In the era of the Human Genome Project, quantitation of gene expression by tumor/host cells is of paramount importance to investigate gene patterns responsible for cancer development, progression and response/resistance to treatment. Quantitative real-time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that support its use as a routine bioinstrumentation for gene level measurement. Several applications have been already implemented in the field of cancer research, and others are being validated, showing that this molecular biology tool can provide both researchers and clinicians with precious information concerning the behaviour of tumors. The knowledge of the biochemical principles underlying this biotechnology can be of great value to correctly interpret qrt-PCR data.
The study aimed at development of a multiplex PCR system for amplification of three Y-chromosome STR loci: DYS390, DYS392 and DYS393, and its application in haplotype polymorphism analysis in the population of northern Poland. Due to interactions between originally published primers, a new DYS392 primer pair was proposed. In a population of 158 unrelated males, 28 different haplotypes could be observed, 12 of which were seen only once. The haplotype diversity is 0.805. Distribution of haplotypes of the studied loci is specific to the population of northern Poland and distinguishes it from compared West-European populations. To our knowledge, this is the first report on a Y-STR multiplex system that can be analysed on native polyacrylamide gels.
This study compares a number of parameters that are important in the ligation of the polymerase chain reaction-amplified DNA inserts into plasmid vectors and their efficient transformation to bacterial cells. The parameters covered were: T4 polynucleotide kinase treatment followed by either the large fragment of E. coli DNA polymerase or T4 DNA polymerase reactions, the amount of T4 DNA ligase, temperature and duration of ligation, molar ratio of insert to vector as well as the total DNA concentration. The results show that the T4 polynucleotide kinase-treated group without further enzymatic manipulation, at an insert to vector ratio of 3:1 gave the highest recombination efficiency when 10 μg/ml DNA and 20 units T4 DNA ligase were applied for ligation for 12 h at 4°C.
Flowering is a crucial turning point in the life cycle of most plants. The process of flowering is controlled by external factors such as light and temperature. Floral induction is the first step in the transition from the vegetative to reproductive stage of development. In the photoperiodically sensitive plants this process is regulated by the duration of light and darkness during a 24-h cycle. The aim of our study was to determine whether the undifferentiated callus tissue obtained from cotyledons, is suitable for molecular investigations on the mechanisms of flower induction. The callus tissue was obtained from cotyledons of Pharbitis nil plants, which were cultivated in inductive or non-inductive conditions. The callus obtained after two subcultures was used for isolation of RNA. The total RNA was extracted as described by Chomczynski (1993). We have examined the changes in the pattern of RNA in these two types of callus, using the technique of differential display by the polymerase chain reaction (PCR). Differential display is a method for the identification and cloning of differentially expressed eucaryotic genes. We have found the differences between patterns of RNA derived from callus tissue cultivated under non-inductive conditions and callus tissue cultivated under inductive conditions. In conclusion we can suggest that the tested callus preserved the information on the photoperiodic induction in cells. The process of undifferentiation did not result in the loss of the properties acquired by cotyledon tissue during the photoperiodic treatment.
Objective. A tropism to epithelial cells and lymphocytes, an inhibition of apoptosis in host cells, an ability to occurrence in persistent form resistant to antibiotic treatment are the features of Chlamydia pneumoniae, which can have connection with chronic inflammation of an adenoid tissue and adenoid hypertrophy. This study aimed to (1) detect the C. pneumoniae in an adenoid in children undergoing adenoidectomy, (2) estimate a connection between C. pneumoniae occurrence and the size of adenoid, (3) demonstration in which of adenoid cells C. pneumoniae occurs most often. Material and methods. The examined group consisted of 200 children aged from 2 to 16 years (mean age 6,4) undergoing adenoidectomy. In all children during qualification for adenoidectomy a fiberoscopic examination of the nasopharynx was performed. A part of removed adenoid tissue was analysed by real-time PCR for C. pneumoniae. Adenoids from children with positive PCR examination and from 10 children with negative PCR examination were examined using immunohistochemistry (IHC). Results. C. pneumoniae in the adenoid was present in 5,5% children. Positive results were obtained most frequently (24,14%, 7/29) in the eldest group (10-16 years). A statistical analysis demonstrated the correlation between C. pneumoniae occurrence in an adenoid tissue and the size of adenoid. In immunohistochemistry C. pneumoniae was found the most frequently in lymphocytes and in epithelial cells. Conclusions. A presence of C. pneumoniae in lymphocytes and epithelial cells of the adenoid first of all in older children with adenoid hypertrophy confirms the participation of this bacteria in adenoid pathology.
STRESZCZENIE: Wstęp. Powinowactwo do komórek nabłonka i limfocytów, zdolność do hamowania apoptozy zakażonych komórek gospodarza oraz występowanie w formie przetrwałej niewrażliwej na antybiotyki – to cechy Chlamydia pneumoniae, które mogą być związane z przewlekłym stanem zapalnym migdałka gardłowego i jego przerostem. Celem pracy była odpowiedź na pytania: 1) Czy w migdałku gardłowym u dzieci zakwalifikowanych do adenoidektomii występuje C. pneumoniae? 2) Czy występuje zależność między obecnością C. pneumoniae w migdałku gardłowym a wielkością migdałka gardłowego? 3) W których komórkach migdałka gardłowego najczęściej występuje C. pneumoniae? Materiał i metody. Grupę badaną stanowiło 200 dzieci w wieku od 2 do 16 lat (średni wiek – 6,4) wybranych spośród pacjentów zakwalifikowanych do adenoidektomii. U wszystkich podczas kwalifikacji do zabiegu wykonywano badanie fiberoskopowe nosogardła. Tkankę usuniętego migdałka gardłowego badano metodą real-time PCR w kierunku C. pneumoniae. Migdałki pobrane od dzieci z dodatnim wynikiem badania metodą PCR oraz od 10 losowo wybranych dzieci z ujemnym wynikiem tego badania, oceniano stosując badanie immunohistochemiczne (IHC). Wyniki. DNA C. pneumoniae w usuniętym migdałku gardłowym stwierdzono u 5,5% dzieci. Dodatnie wyniki uzyskiwano najczęściej w grupie wiekowej od 10. do 16. roku życia (24,1%, 7/29). Wykazano zależność między występowaniem C. pneumoniae w migdałku gardłowym a wielkością migdałka gardłowego. U wszystkich dzieci z dodatnim wynikiem badania metodą PCR potwierdzono obecność C. pneumoniae w migdałku gardłowym przy zastosowaniu IHC. Najczęściej wykrywano C. pneumoniae w limfocytach oraz w komórkach nabłonka. Wnioski. Obecność C. pneumoniae w limfocytach i komórkach nabłonka migdałka gardłowego głównie u dzieci starszych z przerostem migdałka gardłowego potwierdza udział tej bakterii w procesach patologicznych tkanki migdałka gardłoweg
STRESZCZENIE: Wstęp. Powinowactwo do komórek nabłonka i limfocytów, zdolność do hamowania apoptozy zakażonych komórek gospodarza oraz występowanie w formie przetrwałej niewrażliwej na antybiotyki – to cechy Chlamydia pneumoniae, które mogą być związane z przewlekłym stanem zapalnym migdałka gardłowego i jego przerostem. Celem pracy była odpowiedź na pytania: 1) Czy w migdałku gardłowym u dzieci zakwalifikowanych do adenoidektomii występuje C. pneumoniae? 2) Czy występuje zależność między obecnością C. pneumoniae w migdałku gardłowym a wielkością migdałka gardłowego? 3) W których komórkach migdałka gardłowego najczęściej występuje C. pneumoniae? Materiał i metody. Grupę badaną stanowiło 200 dzieci w wieku od 2 do 16 lat (średni wiek – 6,4) wybranych spośród pacjentów zakwalifikowanych do adenoidektomii. U wszystkich podczas kwalifikacji do zabiegu wykonywano badanie fiberoskopowe nosogardła. Tkankę usuniętego migdałka gardłowego badano metodą real-time PCR w kierunku C. pneumoniae. Migdałki pobrane od dzieci z dodatnim wynikiem badania metodą PCR oraz od 10 losowo wybranych dzieci z ujemnym wynikiem tego badania, oceniano stosując badanie immunohistochemiczne (IHC). Wyniki. DNA C. pneumoniae w usuniętym migdałku gardłowym stwierdzono u 5,5% dzieci. Dodatnie wyniki uzyskiwano najczęściej w grupie wiekowej od 10. do 16. roku życia (24,1%, 7/29). Wykazano zależność między występowaniem C. pneumoniae w migdałku gardłowym a wielkością migdałka gardłowego. U wszystkich dzieci z dodatnim wynikiem badania metodą PCR potwierdzono obecność C. pneumoniae w migdałku gardłowym przy zastosowaniu IHC. Najczęściej wykrywano C. pneumoniae w limfocytach oraz w komórkach nabłonka. Wnioski. Obecność C. pneumoniae w limfocytach i komórkach nabłonka migdałka gardłowego głównie u dzieci starszych z przerostem migdałka gardłowego potwierdza udział tej bakterii w procesach patologicznych tkanki migdałka gardłoweg
Wheat bread-making quality is closely correlated with composition and quantity of gluten proteins, in particular with high-molecular weight (HMW) glutenin subunits encoded by the Glu-1 genes. A multiplex polymerase chain reaction (PCR) method was developed to identify the allele composition of HMW glutenin complex Glu-1 loci (Glu-A1, Glu-B1 and Glu-D1) in common wheat genotypes. The study of multiplex PCR to obtain a well-balanced set of amplicons involved examination of various combinations of selected primer sets and/or thermal cycling conditions. One to three simultaneously amplified DNA fragments of HMW glutenin Glu-1 genes were separated by agarose slab-gel electrophoresis and differences between Ax1, Ax2* and Axnull genes of Glu-A1 loci, Bx6, Bx7 and Bx17 of Glu-B1, and Dx2, Dx5 and Dy10 genes of Glu-D1 loci were revealed. A complete agreement was found in identification of HMW glutenin subunits by both multiplex PCR analysis and SDS-PAGE for seventy-six Polish cultivars/strains of both spring and winter common wheat. Rapid identification of molecular markers of Glu-1 alleles by multiplex PCR can be an efficient alternative to the standard separation procedure for early selection of useful wheat genotypes with good bread-making quality.
We have analysed allele distribution at the highly polymorphic variable number of tandem repeats (VNTR) locus D1S80 (pMCT118) in the Polish population using the polymerase chain reaction (PCR) technique. Characteristics of the D1S80 locus makes it a very useful marker for population genetic research, genetic linkage studies and forensic identification of individuals. During our routine application of the D1S80 marker to paternity testing in several cases of homozygosity detected by polyacrylamide gel electrophoresis, heteroduplex formation for alleles 18 and 24 was also observed. Direct sequencing of PCR products revealed that alleles 18 and 24 of locus D1S80 actually represent a mixture composed of different sequences. Our observations indicate that identification of some 18 and 24 VNTR alleles based only on size estimated in electrophoretic analyses could lead to errors in paternity testing and DNA profiling.
The article contains information regarding the similarities and differences between proteins of the thiol-activated cytolysins family (TACY). Members of this group of haemolysins are produced by several species of Gram-positive bacteria (Listeria spp., Streptococcus spp., Bacillus spp., Clostridium spp.,) and also by Gram-negative Klebsiella spp.. Their cytolytic activity is sensitive to oxygen and can be restored by adding reducing compounds. TACY bind to cholesterol-containing membranes to form pores. Preincubation of the toxin with small amounts of cholesterol inhibits hemolytic activity as well as cytolysis of eucaryotic cells. Members of the group show 40-70% similarity in amino acid sequence, and contain an almost invariant Trp-rich undecapeptide sequence (ECTGLAWEWWR). The TACY are important virulence factors. Recently increased use has been made of molecular methods (PCR, hybridization) in the detection and identification of the TACY producing bacteria.
Current information on barley resistance genes available from scientific papers and on-line databases is summarised. The recent literature contains information on 107 major resistance genes (R genes) against fungal pathogens (excluding powdery mildew), pathogenic viruses and aphids identified in Hordeum vulgare accessions. The highest number of resistance genes was identified against Puccinia hordei, Rhynchosporium secalis, and the viruses BaYMV and BaMMV, with 17, 14 and 13 genes respectively. There is still a lot of confusion regarding symbols for R genes against powdery mildew. Among the 23 loci described to date, two regions Mla and Mlo comprise approximately 31 and 25 alleles. Over 50 R genes have already been localised and over 30 mapped on 7 barley chromosomes. Four barley R genes have been cloned recently: Mlo, Rpg1, Mla1 and Mla6, and their structures (sequences) are available. The paper presents a catalogue of barley resistance gene symbols, their chromosomal location and the list of available DNA markers useful in characterising cultivars and breeding accessions.
Numerous hematological diseases, in particular leukemias, can be treated successfully with allogeneic bone marrow transplantation (allo-BMT). Highly polymorphic microsatellite markers and X, Y-chromosome-specific sequences provide useful genetic markers for detection of complete or mixed chimerism in patients after allo-BMT. Chimerism can be monitored successfully using polymerase chain reaction technique (PCR) and cytogenetic analysis, especially fluorescent in situ hybridization (FISH). It is still unclear whether individuals with mixed chimerism after bone marrow transplantation have an increased risk of developing leukemic relapse or graft rejection. Molecular study of cellular chimerism can also be used for quantitative assessment of the amount of donor's cells in a recipient after bone marrow transplantation and for monitoring of minimal residual disease (MRD) or disease relapse. We report application of three different DNA-typing techniques: automated DNA sizing technology, fluorescent in situ hybridization and also Y-specific DNA probing for analysis of post-BMT chimerism in a case of sex-mismatched bone marrow transplantation. Key words: allo-BMT, chimerism, FISH, PCR.
In search for new markers of insulin-dependent diabetics (IDDM) susceptibility we studied the CATT tetranucleotide repeat in intron 1 of tyrosine hydroxylase (TH) gene on chromosome 11p, the CA repeat at T-cell receptor alpha chain (TCRA) locus on chromosome 14q region containing glutamic acid decarboxylase (GAD2) gene.Alleles at these microsatellite loci were indentified in a population of diabetic children and unrelated healthy controlos originationg from Wielkopolska, a midwestern region of Poland.We found significant association of certain alleles at TH, TCRA and D10S211 loci with diabetes in the population under study.On the contrary, none of the alleles at D10S213 locus was associated with the disease.Our findings indicate that typing of microsatellite markers may represent useful additional tool for identifying individuals at high risk of developing IDDM.Regarding loci on chromosome 10 our data and data publishing by other autors may suggest the existence of two separate regions of associatin with IDDM susceptibility on this chromosome.
In this paper we present an attempt to identify genetic modification of plants via PCR. Plants and plant products commercially available in Poland were tested. The results show that some of them are genetically engineered, however they are not marked as GMO.
The purpose of this study was to compare hybrid capture assay with PCRs using different primers for the L1, E6-E7 regions for the detection of human papillomavirus (HPV) genome. One hundred twenty-five cervical smears with normal (n = 42) and abnormal (n = 83) cytology were investigated. Those at high-risk for HPV were studied by hybridization antibody capture assay and PCR with the pU-1M/pU-2R primers. Target DNA from the HPV L1 region was amplified by SPF10 primer set and home-PCR with MY09/MY11 primers. The presence of HPV DNA in cervical smears was detected by SPF10 (in 72% of cases), MY09/MY11 (58%), hybrid capture (55%) and pU-1M/pU-2R (39%). Results obtained with the SPF10 and MY09/MY11 consensus primer sets as well as hybrid capture and pU-1M/pU-2R specific for high-risk types differed significantly (χ2, P < 0.0005). The correlation between assays with the use of SPF10 and MY09/MY11 was 86% and between hybrid capture and the pU-1M/pU2R technique - 78%. In 49% of samples HPV DNA was detected by the four methods, whereas in 12% only by the SPF10 primers. The most sensitive technique was found to be PCR with the use of SPF10 primers, while the most specific - the MY09/11 PCR method. It seems that home-PCR with MY09/MY11 primers could be applied in screening tests.
Conventional methods to identify fungi have often relied on identification of disease symptoms, isolation and culturing of environmental organisms, and laboratory identification by morphology and biochemical tests. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. In this review, some of the molecular approaches to fungal diagnostics based on polymerase chain reaction (PCR) and DNA/RNA probe technology are discussed. This includes several technological advances in PCR-based methods for the detection, identification and quantification of fungi including real-time PCR which has been successfully used to provide rapid, quantitative data on fungal species from environmental samples. PCR and probe based methods have provided new tools for the enumeration of fungal species, but it is still necessary to combine the new technology with more conventional methods to gain a fuller understanding of interactions occurring in the environment. Since its introduction in the mid 1980?s PCR has provided many molecular diagnostic tools, some of which are discussed within this review, and with the advances in micro-array technology and real-time PCR methods the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual fungal species but also on whole communities.
Real-time PCR has become one of the most widely used methods of gene quantitation in molecular biology and medical diagnostics. This technique combines PCR amplification and the detection of the PCR product into a single step. In real-time PCR, the amount of product formed is measured during the course of the experiment by monitoring the fluorescence of dyes or probes introduced into the reaction. Fluorescence data are generated by the use of intercalating dyes such as SYBR Green I or molecular probes, the most important of which are: TaqMan, Molecular Beacons, Hybridization Probes, and Scorpion Probes. The real-time PCR reactions are characterized by the PCR cycle in which the target amplification is first detected. This cycle is referred to as a treshold cycle (Ct), at which fluorescence intensity becomes greater than background fluorescence. Consequently, the greater the quantity of target DNA in the starting material, the faster the significant increase in fluorescence intensity will appear, yielding lower Ct. The relation between Ct and the concentration of the target sequence allows for a precise quantitation of the genetic material in the sample.
The polymerase chain reaction (PCR) method has been applied to the detection of FMD viral RNA in samples taken during the clinical stage of FMD from calf. Total RNA was extracted with guanidinum thiocyanate-phenol-chloroform method and reversety transcribed using AMV-reverse transcriptase. cDNA was used as a template for the amplification by PCR of the 672 bp of the VP1 coding sequence.The amplified fragment of cDNA was cloned in the phagemid pBS(+). The first DNA strand was sequenced and consistently an amino acid sequence was established. Comparison between VP1 amino acid sequence of FMDV types C and earlier described type O was performed.
Contamination of cereals with mycotoxins produced by Fusarium is a worldwide problem requiring rapid and sensitive detection methods. This paper describes the development of a PCR protocol facilitating the detection of F. tricinctum, which belongs to the FHB (Fusarium Head Blight) complex responsible for contamination of cereal grains with enniatins and moniliformin. Sequence alignment of partial IGS rDNA revealed a single nucleotide polymorphism, which was used to design primers differentiating F. tricinctum from other members of the FHB complex. The specificity of the assay was tested on 68 isolates belonging to 21 Fusarium species originating from different parts of the world and hosts/substrates. Positive PCR results were obtained from all 12 F. tricinctum isolates tested; however, unexpected amplicons were amplified from the templates of F. acuminatum (CBS 618.87) and F. nurragi (CBS 393.96). No cross reactivity was found with any other Fusarium species tested. The PCR assay was tested on 24 asymptomatic wheat seed samples originating from Northern Poland and resulted in 13 positive samples, of which 11 samples were contaminated with moniliformin and/or antibiotic Y.
In this report we demonstrate a simple, effective and reliable diagnostic test of BLAD carrier detection based on specific PCR amplification of a 367 bp CD18 gene fragment and RFLP analysis using Taq I restriction enzyme. In a non random population of 220 animals we found 48 BLAD carriers. Within the amplified PCR fragment an unknown intron sequence of 159 bp was identified.
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