Polymerase Chain Reaction Conditions Research Articles

Standardization of methods for detection of Ralstonia solanacearum in potato*

In support of EU Council Directive 98/57/EC on the control of potato brown rot disease, caused by Ralstonia solanacearum, a ring test was conducted to evaluate and standardize currently recommended methods for official testing of potato tubers. Test tubers were either naturally infected (symptomatic) or vacuum‐infiltrated with different R. solanacearum suspensions or water. Testing was performed independently in 19 EU plant health laboratories using standardized protocols based on those annexed to the Directive. With high pathogen concentrations, fully reliable results were obtained with dilution plating on a selective medium, immunofluorescent antibody staining (IFAS), enzyme‐linked immunosorbent assay (ELISA), fluorescent in situ hybridization and a tomato bioassay. The reliability of diagnoses across all laboratories decreased with inoculum concentration in asymptomatic tubers, irrespective of the methods used. Comparable results were obtained with 3 commercial antisera in IF AS or with 2 antisera in ELISA but the IFAS method was slightly more reliable. In several laboratories, diagnoses obtained using polymerase chain reaction (PCR) protocols were less reliable than those using the other methods. Independent optimization of PCR conditions in each laboratory and the introduction of internal positive PCR controls are recommended.

Competitive PCR–DGGE analysis of bacterial mixtures: an internal standard and an appraisal of template enumeration accuracy

Analysis of polymerase chain reaction (PCR) amplified 16S rDNA fragments from environmental samples by denaturing gradients of chemicals or heat [denaturing gradient gel electrophoresis (DGGE) and thermal gradient gel electrophoresis (TGGE)] within polyacrylamide gels is a popular tool in microbial ecology. Difficulties in acceptance of the technique and interpretation of the results remain, due to its qualitative nature. In this study we have addressed this problem by the construction and evaluation of a quantitative standard for incorporation into test DNA samples. The standard was based on a naturally occurring 16S rRNA gene carried by the X-endosymbiont of the psyllid Anomoneura mori, a γ-proteobacterium. This sequence is the most AT-rich 16S rDNA gene recovered from any cultured organism or environmental sample described to date, and a specifically amplified rDNA fragment denatured under exceptionally low stringency denaturing conditions. The native sequence was modified to incorporate perfect matches to the PCR primers used. The efficiency of amplification of this standard in comparison to a range of 16S rDNA sequences and the errors involved in enumerating template molecules under a range of PCR conditions are demonstrated and quantified. Tests indicated that highly accurate counts of released target molecules from a range of bacterial cells could be achieved in both laboratory mixtures and compost.

Genomic variation within Monilinia laxa, M. fructigena and M. fructicola, and application to species identification by PCR.

Brown rot and twig canker of fruit trees are caused by Monilinia laxa, M. fructigena and M. fructicola. The Internal Transcribed Spacer (ITS) between the 18S and the 28S rRNA genes of four M. laxa and four M. fructigena isolates collected in France was amplified by Polymerase Chain Reaction (PCR) using universal primers and sequenced. Multiple alignment of the ITS sequences and comparison with published sequences revealed very little intraspecific variation and a low interspecific polymorphism clustered in two regions. Species-specific PCR primers were designed to amplify a 356 bp fragment for each of the three species. The specificity of the three primer pairs was successfully tested with a collection of 17 M. laxa, 18 M. fructigena and 6 M. fructicola isolates collected from different hosts and different countries, unequivocally confirming the identification of each isolate based on morphological and cultural traits. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assays was also successfully confirmed with DNA extracted from different fungal species, either phylogenetically close to the genus Monilinia or commonly found on diseased fruits. Using this new reliable technique, doubtful isolates can be directly identified in a single PCR run. Moreover, detection and identification of the Monilinia species were successfully achieved directly on diseased fruits. This simple and rapid method can be particularly useful to detect M. fructicola which is a listed quarantine fungus in all European countries.

A sequence of progesterone receptor homologue in freshwater crocodile (Crocodylus siamensis)

A sequence of progesterone receptor homologue in freshwater crocodile (Crocodylus siamensis)

Detection and quantification of Phytophthora species which are associated with root‐rot diseases in European deciduous forests by species‐specific polymerase chain reaction

SummaryOligonucleotide primers were developed for the polymerase chain reaction (PCR)‐based detection of selected Phytophthora species which are known to cause root‐rot diseases in European forest trees. The primer pair CITR1/CITR2, complementing both internal transcribed spacer regions of the ribosomal RNA genes, gave a 711 bp amplicon with Phytophthora citricola. The Phytophthora cambivora specific primer pair CAMB3/CAMB4, producing a 1105bp amplicon, as well as the Phytophthora quercina specific primer pair QUERC1/QUERC2, producing a 842 bp amplicon, were derived from randomly amplified polymorphic DNA (RAPD)‐fragments presented in this paper. All three primer pairs revealed no undesirable cross‐reaction with a diverse test collection of isolates including other Phytophthora species, Pythium, Xerocomus, Hebeloma, Russula, and Armillaria. Under the PCR conditions described the detection of a well discernable amplicon was possible down to 100 pg (P. cambivora), 4pg (P. quercina), and 2pg (P. citricola) target DNA. This diagnostic PCR system was able to detect P. citricola, P. quercina, and P. cambivora in seedlings of pendunculate oak (Quercus robur) and European beech (Fagus sylvatica) which were artificially infected under controlled conditions.

Cloning and nucleotide sequence of retroposons specific to hominoid primates derived from an endogenous retrovirus (HERV-K).

595 ENDOGENOUS RETROVIRUSES comprise approximately 1% of the human genome.1 Although most human endogenous retroviruses (HERVs) are defective, one class, HERV-K (homologous to mouse mammary tumor virus [MMTV]), encodes viral proteins and particles3 and is highly expressed in germ cell tumors. Retroposons (a retroelementso and a retrovirus-like elements,o which multiply in the genome by retrotransposition) are distinguished from retroviruses by the absence of one or both long terminal repeats (LTRs). These repetitive elements are classified into two groupsÐshort (up to 600 bp) interspersed nuclear elements (SINEs) and long (about 6 kg) interspersed nuclear elements (LINEs)Ð that include a number of families that have diverged in the course of mammalian evolution.4 One class of SINEs, the Alu sequences, is specific to primates. Another class, termed SINE-R (for retrovirus), which is derived from the HERV-K sequence, has apparently undergone modification in the hominoid lineage.5 Such elements are a potential agency of genome change.1,6,7 The human-specific retroposon SINE-R.C2, a member of the SINE-R retroposon family (present at 4000 to 5000 copies per haploid human genome), was discovered in an investigation of the variable number of tandem repeats (VNTR) locus within intron 3 of the gene for C2, the second component of complement, located in class III of the major histocompatibility complex on the short arm of human chromosome 6.8,9 Other members of the SINE-R family in the human genome, SINER11, -14, and -19, were isolated by colony blot hybridization with an HERV-K10 probe. By BLAST search, schizophrenic brain S11 cDNA (GenBank, accession No. AA772777) was also found to have high homology with the 39 LTR of HERV-K10. Therefore, specific polymerase chain reaction (PCR) primers, JS902 and JS903, were designed from S11 cDNA. Using these primers, the sequences HS307 (GenBank, accession No. AB016781) and HS408 (GenBank, accession No. AB016782), which have a high degree of homology to the SINE-R.C2, -R11, -R14, and -R19 retroposons, were identified in an investigation of the Xq21.3 region that has been tentatively linked to schizophrenia and schizo-affective disorder by Laval et al.10 and Kim et al. Each of these retroposons derives from the 3 9 long terminal repeat and the small upstream flanking regions of the human endogenous retrovirus HERV-K10,5 the entire sequence of which has been reported as a candidate autoimmune gene in type I diabetes.11 The HERV-K10 (present at 50 copies per haploid human genome) has been found to be present in all primates except for New World monkeys, whereas SINE-R.C2 is present only in Homo sapiens. Other members of the SINER family have been found to be present in the human, chimpanzee, and gorilla genomes by Southern blot analysis.12 To obtain sequence information relating to SINE-R-type retroposons in hominoid primates, genomic DNAs from the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and agile gibbon (Hylobates agilis) were amplified by PCR using primers JS902 (5 9 -GAGA ATGGGCCATGATGAC-3 9 , bases 4±22) and JS903 (59 GATCATTCTTGGATGTTTCT-3 9 , bases 466±485) derived from schizophrenic brain S11 cDNA (GenBank, accession No. AA772777). The PCR conditions followed were those of Kim et al. with an annealing temperature of 56°C. PCR products were separated on a 1.8% agarose gel, purified with the QIAEX II gel extraction kit (Qiagen, Chatsworth, CA) and cloned into the T-khs307 vector.14 Plasmid DNA was isolated by the alkali lysis method, using the High Pure plasmid isolation kit (Boehringer Mannheim, Indianapolis, IN). Individual plasmid DNAs were screened for inserts by PCR and positive samples were subjected to sequence analyses on both strands with T7 and M13 reverse primers using an automated DNA sequencer (model 373A; Applied Biosystems, Foster City, CA) and the DyeDeoxy terminator kit. Sequence analyses were done with the aid of GAP and PILEUP from the GCG program. The neighbor-joining phylogenetic analysis was performed with the CLUSTAL W program.15 We searched for sequence homology using the BLAST network server.16 Using genomic DNA from hominoid primates, SINE-R-type retroposons were amplified and nucleotide sequences deter-

Dominant Negative Isoform of Rat Norepinephrine Transporter Produced by Alternative RNA Splicing

We have cloned from rat brain a family of alternatively spliced cDNAs from a single gene, which encodes a norepinephrine transporter (NET) having variations at the 3'-region including both coding and noncoding regions. This produces two transporter isoforms, rNETa and rNETb, which differ at their COOH termini. The rNETa isoform reveals a COOH terminus homologous to human NET and transports norepinephrine. In contrast, rNETb revealed no detectable transport function but reduced functional expression of rNETa when both isoforms were expressed in the same cell. Thus, rNETb potentially functions as a dominant negative inhibitor of rNETa activity. Co-expression of rNETb with a gamma-aminobutyric acid transporter (rGAT1), a serotonin transporter (rSERT), and a dopamine transporter (rDAT) reduced their transport activity. No reduction was found with the glutamate/aspartate transporter (rGLAST). Alternative RNA splicing of NET suggests a novel mechanism for the regulation of synaptic transmission.

Efficiency of Randomly Amplified Polymorphic DNA to Sequence Characterized Amplified Region Marker Conversion and their Comparative Polymerase Chain Reaction Sensitivity in Cucumber

The conversion of randomly amplified polymorphic DNA (RAPD) markers to sequence characterized amplified region (SCAR) markers, and the effects of differing polymerase chain reaction (PCR) conditions were studied in cucumber (Cucumis sativus L.). Attempts were made to clone and sequence 75 RAPD PCR products to produce SCAR primers (16 to 22 nucleotides) designed to amplify original RAPD PCR products. The influence of template DNA source, purity, and concentration, MgCl2 concentration, Taq polymerase source, and type of thermocycler upon RAPD and SCAR marker performance was evaluated. Conversion of RAPD to SCAR markers was not universally successful, and SCAR primers reacted differently to varying PCR conditions. Only 48 (64%) of 75 RAPD markers were successfully converted to SCAR markers and 11 (15%) of these reproduced the polymorphism observed with the original RAPD PCR product. Moreover, some SCAR primer pairs produced multiple polymorphic PCR products. The band intensity of SCAR markers were brighter (P = 0.05) than their corresponding RAPD markers with only one exception. The SCAR markers examined were less influenced (P = 0.05) by MgCl2 concentration than their corresponding RAPD markers. However, some SCAR markers were more sensitive to reaction impurities than their RAPD counterparts and SCAR markers tended to be less readily visualized (decrease in frequency of visible PCR product) with low concentrations (1 and 2 mm) of template DNA than their corresponding RAPD markers. Neither the source of Taq nor the type of thermocycler used affected the performance of SCAR and RAPD markers. These data suggest that although SCAR markers may demonstrate enhanced performance over the RAPD markers from which they are derived, careful consideration must be given to both the costs and potential benefits of SCAR marker development in cucumber.

Origin of spurious multiple bands in the amplification of microsatellite sequences.

Multiple band artifacts are seen commonly in the analyses of short repetitive sequences, also known as microsatellites, using the polymerase chain reaction (PCR). In this study, the conditions of PCR...

A multiplex PCR for Massachusetts and Arkansas serotypes of infectious bronchitis virus

Infectious bronchitis virus (IBV), the prototype of the coronavirus family, is an enveloped, single-stranded RNA virus with a genome size of approximately 27·6 kilobase. Infectious bronchitis virus causes an acute, highly contagious respiratory and urogenital disease of chickens which results in significant economic losses in commercial broilers, layers and breeders. A rapid, highly sensitive and specific method is needed in the differential diagnosis of infections of different serotypes. A multiplex polymerase chain reaction (PCR) method was developed and optimized to simultaneously detect Massachusetts (Mass) and Arkansas (Ark) serotypes of IBV. One common primer and two serotype specific primers were chosen from the S1 gene sequences of IBV and used in one PCR reaction. Under optimized PCR conditions, two serotype specific PCR products, 1026 bp for Mass and 896 bp for Ark, respectively, were amplified and detected by agarose gel electrophoreses. The specificity of the technique was verified by using 20 different strains and isolates of IBV, and other avian bacterial and viral pathogens. Using a serial 10-fold dilution of the artificial mixture of both Mass and Ark samples, the detection limit was found to be 5 pg RNA after 35 cycles of PCR. The multiplex PCR was able to detect and differentiate both serotypes in embryonated eggs that were co-infected with different EID50virus titers of Mass 41 and Ark 99. The multiplex PCR developed in this study will be valuable for rapid identification, differential diagnosis, and epidemiological studies of these two serotypes of IBV infections.

Nucleotide diversity in three different genomic regions of Venezuelan HIV type 1 isolates: a subtyping update.

73 HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) infection continues its rapid global spread. A short time ago a few HIV-1 subtypes based on env sequences were identified, and outside the African continent most HIV-1 isolates belonged to subtype B. However, the rapid genetic diversification of HIV1 is reflected in the current separation of isolates into two groups (M and O), and an increasing number of subtypes within each group. New subtypes are finding their way outside the geographical areas where they were first isolated.1,2 Continuous reports of additional a non-subtype Bo HIV-1 infections around the world underscore the need for surveillance to track HIV-1 variants and to further define the molecular basis of their genetic variation. In the American continent and the Caribbean region, HIV-1 subtype B is dominant. However, the detection of HIV-1 of different subtypes in this area (i.e., subtypes A, C, D, E, and F, the group O) exemplifies the spread of HIV1 clades well beyond their original geographical locations.4±6 In addition, with more than 1.5 million estimated HIV infections, Latin America and the Caribbean follow Africa and southern Asia as the macroregion with the highest number of estimated HIV infections. We have undertaken the first molecular characterization of HIV-1 isolates from Venezuela.3 These initial viruses (VE1 to VE8) were isolated in 1991, and an analysis of pol and env genomic segments identified them as belonging to subtype B. In the present report 20 additional isolates of HIV-1 from Venezuela have been molecularly characterized on the basis of three distinct, noncontiguous genomic regions: the long terminal repeat (LTR), the first exon of the tat gene, and the env region encoding the C2V3 domain of gp120, with the aim of determining the extent of their genetic variation, and to characterize sequence relationships and evolutionary pattern in recent years. Twenty-eight blood samples were obtained in two periods: a first group of 8 samples (VE1 to VE8) was collected in 1991,3 and the second group includes samples from 1995. Epidemiological and clinical data are provided in Table 1. Proviral DNA was extracted as previously described. Three genomic regions were amplified for each DNA sample: 560 nucleotides of the HIV-1 LTR (positions 2 285 to 1 275 from the transcription start site), the first exon of the tat gene (216 bp), and an internal fragment of 1.2 kb spanning the gp120 V1±V5-coding region of env. Nested polymerase chain reaction (PCR) was used for all amplifications. PCR conditions and primers used to amplify the LTR region and the first exon of tat have been previously described.7 To amplify the V1±V5-coding region of env, the primers described in Delwart et al.8 were used. The PCR products of env were compared with those of 14 previously characterized HIV-1 strains1 by a heteroduplex mobility assay (HMA) as previously described. Nucleotide sequencing was performed using the fmol method (Promega, Madison, WI), followed by treatment with terminal deoxynucleotidyl transferase, as described.9 LTR (nucleotides 170 to 730 of the CAM1 genome1) and tat (nucleotides 5832 to 6047 of the CAM1 genome1) were sequenced using the inner primers LTR3/LTR4, and TAT3/TAT4, respectively. The C2V3-coding region (nucleotides 6817 to 7218 of the CAM1 genome) was sequenced as described previously.9 Initial subtyping experiments involved characterization of the new isolates by heteroduplex mobility assay,8 using reference plasmid DNA corresponding to HIV-1 subtypes A to H (heteroduplex mobility analysis HIV-1 env subtyping kit protocol version 3; NIH AIDS Research and Reference Reagent Program). Comparison of 28 Venezuelan samples with standard probes of several subtypes showed unequivocal electrophoretic patterns, corresponding to subtype B (data not shown). The epidemiological relationships of HIV-1 isolates from Venezuela with other HIV-1 isolates were further analyzed by phylogenetic analyses based on nucleotide sequences of the LTR region, the first exon of tat, and the C2V3 domain of the gp120coding region. Venezuelan HIV-1 isolates clustered with subtype B viruses in all cases (Fig. 1).10±14 Nucleotide diversities among Venezuelan samples were similar for the three genomic segments analyzed. Pairwise comparisons showed an average divergence of 6.6% (range of 1.4 to 11.0%), 8.0% (1.2 to 14.9%), and 9.4% (1.5 to 13.8%) for LTR, tat, and env, respectively. Average genetic distances be-

15] READS: A method for display of 3′-end fragments of restriction enzyme-digested cDNAs for analysis of differential gene expression

Publisher Summary This chapter discusses a gel-based method for analysis of differential gene expression called READS 2 or restriction endonucleolytic analysis of differentially expressed sequences. This method uses stringent polymerase chain reaction (PCR) conditions to amplify only the extreme Y-end fragment of a restriction endonuclease-digested cDNA molecule without employing methods for physical separation of the fragments before or after PCR amplification. A comprehensive analysis of differentially expressed mRNAs between two physiological states can be done by comparing the gel patterns of their 3'-end cDNA restriction fragments. One of the important features of READS is the reproducibility of gel patterns in repeat experiments. Because the method relies on specific amplification of only the 3'-end restriction fragments under stringent PCR conditions, reproducibility of gel patterns can be expected because the set of cDNA molecules synthesized from a particular total RNA by a given anchored primer will always be the same and will, therefore, always generate the same pattern of 3'-end restriction fragments. The chapter provides a detailed description of the method used to perform READS analysis on control and activated Jurkat T cells.

Development of a PCR-based method coupled with a microplate colorimetric assay for the detection of Porcine Parvovirus and application to diagnosis in piglet tissues and human plasma

A new method for Porcine Parvovirus (PPV) diagnosis was developed. The method is based on polymerase chain reaction (PCR) amplification followed by hybridization and colorimetric detection of PCR products in microwell plates. A highly specific and sensitive amplification step was ensured by primers carefully selected in the VP2 structural gene and optimized PCR conditions. Uracyl-DNA-Glycosylase (UDG) in combination with dUTP was used to avoid false-positive results, and 100 copies of internal control (IC) were added to each PCR reaction to reveal any false-negative samples. Biotinylated amplified fragments were hybridized on specific capture probes covalently linked to microwell plates. Finally, the detection of hybridized PCR products was performed by means of a colorimetric reaction, which was automated. The method permitted the detection of 103copies (6 fg) of replicative form DNA (RF-DNA) in 20 mg of lung sample, and 500 copies (3 fg) in 100μl of plasma. It was used to analyse 24 field piglet tissue samples, and 35 human plasma or serum specimens collected from patients treated with porcine Factor VIII concentrates.

Mitochondrial DNA rearrangements, including partial duplications, occur in young and old rat tissues

Using polymerase chain reaction (PCR) with back-to-back primers, 85 different mitochondrial DNA (mtDNA) rearrangements, consisting of partial duplications or mini-circles, were detected in brain, liver, and heart tissue from Fischer 344 rats. The regions around the mitochondrial tRNA Leu (UUR) gene, the cluster of three tRNA genes [His, Ser(AGY), Leu(UUC)], as well as the region of the displacement loop were analyzed separately with different primer sets. Rearrangements were detected in all regions analyzed in samples taken throughout the animal life span, ranging from 1 day old to 33 months of age (senescent). Two-thirds of the rearrangements terminated at short (3–9-bp) direct repeats. Three of the different rearrangements were detected in more than one animal; the most common rearrangement was found in nine different template preparations. Two loci (hot spots) were found to be particularly susceptible to rearrangement, and both were located at sequences that exhibited highly conserved potential for secondary structure formation. The displacement loop region of 10 samples exhibited the presence of multiple tandem duplications ranging between 324 and 449 bp in length. One of these consisted of heterologous, but overlapping, repeating units. Identical PCR protocols were carried out in control experiments using a cloned fragment of mtDNA that encompassed the most common hot spot sequence. The results showed that this fragment did not artifactually generate a rearrangement junction under our PCR conditions and suggested that this sequence does not promote rearrangement mutations in bacteria during the cloning process.

Differential display of mRNA.

Differential display of mRNA (DD) is a technique in which mRNA species expressed by a cell population are reverse transcribed and then amplified by many separate polymerase chain reactions (PCR). PCR primers and conditions are chosen so that any given reaction yields a limited number of amplified cDNA fragments, permitting their visualization as discrete bands following gel electrophoresis. This robust and relatively simple procedure allows identification of genes that are differentially expressed in different cell populations. Here we review DD including some recent modifications, and compare it with other techniques for analyzing differential mRNA expression.

Simultaneous TD/RT-PCR detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus with a single pair of primers

Cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) are the two most prevalent and economically important orchid viruses which have attained a world-wide distribution. We have designed one pair of common primers, which were able to detect, simultaneously, both viruses by a single polymerase chain reaction (PCR). Sequence alignment and primer analyses were employed in primer design. The effect of touch-down PCR, primer annealing temperature, number of thermal cycles, MgCl 2 concentration and the degree of primer degeneracy, were investigated and applied to the optimisation of PCR conditions in the detection of both viruses in order to achieve equal amplification efficiencies. This is the first successful attempt at using a single pair of PCR primers to detect two viruses belonging to different taxonomic groups simultaneously.

N-cadherin expression in human adult T-cell leukemia cell line.

Adult T-cell leukemia/lymphoma (ATL) has been reported as a new category of T-cell malignancy [1]. Human T-cell lymphotropic virus I (HTLV-I) has been linked etiologically with the disease [2]. Both ATL and cutaneous T-cell lymphoma (CTCL) show a helper/inducer T-cell phenotype (CD4+CD8–), but there are differences between these phenotypes, e.g. the expression of IL-2 receptor (CD25) is higher in ATL than in CTCL [3]. Aiba et al. [4] reported that some populations of T cell express E-cadherin, and Cepek et al. [5] and Ashton et al. [6] have shown that a cadherin molecule other than E-cadherin is expressed in T-cell lymphoma and T lymphocytes by immunoblotting using anti-pan-cadherin and anti-E-cadherin antibodies. Recently N-cadherin expression in some human leukemia cell lines has been reported by Tsutsui et al. [7]. ATL cells sometimes infiltrate into the epidermis and form microabscesses as well as T-cell lymphoma, which suggests the existence of a cadherin molecule, but the type of cadherin expressed in ATL cells is still unclear. To identify the cadherin molecule expressed in ATL cell lines, we performed a polymerase chain reaction (PCR) using the degenerate primers 5′-GAATTCACNGCNCCNCCNTAYGA and 5′-GAATTCTCNGCNARYTTYTTRAA to amplify multiple cadherin subtypes with the first strand of cDNAs generated by reverse transcription of the mRNAs of Hut102 cells [8]. The PCR conditions were essentially the same as those described by Matsuyoshi et al. [9]. After PCR, the reaction products were separated by 2% agarose gel electrophoresis, and the DNA of about 160 bp in size was extracted using a Geneclean Kit (Funakoshi, Tokyo, Japan) and ligated with a TA vector (Invitrogen, San Diego, Calif.) for sequencing. The sequencing of the DNA was done using an ALFred DNA Sequencer (Pharmacia Biotech, Uppsala, Sweden) which is designed for the automated electrophoresis and analysis of sequencing reactions by the direct detection of fluorescently labeled DNA molecules. We identified a fragment homologous with cadherins from Hut102. The deduced amino acid sequence of the fragment was identical to human N-cadherin (Fig. 1A,B). To confirm the expression of N-cadherin at the mRNA level and to confirm the existence of E-cadherin mRNA in Hut102, we carried out RT-PCR with the first strand of cDNA generated by reverse transcription of the mRNA of Hut102 cells, normal human epidermal keratinocytes and human fibroblasts using specific primers for N-cadherin (5′-GTGCCATTAGCCAAGGGAATTCAGC) and (5′-GCGTTCCTGTTCCACTCATAGGAGG), and E-cadherin Norihisa Matsuyoshi · Ken-ichi Toda · Sadao Imamura

Polymerase chain reaction detection of Haemophilus ducreyi DNA.

OBJECTIVES: To develop a polymerase chain reaction (PCR) method to detect Haemophilus ducreyi DNA in cultured isolates and clinical material. METHODS: Primers specific to the H ducreyi 16s rRNA gene...

An improved polymerase chain reaction (PCR) protocol for unambigous detection of growth hormone gene deletions.

hGH-1 gene deletions are detected by simultaneous PCR amplification along the two homologous DNA sequences flanking the hGH-1 gene on both sides and are differentiated by SmaI restriction enzyme digestion. We have observed that among the SmaI digested PCR products from normal homozygous subjects, from those heterozygous for the 7.6 kb deletion and from those heterozygous for a 6.7 kb deletion, along with the expected fragments there is an unexpected 1470 bp fragment. This fragment arises from the co-amplification of a third homologous sequence located downstream from the hGH-1 gene and it confuses differentiation between normal homozygous and heterozygous for 7.6 kb subjects from the 6.7 kb heterozygous subjects. To overcome this problem we have improved PCR conditions using a different reverse primer. These changes avoid the interaction of the primers with the third homologous sequence located downstream from the hGH-1 gene and prevent the appearance of this additional band that complicates the interpretation of the results. We conclude that the new reverse primer sequence avoids the amplification of the downstream hGH-1 gene sequence and the production of the 1474 bp band after SmaI endonuclease enzyme digestion and makes it possible to differentiate homozygous normal subjects and those who are heterozygous for a 7.6 kb deletion from those who are heterozygous for a 6.7 kb deletion.

Improved polymerase chain reaction conditions for quick diagnostics of Huntington disease

Huntington disease (HD) belongs to a growing list of neurodegenerative disorders (fragile X syndrome [6], myotonic dystrophy [1], spino-bulbar muscular atrophy [2]etc.) characterized by unstable expanded trinucleotide repeats (so-called `dynamic mutations'). The dynamic mutation causing HD represents the expansion of CAG triplets in the first exon of a gene IT15 (chromosome 4) coding for huntingtin. This trinucleotide stretch is varying in the range of 11–34 in normal chromosomes and 39–121 in HD chromosomes. The most direct diagnostic approach is to amplify the proximal region of IT15 gene (from patients genomic DNA) by polymerase chain reaction (PCR) and estimate the number of CAG triplets. All protocols published to date are difficult to reproduce because amplification is inefficient giving additional non-specific products. The strategy of our experiment is shown in Fig. 1. We designed one new primer, primer No. 2 (another primer was primer No. 1) and novel PCR conditions. Primer No. 2 is located closer to CAG triplets and its extension is not including the GC rich region. PCR amplified products, using primer Nos. 1 and 2, thus do not include the GC rich region and, therefore, are much more efficiently amplified (compared to the products of amplification with primer Nos. 1 and 3).