Size Of DNA Fragments Research Articles

Plasma DNA aberrations in systemic lupus erythematosus revealed by genomic and methylomic sequencing.

We performed a high-resolution analysis of the biological characteristics of plasma DNA in systemic lupus erythematosus (SLE) patients using massively parallel genomic and methylomic sequencing. A number of plasma DNA abnormalities were found. First, aberrations in measured genomic representations (MGRs) were identified in the plasma DNA of SLE patients. The extent of the aberrations in MGRs correlated with anti-double-stranded DNA (anti-dsDNA) antibody level. Second, the plasma DNA of active SLE patients exhibited skewed molecular size-distribution profiles with a significantly increased proportion of short DNA fragments. The extent of plasma DNA shortening in SLE patients correlated with the SLE disease activity index (SLEDAI) and anti-dsDNA antibody level. Third, the plasma DNA of active SLE patients showed decreased methylation densities. The extent of hypomethylation correlated with SLEDAI and anti-dsDNA antibody level. To explore the impact of anti-dsDNA antibody on plasma DNA in SLE, a column-based protein G capture approach was used to fractionate the IgG-bound and non-IgG-bound DNA in plasma. Compared with healthy individuals, SLE patients had higher concentrations of IgG-bound DNA in plasma. More IgG binding occurs at genomic locations showing increased MGRs. Furthermore, the IgG-bound plasma DNA was shorter in size and more hypomethylated than the non-IgG-bound plasma DNA. These observations have enhanced our understanding of the spectrum of plasma DNA aberrations in SLE and may provide new molecular markers for SLE. Our results also suggest that caution should be exercised when interpreting plasma DNA-based noninvasive prenatal testing and cancer testing conducted for SLE patients.

Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay

Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV.

Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing.

Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.

Determining the Quantitative Dynamics of Nucleic Acids in Live Cells through Rics and iMSD Approaches

361-Pos Board B116 Determining the Quantitative Dynamics of Nucleic Acids in Live Cells through Rics and iMSD Approaches Stephen P. Mieruszynski 1 , Michelle A. Digman 2 , Enrico Gratton 2 , Mark Jones 1 . Health and Science, University of Western Sydney, Richmond, Australia, Biomedical Engineering Department, Laboratory for Fluorescence Dynamics, University of California, Irvine, CA, USA. Addressing the mobility and dynamics of DNA within live cells requires the characterization of individual particles in real time. Two such techniques enabling the quantification of the dynamics and means of motion of fluorescently labeled particles include the Raster Image Correlation Spectroscopy (RICS) and the image-based Mean Square Displacement (iMSD) approaches. In this study, the RICS and iMSD approaches were applied in order to elucidate the mobility and means of motion of DNA lipoplexes formed from varying sized DNA (20bp- 5.5kbp). The RICS approach demonstrated that two species of mobility were pre- sent within the cytoplasm. Across the DNA fragment sizes, the slower species was not size dependent and were statistically the same. Whereas, faster moving particles which demonstrated a size dependent mobility. Nuclear localized DNA demonstrated to be significantly dynamic compared to other previously pub- lished work, ranging from 1.22 mm2/s (5.5kbp) to 3.67 mm2/s (21bp), consistent to nuclear proteins and RNA. The iMSD approach applied enabled the discrim- ination between different means of motion including random diffusion (RD), active transport (AT), confined diffusion (CD), anomalous subdiffusion (AS) and transient confinement (TC). Motion through RD and TC had demonstrated a size dependency across the DNA sizes assessed. Whereas, motion through AT, CD and AS did not demonstrate size dependency additionally these other means of motion had a higher mobility rate than that of RD.

Species-specific multiplex real-time PCR assay for identification of deer and common domestic animals

A multiplex real-time PCR method for discriminating deer and common domestic species, including cattle, goat, horse, donkey, pig, and chicken was developed. Species-specific primer pairs were designed and used to produce different size DNA fragments with diverse melting temperature (T m ) values. The specificity and sensitivity of these primer pairs were separately confirmed using simplex real-time PCR analysis. Multiplex real-time PCR analysis was performed using combined primers, yielding distinct melting curve profiles for each species. The sensitivity limit of the multiplex PCR method was evaluated. Trace DNA of other species in deer DNA could be identified. Common deer products, including blood, meat, and antler were tested using this multiplex PCR method and different species of deer and domestic animals were identified. The rapid multiplex real-time PCR assay described herein is a specific, sensitive, and reliable method for high-throughput authentication of deer and domestic animal products.

Occurrence and Genome Analysis of Cucurbit chlorotic yellows virus in Iran

In 2011 and 2012, several cucurbit-growing regions of Iran were surveyed and samples with symptoms similar to those induced by Cucurbit chlorotic yellows virus (CCYV) were collected. The pathogen was transmitted to cucumber and melon under greenhouse conditions by whiteflies (Bemisia tabaci). RT-PCR using designed CCYV-specific primer pair (CCYV-F/CCYV-R) resulted in amplification of the predicted size DNA fragment (870 bp) for the coat protein (CP) gene in samples collected from Boushehr, Eyvanakay and Varamin. Nucleotide sequences of the CP of the three Iranian CCYV isolates were compared with five CCYV isolates obtained from GenBank and analysed. Phylogenetically, all CCYV isolates clustered in two groups; Group I is composed of five non-Iranian isolates from China, Lebanon, Japan, Sudan and Taiwan, and the three Iranian isolates formed Group 2. Among Iranian isolates, the Eyvanakay isolate clustered in a distinct clade with the Boushehr and Varamin isolates. A phylogenetic tree based on amino acid identity of CP showed that CCYV was closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) and Cucurbit yellow stunting disorder virus (CYSDV). This is the first report of CCYV in Iran.

Catfish Species Identification Using Lab-On-Chip PCR-RFLP

Lab-on-a-chip based Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) technology was developed for the identification of seven catfish species including an Ictalurid hybrid. RFLP profiles of mitochondrial cytochrome b fragments digested by three different restriction endonucleases (DdeI, HaeIII, and NlaIII) were constructed from morphologically verified catfish samples on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). High sensitivity and ease of handling through end-point microchip-based capillary electrophoresis increased resolution and accuracy of DNA fragment sizing. RFLP profiles derived from a combination of all three enzymes produced consistent species-specific identification profiles. Observed restriction fragment patterns for Clarias batrachus and Ictalurus punctatus were similar, but both of these species could be consistently differentiated using a single band of the HaeIII restriction site. Assay advantages due to shorter assay times, assay ease, and minimum usage of harmful solvents and chemicals when compared to traditional DNA barcoding were counterbalanced by the need to develop and optimize specific restriction digest profiles for all potential species of interest. With further development, this method may be utilized in testing of catfish products to ensure the enforcement of seafood labeling regulations.

Assessment of five soil DNA extraction methods and a rapid laboratory-developed method for quality soil DNA extraction for 16S rDNA-based amplification and library construction

Extraction of DNA from soil samples using standard methods often results in low yield and poor quality making them unsuitable for community analysis through polymerase chain reaction (PCR) due to the formation of chimeric products with smaller template DNAs and the presence of humic substances. The present study focused on the assessment of five different methods for metagenomic DNA isolation from soil samples on the basis of processing time, purity, DNA yield, suitability for PCR, restriction digestion and mDNA library construction. A simple and rapid alkali lysis based on indirect DNA extraction from soil was developed which could remove 90% of humic substances without shearing the DNA and permits the rapid and efficient isolation of high quality DNA without the requirement of hexadecyltrimethylammonium bromide and phenol cleanup. The size of DNA fragment in the crude extracts was >23kb and yield 0.5–5μg/g of soil. mDNA purification using Sephadex G-50 resin yielded high concentration of DNA from soil samples, which has been successfully used for 16S rDNA based amplification of a 1500bp DNA fragment with 27F and 1492R universal primers followed by restriction digestion and mDNA library construction.

Integron mediated multidrug resistance in extended spectrum beta-lactamase producing clinical isolates of Klebsiella pneumoniae

The present study describes integron mediated multiple antibiotic resistance in extended-spectrum β-lactamase producing clinical isolates of Klebsiella pneumoniae. One hundred and four clinical isolates of K. pneumoniae from two Iranian hospitals were screened for extended-spectrum β-lactamase production and susceptibility of the extended-spectrum β-lactamase producing isolates was determined to 17 antibiotics by disc diffusion. Presence of integron classes 1, 2 and 3 was detected by PCR and integrase specific primers. Isolates harboring class 1 integron were then screened for variable regions using PCR. Fifty isolates (48%) produced extended-spectrum β-lactamases among which, 22 (44%) harbored class 1, 3 (6%) carried class 2 and none contained class 3 integons. Integron carriage was significantly associated with higher rates of multiple antibiotic resistance in extended-spectrum β-lactamase producing clinical isolates of K. pneumoniae. Integron harboring isolates were more resistant to aztreonam (51.3%), ceftazidime (42.6%), cefotaxime (43.3%), cefepime (24.6%), kanamycin (43.2%), tobramycin (30.7%), norfloxcacin (32%) and spectinomycin (25.6%) compared to the organisms without integrons. On the other hand, resistance to nitrofurantoin and streptomycin was significantly higher among the integron negative isolates. PCR amplification of class1 integron variable regions revealed 9 different sized DNA fragments and isolates with similar profiles for class 1 integron variable regions showed the same antibiotic resistance phenotypes.

Proof-of-concept study for successful inter-laboratory comparison of MLVA results

Multiple-locus variable-number of tandem repeats analysis (MLVA) is widely used for typing of pathogens. Methods such as MLVA based on determining DNA fragment size by the use of capillary electrophoresis have an inherent problem as a considerable offset between measured and real (sequenced) lengths is commonly observed. This discrepancy arises from variation within the laboratory set-up used for fragment analysis. To obtain comparable results between laboratories using different set-ups, some form of calibration is a necessity. A simple approach is to use a set of calibration strains with known allele sizes and determine what compensation factors need to be applied under the chosen set-up conditions in order to obtain the correct allele sizes. We present here a proof-of-concept study showing that using such a set of calibration strains makes inter-laboratory comparison possible. In this study, 20 international laboratories analysed 15 test strains using a five-locus Salmonella enterica serovar Typhimurium MLVA scheme. When using compensation factors derived from a calibration set of 33 isolates, 99.4% (1,461/1,470) of the MLVA alleles of the test strains were assigned correctly, compared with 64.8% (952/1,470) without any compensation. After final analysis, 97.3% (286/294) of the test strains were assigned correct MLVA profiles. We therefore recommend this concept for obtaining comparable MLVA results.

A new method for the determination of critical polyethylene glycol concentration for selective precipitation of DNA fragments

Separation strategies based on size-selective precipitation of DNA fragments with polyethylene glycol (PEG) have been used for achieving desired DNA interval in automated sample preparation for next-generation sequencing. By varying PEG concentration, DNA fragments of different sizes can be precipitated onto surfaces of carboxyl-coated paramagnetic particles selectively, and therefore, the desired DNA interval can be obtained. However, one of the crucial points in this approach is to determine the critical PEG concentration for DNA fragment of a certain size. The aim of this work was to develop a convenient and reliable method for accurately determining the critical PEG concentration. In our method, at a fixed concentration of sodium chloride (NaCl), recovered DNA samples obtained with different PEG concentrations were directly quantified, and their concentrations as a function of the PEG concentration were fitted by the logistic function. The critical PEG value was easily and accurately determined from the fitted logistic function. The repeatability and stability of the critical PEG value were assessed, showing an excellent reliability of the method. Based on this method, critical PEG values of different-size DNA fragments were determined at different NaCl concentrations. The effectiveness of the method was also demonstrated by selective precipitation of DNA fragments.

Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment

Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F.columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F.columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.

English

Three molecular techniques random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and restriction fragment length polymorphism (RFLP) were employed for identification and to study the genetic relationship among six species of dermatophytes and three species of yeasts isolated from Egyptian and Libyan patients with skin mycosis. Each species was represented by two isolates, one from Egyptian patients and the second from Libyan. RAPD in which four random 10-mer primers and two ISSR primers were used to amplify the DNA fragments of target fungi and RFLP in which two universal primers (ITS1 and ITS4) were used to amplify the internal transcribed spacer (ITS) regions of the ribosomal (rRNA) gene in fungal isolates followed by digestion with HinfI and HaeIII endonucleases was carried out. Three molecular marker techniques showed considerable potential for identifying and discriminating dermatophytes and Candida species and the achieved results confirmed identification based on conventional morphological methods. Results of RAPD and ISSR markers revealed 78.7% genetic similarity (GS) betweenMicrosporum canis and other tested fungi reflecting a relatively longer genetic distance from other isolates of dermatophytes and yeasts. Candida krusei andCandida tropicalis were closely related showing 93.3% GS. C. albicans showed 90.9% similarity with other species of Candida. Epidermophyton floccosum was easily separated from all Trichophyton species showing 87.3% similarity. Unique bands were displayed by certain fungi and can be taken as a positive marker for isolate identification and discrimination. RFLP technique revealed differences in the number (1 to 5) and size (8 to 378 base pairs) of DNA fragments depending on the fungal isolate and restriction enzyme used. Within each fungal species, different isolates of dermatophytes and Candida from Egypt and Libya showed close relationship. Seven isozyme systems namely esterase, peroxidase, malate dehydrogenase, acid phosphatase, glutamate-oxalo-acetate transaminase, Urease and protease were studied to detect the gene expression and genetic variability among the different isolates of dermatophytes and Candida. Key words: Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), restriction fragment length polymorphism (RFLP), dermatophytes,Candida, isozymes.

Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast–embryonic stem cell fusion

Introducing human genes into mice offers the opportunity to analyze their in vivo function or to obtain therapeutic molecules. For proper gene regulation, or in case of multigene families, megabase (Mb)-sized DNA fragments often have to be used. Yeast artificial chromosome (YAC)-mediated transgenesis is irreplaceable for this purpose, because alternative methods such as the use of bacterial artificial chromosomes (BACs) cannot introduce DNA fragments larger than 500 kb into the mouse germ line. However, YAC libraries often contain only partial gene loci. Time-consuming reconstruction of YACs, genetic instability and the difficulty in obtaining intact YAC DNA above a certain size impede the generation of humanized mice. Here we describe how to reconstruct YACs containing Mb-sized human DNA, such as the T cell receptor-α (TRA) gene locus, thus facilitating the introduction of large DNA fragments into the mouse germ line. Fusion of YAC-containing yeast and embryonic stem (ES) cells avoids the need for YAC DNA purification. These ES cells are then used to stably introduce the functional TRA gene locus into the mouse germ line. The protocol takes ∼1 year to complete, from reconstruction of the entire TRA gene locus from YACs containing partial but overlapping TRA regions to germline transmission of the YAC.

The effectiveness of ISSR profiling for studying genetic diversity of Aspergillus flavus from peanut-cropped soils in China

We used markers based on inter-simple sequence repeats (ISSR) to examine the genetic diversity of Aspergillus flavus from peanut-cropped soils in China. Of the 100 primers, 22 primers produced clear and reproducible ISSR bands, and the di-nucleotide accounted for 73% of those primers. The size of DNA fragments ranged from 100 to 2000 bp. The primer UBC 834 produced the largest number of polymorphic bands (10), followed by UBC 809, UBC 817, UBC 895, and UBC 899, which all amplified 7 polymorphic bands. Using the five primers, the tested strains were clearly separated based on genetic similarity coefficients (GSC). The range of GSC was from 0.59 to 0.90. In unweighted pair-group method with arithmetic averages (UPGMA) analysis, the A. flavus samples grouped in five clusters. The study showed that the ISSR technology is an effective molecular approach for studying diversity of A. flavus from peanut-cropped soils in China.

The effect of predator identity on post-feeding prey DNA detection success in soil-dwelling macro-invertebrates

Soils harbour an extraordinary diversity of animals which are functionally connected by their feeding interactions. Determining these feeding links, however, is difficult due to the opaque habitat the animals live in and because many prey remains found in predator gut contents cannot be identified morphologically. DNA-based gut content analysis overcomes these hurdles. This approach opens new possibilities to empirically examine below-ground feeding links by measuring prey DNA detection frequencies in various predators. Comparing prey DNA detection rates between different predator taxa, however, is not straightforward as digestion rates and thus post-feeding prey DNA detection intervals can be affected by predator identity. In the present study we examined the effect of predator identity for a range of soil-dwelling adult and larval beetles as well as lithobiid and geophilid centipedes. These predators were fed with white grubs (scarabaeid beetle larvae) and their post-feeding prey DNA detection intervals were established for small, medium and large prey DNA fragments. In geophilids the 50% detection probability for the medium prey DNA fragment was 98 h post-feeding whereas it ranged between 22 and 34 h in beetles. Among the different beetle species, the magnitude of the differences in detection intervals was strongly affected by the size of the prey DNA fragment targeted: the intervals showed the highest similarity in the medium-sized prey DNA fragment which was most frequently detected. Predator biomass and meal size rarely affected post-feeding prey DNA detection intervals. Our findings suggest that prey DNA detection success is similar within taxonomically related species of soil-dwelling macro-invertebrates such as beetles and centipedes. Between these groups prey detection can be adjusted according to taxon-specific digestion rates for better comparisons. Moreover, the magnitude of the predator identity effect is modulated by assay sensitivity: highly sensitive assays which target small- to medium-sized prey DNA fragments are recommended as these minimize the interspecific differences in post-feeding prey DNA detection intervals.

Discriminatory simplex and multiplex PCR for four species of the genus Sclerotinia

Sclerotinia sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Eriks, and S. homoeocarpa F.T. Benn are the most relevant plant pathogenic species within the genus Sclerotinia because of their large range of economically important hosts, including tomato, peanut, alfalfa, and turfgrass, among others. Species identification based on morphological characteristics is challenging and time demanding, especially when one crop hosts multiple species. The objective of this study was to design specific primers compatible with multiplexing, for rapid, sensitive and accurate detection and discrimination among four Sclerotinia species. Specific primers were designed for the aspartyl protease gene of S. sclerotiorum, the calmodulin gene of S. trifoliorum, the elongation factor-1 alpha gene of S. homoeocarpa, and the laccase 2 gene of S. minor. The specificity and sensitivity of each primer set was tested individually and in multiplex against isolates of each species and validated using genomic DNA from infected plants. Each primer set consistently amplified DNA of its target gene only. DNA fragments of different sizes were amplified: a 264bp PCR product for S. minor, a 218bp product for S. homoeocarpa, a 171bp product for S. sclerotiorum, and a 97bp product for S. trifoliorum. These primer sets can be used individually or in multiplex for identification of Sclerotinia spp. in pure culture or from infected plants. The multiplex assay had a lower sensitivity limit than the simplex assays (0.0001pg/μL DNA of each species). The multiplex assay developed is an accurate and rapid tool to differentiate between the most relevant plant pathogenic Sclerotinia species in a single PCR reaction.

Development of a random genomic DNA microarray for the detection and identification of Listeria monocytogenes in milk

We developed a DNA microarray that contains random genomic DNA fragments of Listeria monocytogenes, validated its diagnostic abilities using cells grown in laboratory media and milk, and established enrichment conditions for detection of a low population of L. monocytogenes in milk. Genomic DNA of L. monocytogenes strain ATCC 19111 was fractionated by agarose gel electrophoresis after being cleaved using several different pairs of restriction enzymes. Sixty DNA fragments of different sizes were randomly selected and spotted onto an amine-coated glass slide. To validate diagnostic ability, probes on the DNA microarray were hybridized with genomic DNA extracted from L. monocytogenes, other Listeria spp., and foodborne pathogenic bacteria belonging to other genera grown in laboratory media. The DNA microarray showed 98–100% positive hybridization signals for the 16 strains of L. monocytogenes tested, 7–85% positive signals for 9 strains of other Listeria spp., and 0–32% positive signals for 13 strains of other types of foodborne pathogens. In milk, the detection limit of the DNA microarray was approximately 8 log CFU/mL. When milk contained L. monocytogenes (3–4 log CFU/mL) with other types of bacteria (Bacillus spp., B. cereus, Salmonella Montevideo, Peudomonas aeruginosa, and Yersinia enterocolitica; ca. 3 log CFU/mL each), L. monocytogenes enriched in UVM modified Listeria enrichment broth at 37°C for 24h was successfully detected by the DNA microarray. Results indicate that the DNA microarray can detect L. monocytogenes and distinguish it from other Listeria spp. and other foodborne pathogens in laboratory media and milk. This platform will be useful when developing a DNA microarray to rapidly and simultaneously detect and identify various foodborne pathogens in foods.

Determination of PCR products by CE with contactless conductivity detection

The use of CE with contactless conductivity detection for the determination of PCR products is demonstrated for the first time. The separation of specific length PCR products according to their size could be achieved using 5% PVP as a sieving medium in a separation buffer consisting of 20 mM Tris and 20 mM 2-(cyclohexylamino)ethansulphonic acid (pH 8.5). A fused silica capillary of 60 cm length and 50 μm id and an applied separation voltage of -15 kV were employed and separations could be completed within 20-50 min. PCR amplified DNA fragments of different sizes obtained from different bacterial plasmid templates as well as a fragment from genomic DNA of genetically modified soybeans could be successfully identified.

RAPD와 ITS 영역에 의한 민자주방망이 버섯의 유전적 변이

A genetic variation of Lepista nuda and two genus Lepista species (L. irina and L. sordida) were analyzed by random amplified polymorphic DNA (RAPD) and internal transcribed spacer (ITS) sequence analysis. In the resulting RAPD analysis, 22 out of 40 random primers amplified polymorphic RAPD fragment patterns, the amplified bands were 355, and DNA fragment sizes were 200-400bp. Intraspecific genetic dissimilarity of the 10 L. nuda strains were calculated to range from 0% to 21.60%, L. sordida from 16.93% to 24.82%, L. irina were 20.62% to 25.54%, and intraspecific genetic dissimilarity of L. sordida and L. irina was 23.49%. The 673 base pairs were sequenced during the analysis of the ITS I and II region; six L. nuda strains intraspecific genetic dissimilarities ranged from 1.58% to 11.47%, L. nuda and L. sordida from 3.83% to 12.88%, L. nuda and L. irina from 7.11% to 15.61%, and intraspecific genetic variation between L. sordida and L. irina was 4.79%. The findings showed that RAPD and ITS sequencing could be used for developing molecular genetic markers and screening of unidentified genus Lepista species.