaction) is the most common method used. This study was purposed to optimize the annealing temperature in amplifying the 507 bp fragment of rpob gene containing RRDR of isolate 86.DNA of MDR-TB isolate 86 was isolated by using Boom method for further amplified by PCR. The oligonucleotide primers used in this study were FrTB and RrTB. Eight different annealing temperature were used to optimize the amplification of rpoB gene : 53, 55, 57, 58, 59, 60, 61, and 63oC. Detection of PCR products was done with 1,5% agarose gel electrophoresis.Agarose gel electrophoresis of PCR products showed that 507 bp of rpoB gene could be produced at all annealing temperatures. A faint amplified product was observed at temperature 53 and 55oC. However, at temperature 58 and 60oC more intense bands were observed. In conclusion, the best annealing temperature was at 60oC in producing the 507 bp fragment.

Rapid authentication of Cordyceps by lateral flow dipstick

Cordyceps (Dongchongxiacao), a valuable traditional Chinese medicine, is composed of the fruiting body of Ophiocordyceps sinensis (Family: Ophiocordycipitaceae) on a caterpillar of ghost-moth species (Family: Hepialidae). Owing to its multiple potential functions, Cordyceps are in great demand and represent significant economic value. Adulterants or substitutes named Cordyceps or Chongcao from related fungi have been reported. In this study, polymerase chain reaction (PCR) coupled with a lateral flow dipstick (LFD) system was developed to distinguish genuine herb O. sinensis from its common adulterant Cordyceps gunnii and Cordyceps militaris. Specific primers (EF-CS-F1-Biotin, EF-CG-F1-Biotin and EF-CM-F1- Biotin) were designed to differentiate the three Cordyceps species. Internal control (EF-F1-b-DIG and EF-R1-FITC) was included to minimize the false signal due to PCR inhibitors or DNA degradation. LFD was then successfully employed for speedy and accurate detection of the respective PCR products.

English

Microsatellites of the X-chromosome have been increasingly studied in recent years as a useful tool in forensic analysis. This review describes some details of X-chromosomal short tandem repeat (STR) analysis. Among them are: microsatellites, amplification using polymerase chain reaction (PCR) of STRs, PCR product evaluation, PCR product purification, separation and detection, data analysis of STR by Identity Software, locus information and allele frequencies for X-chromosomal STR in different populations (DXS101, DXS7423, DXS8377, DXS6789, DXS6807 genetic loci) and advantages of X-chromosomal micro-satellites. In forensic casework and DNA databases, polymorphism in STR is important as a forensic genetic marker. Key words: Short tandem repeat (STR) loci, forensic, interpretation.

Tag/hybridization-based sensitive detection of polymerase chain reaction products

The polymerase chain reaction (PCR) is an important technology to amplify a single copy or a few copies of DNA segment in genomic DNAs, visualizing the segment as DNA fragment. Thus, PCR is frequently used in various examinations such as detection of bacteria and fungi in the food industry. Here, we report a simple and sensitive method for detection of PCR products using single-strand tag sequence and hybridization of the tag sequence to the complementary tag sequence immobilized on solid material (STH). The detection sensitivity was found to be at least 50 times higher than electrophoresis/ethidium bromide (EtBr) visualization for approximately a 500-bp fragment and higher than the ordinary hybridization, that is, hybridization of denatured PCR product to probe sequence immobilized on solid material.

Colorimetric detection of PCR products of DNA from pathogenic bacterial targets based on a simultaneously amplified DNAzyme

A novel strategy was devised for colorimetric analysis of the products of the polymerase chain reaction (PCR). The method takes advantage of simultaneous amplification of a horseradish peroxidase-mimicking DNAzyme (HRPzyme) during the PCR process. It is performed using a DNA specific forward primer and a universal reverse primer containing a complementary HRPzyme sequence. The double-strand PCR products, which include the HRPzyme sequence, are treated with a mixture of hemin and TMB (3,3′,5,5′–tetramethylbenzidine) in the presence of hydrogen peroxide. The resulting HRPzyme/hemin complex then promotes a peroxidase mimicking reaction, which produces the blue colored oxidized TMB. This colorimetric method can be more easily performed than previously developed gel based detection procedures and, as a result, can be conveniently applied to the specific and sensitive colorimetric analysis of DNA sequences arising from pathogenic bacteria. The potentially broad applicability of the new method has been demonstrated by its use in the identification of the 16s rDNA of Salmonella Typhimurium.

Prevalence of Heptavalent Vaccine-related Pneumococcal Serotypes in Nasopharyngeal carrier in children under five years old in Shahrekord, Iran by Multiplex-PCR during 2010- 2011.

Heptavalent pneumococcal vaccine which included pneumococcal serotypes 4, 6B, 9V,14, 18C, 19F and 23F has been regularly used and is effective on preventing invasive pneumococcal infection. This study aimed to determine vaccine-related serotype distribution in nasopharyngeal carrier and healthy children under five years old. In this cross-sectional study from September 2010 to September 2011, 363 nasopharyngeal specimens were collected from healthy children in day care centers. In positive cultures of streptococcus pneumoniae (S. pneumonia) distribution, serotypes were detected by Multiplex polymerase chain reaction (PCR). Electrophoresis of PCR products was used for detection of serotypes of S. pneumoniae. The carrier rate of S. pneumoniae was 29.5% with 95% confidence interval as 24.8- 34.5%. Electrophoresis of PCR products for detection of serotypes of S. pneumonia revealed 430, 220, 753, 189, 573, 304, and 384 bp (s) for 4, 6B, 9V, 14, 18C, 19F, and 23F serotypes of S. pneumoniae, respectively. The frequency of 23F, 6B, 19F, and 18C serotypes were 43%, 34%, 18%, and 5% respectively, but other serotypes (4, 9V and 14) were not detected. Based on the 30% carrier rate and high prevalence of most of heptavalent pneumococcal conjugate vaccine serotypes in our study, this vaccine should be used for prevention of invasive infection in Iranian children.

Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase

An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268–luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF–luciferase fusion protein. By means of the automatic analyzer with ZF–luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0×10 to 1.0×106 copies.

NIPBL, a Cohesion Loading Factor, Is Somatically Mutated in Gastric and Colorectal Cancers with High Microsatellite Instability

Timely segregation of sister chromatids during the cell cycle is essential for maintaining euploidy [1]. Aberrant segregation into daughter cells promotes aneuploidy and chromosome instability, which are features of many tumors [1]. Chromatid cohesion, a physical linkage of replicated sister chromatids, is mediated by a multiprotein complex called cohesin [1]. NIPBL is a cohesion loading factor that is required for cohesin to interact with chromosomes [2, 3]. NIPBL mutations (missense, nonsense, frameshift and deletion mutations) account for approximately 60 % of Cornelia de Lange syndrome, a congenital disorder with multisystem developmental abnormalities [3]. In Cornelia de Lange syndrome, mutated NIPBL interferes with its interaction with its partners in the cohesion complex [3]. Components in the cohesin complex and cohesin regulatory proteins are frequently altered at expression and mutation levels in human cancers [1]. For example, cohesin protector SGOL1 down-regulation leads to chromosomal instability in colorectal cancers (CRC) [4]. Somatic mutations of cohesion regulatory factor PDS5B have been identified in prostate cancers [5]. These data suggest that cohesin-related genes could be considered potential tumor suppressor genes (TSG) [1]. Somatic mutations of the NIPBL gene have been found in CRC with stable miscrosatellite instability (MSI) that may underlie chromosomal instability in CRC [6]. Together, these data suggest a possibility that NIPBL is a candidate TSG. In a public genome database (http://genome.cse.ucsc. edu/), we found that the human NIPBL gene had mononucleotide repeats in its coding sequences that could be targets for frameshift mutation in cancers with high MSI (MSI-H) [7]. To data, however, the data on NIPBL somatic mutation in gastric (GC) and CRC with MSI-H are lacking. To see whether the mononucleotide repeats in the NIPBL gene are mutated in GC and CRC, we analyzed two A7 and one A8 repeats in NIPBL exon 10 by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay with three primer pairs. For this, we used 32 GC with MSI-H, 59 GC with MSS, 47 CRC with MSI-H and 53 CRC with MSS in methacarnfixed tissues. All of the patients of the cancers were Koreans. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosinstained slides by microdissection [8]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP. On the SSCP, we observed aberrant bands of the NIPBL gene in 11 cancers (Fig. 1). DNA from normal tissue Min Sung Kim and Chang Hyeok An contributed equally to this study.

Recommendations for the processing and storage of water samples before polymerase chain reaction (PCR) analysis

A range of options for the storage and partial processing of water samples before polymerase chain reaction (PCR) analysis were evaluated. In addition, temporal storage of extracted DNA at 4 °C was investigated. Filtering the water sample and then storing that filter at −20 °C for up to six months before DNA extraction was equivalent to extracting DNA immediately. Freezing a water sample resulted in an immediate 1 log loss of PCR signal, although thereafter the sample was stable for at least three months. A rapid loss of detectable PCR product was found when storing a water sample at 4 °C for longer than one week. Extracted DNA could be stored at 4 °C for at least six months without considerable loss of PCR signal.