PCR Buffer Research Articles

Decreased Km to dNTPs is an essential M-MuLV reverse transcriptase adoption required to perform efficient cDNA synthesis in One-Step RT-PCR assay.

Personalized medicine and advanced diagnostic tools based on RNA analysis are focusing on fast and direct One-Step RT-PCR assays. First strand complementary DNA (cDNA) synthesized by the reverse transcriptase (RT) is exponentially amplified in the end-point or real-time PCR. Even a minor discrepancy in PCR conditions would result in big deviations during the data analysis. Thus, One-Step RT-PCR composition is typically based on the PCR buffer. In this study, we have used compartmentalized ribosome display technique for in vitro evolution of the Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV RT) that would be able to perform efficient full-length cDNA synthesis in PCR buffer optimized for Thermus aquaticus DNA polymerase. The most frequent mutations found in a selected library were analyzed. Aside from the mutations, which switch off RNase H activity of RT and are beneficial for the full-length cDNA synthesis, we have identified several mutations in the active center of the enzyme (Q221R and V223A/M), which result in 4-5-fold decrease of Km for dNTPs (<0.2 mM). The selected mutations are in surprising agreement with the natural evolution process because they transformed the active center from the oncoretroviral M-MuLV RT-type to the lenitiviral enzyme-type. We believe that this was the major and essential phenotypic adjustment required to perform fast and efficient cDNA synthesis in PCR buffer at 0.2-mM concentration of each dNTP.

DNA Isolation and Optimization of ISSR-PCR Reaction System in Oryza sativa L.

Inter simple sequence repeats (ISSRs) have been utilized widely for molecular markers in analyzing the genetic diversity and phylogenetic and regions in the genome flanked by microsatellite sequences. PCR amplification of these regions using a single primer yields multiple amplification products that can be used as a dominant multilocus marker system for the study of genetic variation in various organisms. For this study provides, DNA isolation, adjusting in six factors (Buffer, MgCl2, dNTPs, ISSR primers, Template DNA and Taq polymerase ) at six levels, and optimization of PCR temperature for the ISSR reaction was 60-45 °C, primers screening on indica rice ( Oryza sativa ). In this research, simple method of DNA isolation by using seedling. The objective of the present investigation was to assess the optimizations and quantification. Has been shown that stalk enhanced the maximum value of genomic. The results show that 100 ISSR primers were examined as well as, 56 ISSR primers was productively amplified. Optimum components for PCR reactions were 5.0 μl of 5X PCR Buffer, 1.5 μl of 25mM MgCl2, 1 μl of 10 mM dNTP, 1 μl of 10 Μm ISSR primers, 2 μl Template DNA, and 0.1 μl of 5 units/ml Taq polymerase. Based on this study, has brought out some information on the relationship between these ISSR primers will be applied further for molecular profiling as well as response evaluation in rice varieties

Efficient identification of gene knockout mutant mediated by CRISPR/Cas9 by CELⅠ crude extracts

CRISPR/Cas9, emerged as an efficient and powerful gene editing technology, has become the mainstream genome editing technology. Constructing mutants using CRISPR/Cas9 system is of great significance to the functional study and breeding application of useful genes. As the basis of the technology, a method for identification of mutation with efficiency and lower cost is needed. In this report, we studied the factors influencing mutation detected by CEL Ⅰ crude extracts, such as the amount of protein, enzyme incubation time, PCR buffers. Under the optimized conditions, we can integrate the mutation detection steps into one-tube reaction. We used this system to examine the mutation types and frequency of rice stn1 mediated by CRISPR/Cas9. We also used this method to identify different mutation types including homozygous, heterozygous and bi-allelic mutations. The accuracy of this method reached 100% verified by sequencing. Altogether, our results showed that using CELⅠ crude extracts was an efficient and low cost method for identification of CRISPR/Cas9 mediated mutation.

Chitosan-Modified Filter Paper for Nucleic Acid Extraction and “in Situ PCR” on a Thermoplastic Microchip

Plastic microfluidic devices with embedded chitosan-modified Fusion 5 filter paper (unmodified one purchased from GE Healthcare) have been successfully developed for DNA extraction and concentration, utilizing two different mechanisms for DNA capture: the physical entanglement of long-chain DNA molecules with the fiber matrix of the filter paper and the electrostatic adsorption of DNA to the chitosan-modified filter fibers. This new method not only provided a high DNA extraction efficiency at a pH of 5 by synergistically combining these two capture mechanisms together, but also resisted the elution of DNA from filters at a pH > 8 due to the entanglement of DNA with fibers. As a result, PCR buffers can be directly loaded into the extraction chamber for "in situ PCR", in which the captured DNA were used for downstream analysis without any loss. We demonstrated that the capture efficiencies of a 3-mm-diameter filter disc in a microchip were 98% and 95% for K562 human genomic DNA and bacteriophage λ-DNA, respectively. The washes with DI water, PCR mixture, and TE buffer cannot elute the captured DNA. In addition, the filter disc can enrich 62% of λ-DNA from a diluted sample (0.05 ng/μL), providing a concentration factor more than 30-fold. Finally, a microdevice with a simple two-chamber structure was developed for on-chip cell lysis, DNA extraction, and 15-plex short tandem repeat amplification from blood. This DNA extraction coupled with "in situ PCR" has great potential to be utilized in fully integrated microsystems for rapid, near-patient nucleic acid testing.

YfilerⓇ Plus population samples and dilution series: stutters, analytic thresholds, and drop-out probabilities.

The Yfiler Ⓡ Plus Amplification Kit amplifies 27 Y chromosomal small tandem repeat (STR) markers. The kit has five-fluorescent dye chemistry and the improved PCR buffer system of modern STR kits. We validated the kit for accredited investigations of crime scene samples by a thorough study of kit dynamics and performance. We determined dye-dependent analytical thresholds by receiver operating characteristics (ROC) and made a customised artefact filter that includes theoretical known artefacts by use of previously analysed population samples. Dilution series of known male DNA and a selection of crime scene samples were analysed with the customised thresholds and artefact filters. The Yfiler Ⓡ Plus Amplification Kit was sensitive giving full profiles down to 70 pg of male DNA. The balances between the fluorescent dyes as well as between loci were very good. The kit was able to produce full Y-STR profiles from crime scene samples containing small amounts of male DNA and large amounts of female DNA (although unspecific reactions were evident for very unbalanced mixtures). A decrease in the drop-out rate was found for both the dilution series and population samples, as well as a small increase in the drop-in rate for population samples, using the customised threshold and artefact filters compared to company-provided thresholds and artefact filters. The additional drop-ins were all of a nature that would be detected by inspection of the results. For the crime scene samples, large amounts of female DNA complicated the analysis by causing drop-ins of characteristic female DNA artefacts. Even though the customised analytical threshold in combination with the custom-made artefact filters gave more alleles, crime scene samples still needed special attention from the forensic geneticist.

Integration of Nucleic Acid Extraction Protocol with Automated Extractor for Multiplex Viral Detection.

The isolation of nucleic acids (NA) is the preliminary step to carry out genetic studies and DNA biosensor development. The presence of inhibitors in the purified NA interferes with the downstream application. These salts and other organic contaminations particularly challenge the analytical sensitivity of DNA biosensors. The detailed study was carried out to optimize the factors which might affect viral nucleic acid purification. The results suggested that 6 M guanidinium hydrochloride salt concentration was critical for NA isolation. The inverse relation has been found in the pH of the lysis buffer and quality and quantity of NA. The NA yield was relatively stable at pH 4–5. It has been observed that the use of carrier RNA was indispensable for viral genome isolation. The addition of ethanol to lysate in 1:1 ratio greatly improved NA recovery. The elution efficiency of DNase and RNase free water, 1× TE buffer and 1× PCR buffer was compared. The carrier RNA was best eluted in DNase and RNase free water and 1× TE buffer. It was further demonstrated that this method can be automatized for high throughput detection. A simple experiment was conducted to optimize the different parameters of an automated NA extractor to simultaneously extract HBV DNA and HCV RNA. The purified NA was successfully amplified in PCR and RT-PCR to verify the reliability of the established protocol. Thus a semi-automated system for the simultaneous detection of multiple viruses has been demonstrated.

MPS analysis of the mtDNA hypervariable regions on the MiSeq with improved enrichment.

The non-coding displacement (D) loop of the human mitochondrial (mt) genome contains two hypervariable regions known as HVR1 and HVR2 that are most often analyzed by forensic DNA laboratories. The massively parallel sequencing (MPS) protocol from Illumina (Human mtDNA D-Loop Hypervariable Region protocol) utilizes four sets of established PCR primer pairs for the initial amplification (enrichment) step that span the hypervariable regions. Transposase adapted (TA) sequences are attached to the 5'-end of each primer, allowing for effective library preparation prior to analysis on the MiSeq, and AmpliTaq Gold DNA polymerase is the enzyme recommended for amplification. The amplification conditions were modified by replacing AmpliTaq Gold with TaKaRa Ex Taq® HS, along with an enhanced PCR buffer system. The resulting method was compared to the recommended protocol and to a conventional non-MPS approach used in an operating forensic DNA laboratory. The modified amplification conditions gave equivalent or improved results, including when amplifying low amounts of DNA template from hair shafts which are a routine evidence type in forensic mtDNA cases. Amplification products were successfully sequenced using an MPS approach, addressing sensitivity of library preparation, evaluation of precision and accuracy through repeatability and reproducibility, and mixture studies. These findings provide forensic laboratories with a robust and improved enrichment method as they begin to implement the D-loop protocol from Illumina. Given that Ex Taq® HS is a proofreading enzyme, using this approach should allow for improved analysis of low-level mtDNA heteroplasmy.

MPL mRNA Expression in Venous Blood Leukocytes Is Reduced in Patients with Hematologic Malignancies Excluding MPN

Background:Expression of mRNA thrombopoietin receptor (MPL) is detected in various tissues, but peripheral blood cells mature thrombopoietin receptor found in the platelet and mononuclear cells only (http://www.genecards.org/). It has previously been shown MPL mRNA regulates mobilization of bone marrow stem cells (Xiao N. et al., Blood, 2015) and activation of Th2-lymphocytes (Takyar S. et al., J Exp Med, 2013). MPL mRNA expression in bone marrow is associated with the progression of AML (Wetzler M. et al., J Clin Oncol, 1997) and increase of it expression in bone marrow megakaryocytes in Ph- negative myeloproliferative neoplasm (MPN) more pronounced in patients with primary myelofibrosis (PMF) (Bock O. et al., J Pathol, 2004) has been described. MPL mRNA level in venous blood leukocytes in patients with hematologic malignancies is unknown.Aims:Evaluate the MPL mRNA expression in venous blood leukocytes in patients with certain hematologic malignanciesMethods: Real-time PCR was performed to detect of MPL mRNA transcripts levels in white blood cells 21 healthy volunteers, 28 patients with chronic myeloid neoplasm (MPN: PMF - 2, PV- 21 and ET - 5), 27 patients with chronic lymphocytic leukemia (CLL), 48 patients with chronic myeloid leukemia (CML), 3 patients with acute lymphocytic leukemia (ALL) and 8 patients with acute myeloid leukemia (AML). Venous blood were collected in tube with RNAse inhibitor. Total RNA was isolated using "Ribo-zol-D" (Aplisens). 10 µg of total RNA were transcribed using "Reverta-L" (Aplisens). PCR was optimized for the thermocycler iQ5 (Bio-Rad) under following condition: 40 cycle of 95 C for 15 sec and 60 C for 60 sec, reaction mixture contained 1X KCl PCR buffer, 2 mM MgCl2, 400 uM dNTP, 300 uM forward and reverse primers, 200 uM probe and 1 unit Taq-polymerase. The results were calculated utilizing the delta Ct method in the software package of "R". The threshold cycles (Ct) MPL gene and housekeeping gene GAPD determined using Cy0 method. The results was normalization with GAPDH reference gene and are presented as median and quartiles (25% and 75%).Results:mRNA MPL expression level in leukocytes venous blood decreased among PMF (0.024, 0.03) > ET (0.013; 0.003-0.016) = PV (0.010; 0.007-0.018) > CML (0.002; 0.0008-0.005) > CLL (0.0005; 0.0002-0.0016) = AML (0.0021; 0.0006-0.0027) = ALL (0.0018; 0.0009-0.0027). MPL mRNA expression level was no correlated with any blood cells count.Summary and Conclusions:Our study support the hypothesis of the independence certain functions mRNA MPL and functions mature thrombopoetin receptor in blood cells. Measurement of mRNA MPL can used as an additional diagnostic marker for the differentiation of MPN and evaluate progress in hematologic malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A simple and efficient total genomic DNA extraction method for individual zooplankton.

Molecular approaches are widely applied in species identification and taxonomic studies of minute zooplankton. One of the most focused zooplankton nowadays is from Subclass Copepoda. Accurate species identification of all life stages of the generally small sized copepods through molecular analysis is important, especially in taxonomic and systematic assessment of harpacticoid copepod populations and to understand their dynamics within the marine community. However, total genomic DNA (TGDNA) extraction from individual harpacticoid copepods can be problematic due to their small size and epibenthic behavior. In this research, six TGDNA extraction methods done on individual harpacticoid copepods were compared. The first new simple, feasible, efficient and consistent TGDNA extraction method was designed and compared with the commercial kit and modified available TGDNA extraction methods. The newly described TGDNA extraction method, “Incubation in PCR buffer” method, yielded good and consistent results based on the high success rate of PCR amplification (82%) compared to other methods. Coupled with its relatively consistent and economical method the “Incubation in PCR buffer” method is highly recommended in the TGDNA extraction of other minute zooplankton species.

Діагностика вірусів скручування листя винограду і коротковузля в процесі виробництва сертифікованого садивного матеріалу винограду

Діагностика вірусів скручування листя винограду і коротковузля в процесі виробництва сертифікованого садивного матеріалу винограду

PM 7/60 (2) Pantoea stewartii subsp. stewartii

Specific scopeThis Standard describes a diagnostic protocol for Pantoea stewartii subsp. stewartii.Specific approval and amendmentApproved in 2005‐09.Revised in 2016‐04.

High-throughput malaria parasite separation using a viscoelastic fluid for ultrasensitive PCR detection.

A novel microfluidic device for high-throughput particle separation using a viscoelastic fluid, which enables the rapid detection of extremely rare malaria parasites by using PCR analysis, is proposed. Our device consists of two segments: the 1st stage for sheathless pre-alignment and the 2nd stage for separation based on size-dependent viscoelasticity-induced lateral migration. The use of a high-aspect ratio channel and a viscoelastic polymer solution with low viscosity enables high-throughput processing. The device performance was first optimized using synthetic particles. A mixture of 2 and 10 μm particles was focused at the center plane in the 1st stage. The smaller particles, serving as surrogates for malaria parasites, were subsequently separated in the 2nd stage with a recovery rate of ∼96% at 400 μl min(-1). Finally, separation of the malaria parasites from the white blood cells was performed. At 400 μl min(-1), almost all white blood cells were removed and the malaria parasites were separated with a ∼94% recovery rate and ∼99% purity. Although the initial concentration of the malaria parasites was too low to be detected by PCR analysis, WBC depletion and buffer removal increased the parasite concentration sufficiently such that PCR detection was possible.

A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria.

In this study, we developed a centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria contaminated milk samples. The microdevice was designed to contain identical triplicate functional units and each unit has four reaction chambers, thereby making it possible to perform twelve direct-RPA reactions simultaneously. The integrated microdevice consisted of two layers: RPA reagents were injected in the top layer, while spiked milk samples with food poisoning bacteria were loaded into sample reservoirs in the bottom layer. For multiplex bacterial detection, the target gene-specific primers and probes were dried in each reaction chamber. The introduced samples and reagents could be equally aliquoted and dispensed into each reaction chamber by centrifugal force, and then the multiplex direct-RPA reaction was executed. The target genes of bacteria spiked in milk could be amplified at 39 °C without a DNA extraction step by using the direct-RPA cocktails, which were a combination of a direct PCR buffer and RPA enzymes. As the target gene amplification proceeded, the increased fluorescence signals coming from the reaction chambers were recorded in real-time at an interval of 2 min. The entire process, including the sample distribution, the direct-RPA reaction, and the real-time analysis, was accomplished with a custom-made portable genetic analyzer and a miniaturized optical detector. Monoplex, duplex, and triplex food poisoning bacteria (Salmonella enterica, Escherichia coli O157:H7, and Vibrio parahaemolyticus) detection was successfully performed with a detection sensitivity of 4 cells per 3.2 μL of milk samples within 30 min. By implementing the direct-PRA on the miniaturized centrifugal microsystem, the on-site food poisoning bacteria analysis would be feasible with high speed, sensitivity, and multiplicity.

Frequency of MTHFR GENE C677T Polymorphism for Non-Syndromic Autism Spectrum Disorder Patients

Background: The folate metabolism is a pathway that may involve in the non-syndromic Autism Spectrum Disorder (ASD). Methylenetetrahydrofolate reductase enzyme has a key role in folate metabolism. The C677T polymorphism of MTHFR gene could reduce the effectiveness of the enzyme.Objectives: To evaluate the frequency of MTHFR geneC677T polymorphism for non-syndromic ASD patients.Method: Thirty-four DNA samples were taken from each group. PCR mixture was consisted of 1µL DNA, 2.5µL PCR buffer, 0.5µL dNTP, 1.5µL MgCL2, 0.125µLTaqenzyme, 0.5µLofforwardandreverseprimerandaquabidesttoreach a volume of 25 µL. The PCR profiles were initiation 95ºC for 5 min, denaturation 94ºC for 1min, annealing 55ºCfor 45 seconds, and elongation 72ºC for30 seconds. The cycles were done in 35 times an dfinal elongation was at 72ºC for 5min. The PCR product was 198bp, and then digested by the Hinfl enzyme for 16hours at 37°C, and visualized using2%agarosegeland then electrophoresed for 30 minutes at 100 volts.Result: Non-syndromic ASD samples showed none had homozygote mutant type (677TT), 3 (8.8%) samples had heterozygote (677CT)and 31 (91.2%) samples had wild type (677CC). Meanwhile, normal control showed only 1 (2.9%)sample had homozygote mutant type(677TT), 9 (26.5%) samples had heterozygote (677CT)and 24 (70.6%) samples had wild type (677CC).Conclusion: The frequency of MTHFR geneC677T polymorphism in patients with non-syndromic ASD and controls are not significantly different.

Development of a real-time polymerase chain reaction method for the identification of Candida species

Candida species are one of the major causes of nosocomial infections and are the fourth most common agent involved in bloodstream infections. The impact of non-albicans Candida species is increasing, however C.albicans is still the most common species. Since the antifungal susceptibility pattern among Candida spp. may be different, rapid diagnosis and identification of non-albicans Candida spp. are important for the determination of antifungal agents that will be used for treatment. The aim of the study was to describe a real-time polymerase chain reaction (Rt-PCR) assay that rapidly detects, identifies and quantitates Candida species from blood culture samples. A total of 50 consecutive positive blood culture bottles (BACTEC, Beckton Dickinson, USA) identified at our laboratory between June-November 2013, were included in the study. Reference strains of Candida spp. (C.albicans ATCC 10231, C.glabrata ATCC 90030, C.tropicalis ATCC 1021, C.krusei ATCC 6258, C.parapsilosis ATCC 22019 and C. dubliniensis CD36) grown on Sabouraud dextrose agar were used for quality control. BACTEC bottles that were positive for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were also studied to search the cross-reactivity. A commercial kit (Zymo Research, USA) was used for DNA extraction. Real-time PCR was performed on LightCycler 480 (Roche, Germany) with primers and probes specific for 18S rRNA of Candida species. Twenty microlitres of the reaction mix contained 2 μl of extracted DNA, 2 μl of LightCycler Fast Start DNA Master Probe (Roche Diagnostics, Germany), 2 μl of MgCl(2) (5 mmol), 2 μl of 10x PCR buffer (Roche Diagnostics, Germany), 0.5 μl of each primer (0.01 nmol/μl) and 1 μl of each probe (0.1 μmol/μl) (TibMolBiol, Germany). Amplification was performed using the following conditions; 95°C for 10 mins and 50 cycles of denaturation at 95°C for 10 secs, annealing at 62°C for 10 secs and polymerisation at 72°C for 20 secs. A melting curve was created by cooling the producs at 50°C for 30 secs and then heating to 80°C at a rate of 0.1°C/sec measuring of the fluorescence simultaneously. For the quantitation of fungal DNA according to the standard curve, serial dilutions of C.albicans ATCC 10231 DNA from 3 x 10(5) to 3 x 10(2) ng/μl were used. All of the strains were also identified by conventional methods and sequence analysis in order to compare the results obtained by Rt-PCR. In our study, all patient and standard samples could be amplified, identified and quantitated by this developed Rt-PCR method. A total of 50 strains, of them 26 were C.parapsilosis, 15 were C.glabrata, 6 were C.albicans, and 3 were C.tropicalis have been detected and identified among patient samples. The results were completely concordant with the sequencing and conventional methods, so the sensitivity and specificity of this method were estimated as 100 percent. In conclusion, it was novel Rt-PCR developed and evaluated in this study is considered as a rapid, accurate, reproducible, sensitive and specific method for the detection, identification and quantitation of commonly observed Candida spp. strains.

PCR amplification of a triple-repeat genetic target directly from whole blood in 15 minutes as a proof-of-principle PCR study for direct sample analysis for a clinically relevant target.

BackgroundMost PCR-based diagnostics are still considered time- and labor-intensive due to disparate purification, amplification, and detection steps. Advancements in PCR enzymes and buffer chemistry have increased inhibitor tolerance, facilitating PCR directly from crude samples. Obviating the need for DNA purification, while lacking a concentration step, these direct sample methods are particularly apt for human genetic testing. However, direct PCR protocols have traditionally employed thermal cyclers with slow ramp rates and conservative hold times that significantly increase an assay’s time-to-result. For this proof-of-principle study, our objective was to significantly reduce sample preparation and assay time for a PCR-based genetic test, for myotonic dystrophy type 1 (DM1), by pairing an inhibitor-resistant enzyme mix with a rapid thermal cycler to analyze samples directly in whole blood.MethodsDM1 genetic screening was done with an adapted conventional PCR approach that employed the Streck Philisa® Thermal Cycler, the inhibitor-resistant NEBNext® High-Fidelity 2X PCR Master Mix, and agarose gel electrophoresis or an Agilent 2100 Bioanalyzer for detection. The Gene Link™ Myotonic Dystrophy Genemer™ Kit was used as a reference assay kit to evaluate the rapid assay.ResultsIn this work, a rapid and direct PCR assay testing 10% whole blood as template has been developed as an exclusionary screening assay for DM1, a triple-repeat genetic disorder. PCR amplification was completed in 15 minutes using 30 cycles, including in situ hot-start/cell lysis. Out of the 40 donors screened, this assay identified 23 (57.5%) as DM1 negative suggesting no need for further testing. These data are 100% concordant with data collected using the commercially available Gene Link Genemer™ Kit per the kit-specific PCR protocol.ConclusionsThe PCR assay described in this study amplified DM1 short tandem repeats in 15 minutes. By eliminating sample purification and slower conventional PCR protocols, we demonstrated how adaptation of current PCR technology and chemistries can produce a simple-to-use exclusionary screening assay that is independent of up-front sample prep, improving a clinical lab technician’s time-to-result. We envision this direct and rapid methodology could be applied to other conventional PCR-based genetic tests and sample matrices where genomic DNA is targeted for analysis within a given molecular diagnostic platform.

Phylogeography of E1b1b1b-M81 Haplogroup and Analysis of Its Subclades in Morocco

Phylogeography of E1b1b1b-M81 Haplogroup and Analysis of Its Subclades in Morocco Ahmed Reguig, Nourdin Harich, Abdelhamid Barakat, and Hassan Rouba abstract In this study we analyzed 295 unrelated Berber-speaking men from northern, central, and southern Morocco to characterize frequency of the E1b1b1b-M81 haplogroup and to refine the phylogeny of its subclades: E1b1b1b1-M107, E1b1b1b2-M183, and E1b1b1b2a-M165. For this purpose, we typed four biallelic polymorphisms: M81, M107, M183, and M165. A large majority of the Berber-speaking male lineages belonged to the Y-chromosomal E1b1b1b-M81 haplogroup. The frequency ranged from 79.1% to 98.5% in all localities sampled. E1b1b1b2-M183 was the most dominant subclade in our samples, ranging from 65.1% to 83.1%. In contrast, the E1b1b1b1-M107 and E1b1b1b2a-M165 subclades were not found in our samples. Our results suggest a predominance of the E1b1b1b-M81 haplogroup among Moroccan Berber-speaking males with a decreasing gradient from south to north. The most prevalent subclade in this haplogroup was E1b1b1b2-M183, for which differences among these three groups were statistically significant between central and southern groups. key words Y Chromosome, Haplogroup, Northern Africa, Biallelic Markers, Berber Speakers The tree of binary polymorphisms in the nonrecombining portion of the human Y chromosome provides important insights to trace the pattern of demographic history of human populations and to help decipher paternal genetic relationships among human populations. According to the hierarchical phylogenetic nomenclature of the Y chromosome tree reported by Underhill et al. (2001) and updated by the Y Chromosome Consortium (2002) and Karafet et al. (2008), the E major haplogroup was observed in Africa, Europe, and the Near East (Semino et al. 2004). The most widespread and frequent cluster among its clades was E1b1b1-M35 at this level of molecular resolution, and the main derivative haplogroups were E1b1b1a-M78 in eastern Africa, E1b1b1c-M123 in the Near East, and E1b1b1b-M81 among male lineages in northwestern Africa (Semino et al. 2004). The E1b1b1b-M81 haplogroup can be further characterized by unique event polymorphism markers E1b1b1b*, E1b1b1b1 given by M107, and E1b1b1b2 given by M183; at this level we can find E1b1b1b2* and E1b1b1b2a given by M165 (Figure 1). The Berber-speaking ethnic group represents the ancient inhabitants of northwestern Africa. In Morocco, Berber groups speak several dialects; the three main spoken dialects are Tarifit, Tamazight, and Tachelhit, respectively distributed in the northern, central, and southern parts of the country. To further describe the Y-chromosomal E1b1b1b-M81 haplogroup and its subclades among [End Page 105] Moroccan Berber-speaking groups, we typed four biallelic polymorphisms, M81, M107, M183, and M165, in samples of the Berber-speaking groups collected from different regions of Morocco. We also give an overview of the distribution of this lineage in the Mediterranean area in relation to historical events. Click for larger view View full resolution Figure 1. Phylogeny of E1b1b1b-M81 haplogroup. Subjects and Methods Subjects We analyzed samples of 295 unrelated Moroccan men who spoke Berber as a first dialect: 43 men from the northern (Rif) region who spoke Tarifit, 187 men from the central region who spoke Tamazight, and 65 men from the southern region who spoke Tachelhit (Figure 2). These samples were collected from individuals who reported that their four grandparents lived in the same geographic location. DNA was extracted from fresh peripheral blood by standard phenol-chloroform protocols. Appropriate informed consent was obtained from all participants in this study. This study was carried out after obtaining permission from the Ministry of Health of Morocco. Click for larger view View full resolution Figure 2. Geographic locations of the populations studied. Biallelic Polymorphisms Typing All biallelic polymorphisms were amplified by PCR using the primers described by Underhill et al. (2001) (Table 1). The standard PCR protocol was modified to give a final volume of 25 µL; the composition of the reaction mix was the same for all reactions: 400 nM of each primer, 200 µM dNTP, 1 U Taq DNA polymerase, 100 ng nuclear DNA, 1.5 mmol/L MgCl2, and 2.5 µL 10× PCR buffer. The M81, M107, and M165 markers were analyzed by restriction fragment length polymorphism using the restriction enzymes HpyCH4IV (ACG...

Single-tube colony PCR for DNA amplification and transformant screening of oleaginous microalgae

Recently, several colony PCR methods have been developed to simplify DNA isolation procedure and facilitate PCR-based colony screening efforts in microalgae. A main drawback of current protocols is that cell collection, disruption, and genomic DNA extraction are required preceding the PCR step, making the colony PCR process laborious and costly. In the present study, we have developed a novel procedure that eliminates any steps of DNA extraction and allows the colony screening to be performed in a single PCR tube: algal cells (as low as 5,000) from agar plates or liquid cultures were directly transferred into a PCR tube containing 2× PCR buffer and boiled for 5–10 min depending on different algal strains, followed by addition of other PCR components (dNTPs, primers, and polymerase) and then subjected to conventional PCR reaction. The procedure documented here worked well not only for the model alga Chlamydomonas reinhardtii, but also for the thick-walled oleaginous strains such as Chlorella, Haematococcus, Nannochloropsis, and Scenedesmus with its efficacy independent on amplicon sizes and primer pairs. In addition, screening of Chlorella zofingiensis transformants was achieved using this method. Collectively, our single-tube colony PCR is a much simpler and more cost-effective procedure as compared to those previously reported and has broad applications including gene cloning, strain determination, and high-throughput screening of algae colonies and transformants for biomass and biofuel production.

PM 7/117 (1) Hymenoscyphus pseudoalbidus

Specific scopeThis Standard describes a diagnostic protocol for Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea).Specific approval and amendmentApproved as an EPPO Standard in 2013‐09.

Isothermal Strand-Displacement Polymerase Reaction for Visual Detection of the Southeast Asian–Type Deletion of α-Thalassemia

A rapid and reliable screening test for thalassemia carrier couples is the most effective strategy to decrease the risk of conceiving fetuses with severe thalassemia. We present an approach based on the isothermal strand-displacement polymerase reaction and the use of a lateral flow strip for the visual detection of an α-thalassemia Southeast Asian-type deletion. This assay was used to evaluate 86 clinical samples (72 cases of Southeast Asian-type deletions and 14 cases of other types of thalassemia), and the results obtained were 100% consistent with those obtained using conventional gap-PCR. The approach thus provides a simple, sensitive, rapid, and cost-effective method for the diagnosis of thalassemia genotypes.