Direct PCR Approach Research Articles

Diversity and identification of fungi associated with needles of Pinus radiata in Tasmania

Needle fungi have been extensively studied in conifers but rarely in Pinus radiata. Previous studies of P. radiata have been based on fungal isolation and not direct PCR detection from needles. This research was a component of a study examining factors linked to spring needle cast (SNC) in Tasmanian P. radiata plantations and aimed to identify as many as possible of the fungal species commonly associated with P. radiata needles in Tasmanian plantations. Needle samples were collected from 13 sites representative of the range of sites in which P. radiata is grown in Tasmania. Fungi were detected by a direct PCR approach and identified using barcode sequences from a reference collection of isolates from Tasmania, mainland Australia and New Zealand as well as sequences from the international nucleotide sequence databases. The total number of molecular operational taxonomic units (MOTUs) was 152, with 127 detected by direct PCR and sequencing, and only 35 operational taxonomic units (OTUs) isolated by culture-based methods. Dothideomycetes was the most diverse class detected in this study, with many MOTUs detected by direct PCR and not isolated. Leotiomycetes was the second-most diverse class and Sordariomycetes third, with several OTUs frequently isolated but rarely or not at all detected by direct PCR. DNA sequence data facilitated the discrimination and identification of OTUs, but some effort was required to avoid confusion caused by poorly identified isolates in public DNA databases. This is the most comprehensive study yet of fungi associated with pine needles in Australia and highlights the hitherto unrecognised diversity of Teratosphaeriaceae in P. radiata.

The end of bad hair days

DNA typing of a single hair using conventional DNA extraction processes can often result in no DNA profile but here we describe augmenting the direct-PCR approach resulting in an increased number of alleles being generated. The process includes the use of Prep-n-Go™ buffer prior to direct PCR using the recently released GlobalFiler® STR kit. Up-loadable DNA profiles were generated from all anagen hairs tested. Telogen hairs using Prep-n-Go™ resulted in 36% of the hairs generating up-loadable DNA profiles; this compared with 25% if no Pre-n-Go™ was used in the direct PCR. The addition of this step to the direct PCR of single hairs, both anagen and telogen, resulted in improved success in generating alleles using GlobalFiler®.

Direct PCR Improves the Recovery of DNA from Various Substrates.

This study reports on the comparison of a standard extraction process with the direct PCR approach of processing low-level DNA swabs typical in forensic investigations. Varying concentrations of control DNA were deposited onto three commonly encountered substrates, brass, plastic, and glass, left to dry, and swabbed using premoistened DNA-free nylon FLOQswabs(™) . Swabs (n=90) were either processed using the DNA IQ(™) kit or, for direct PCR, swab fibers (~2mm(2) ) were added directly to the PCR with no prior extraction. A significant increase in the height of the alleles (p<0.005) was observed when using the direct PCR approach over the extraction methodology when controlling for surface type and mass of DNA deposited. The findings indicate the potential use of direct PCR for increasing the PCR product obtained from low-template DNA samples in addition to minimizing contamination and saving resources.

Internal transcribed spacer (ITS) evolution in populations of the hyperparasitic European mistletoe pathogen fungus, Sphaeropsis visci (Botryosphaeriaceae): The utility of ITS2 secondary structures

We investigated patterns of nucleotide polymorphism in the internal transcribed spacer (ITS) region for Sphaeropsis visci, a hyperparasitic fungus that causes the leaf spot disease of the hemiparasite European mistletoe (Viscum album). Samples of S. visci were obtained from Hungary covering all major infected forest areas. For obtaining PCR products we used a fast and efficient direct PCR approach based on a high fidelity DNA polymerase. A total of 140 ITS sequences were subjected to an array of complementary sequence analyses, which included analyses of secondary structure stability, nucleotide polymorphism patterns, GC content, and presence of conserved motifs. Analysed sequences exhibited features of functional rRNAs. Overall, polymorphism was observed within less conserved motifs, such as loops and bulges, or, alternatively, as non-canonical G–U pairs within conserved regions of double stranded helices. The secondary structure of ITS2 provides new opportunities for obtaining further valuable information, which could be used in phylogenetic analyses, or at population level as demonstrated in our study. This is due to additional information provided by secondary structures and their models. The combined score matrix was used with the methods implemented in the programme 4SALE. Besides the pseudoprotein coding method of 4SALE, the molecular morphometric character coding also has potential for gaining further information for phylogenetic analyses based on the geometric features of the sub-structural elements of the ITS2 RNA transcript.

IDENTIFIKASI JAMUR ENDOFIT DAUN JARUM Pinus radiata MENGGUNAKAN METODE EKTRAKSI DNA SECARA LANGSUNG

Pinus radiata is the major softwood species planted in New Zealand and Australia. Endophyte fungi that have been extensively studied in conifers needles mainly based on fungal isolation and also rarely conducted in Pinus radiata. This study aimed to observe the optimum condition of P. radiata needles sample for direct extraction of fungal DNA and to identify the fungi associated with P. radiata needles in Plenty plantations by a direct PCR approach. Room temperature dried needles produced clear and consistent DNA amplification compare to fresh needles, and needles dried on drying room or drying machine. Capnodiales was the most diverse group and no fungal pathogen detected in this study. DNA sequence data from direct DNA extraction of P. radiata needles provided sufficient discrimination and identification of fungal species.

Genetic profiling from challenging samples: Direct PCR of touch DNA

We report on a novel direct PCR approach for retrieving touch DNA from handled items. Trace levels of DNA on handled items can be difficult to recover and analyse using standard methodology. The lack of a full nuclear STR DNA profile may be related to insufficient DNA being present initially, the presence of PCR inhibitors, or the loss of nucleic acid during sample collection and processing. The aim of this study was to optimise the swabbing method used to collect DNA and to improve the success rate of STR-based DNA profiling from touched items. A comparison was carried out to evaluate the use of three common swab types, nylon, cotton and foam at retrieving and releasing template DNA for amplification by direct PCR. To test the effectiveness of our method at processing low levels of DNA, complete STR DNA profiles were obtained from fingerprints deposited onto sterile plastic slides, which were swabbed and subjected to direct PCR. Our findings indicate the potential use of direct PCR when analysing samples that contain limited DNA.

Direct multiplex PCR for grapevine genotyping and varietal identification

Grapevine cultivar identification is based mainly on two complementary methodologies: microsatellite (simple sequence repeat (SSR)) DNA analysis and traditional ampelography. Here, we report a direct multiplex PCR approach that allows the simultaneous amplification of 11 SSRlocifrom crude samples, i.e. bypassing DNA extraction, by using an engineered DNA polymerase improved to tolerate plant PCR inhibitors. Many different plant tissues were successfully amplified: leaf, root, wood, berry flesh and skin, stalk and must, from wine and table grape varieties, and rootstocks. The direct multiplex PCR that we propose is quicker and cheaper than the methodologies used until now, and provides accurate results. Thus, SSR DNA analysis becomes economically more accessible to a larger number of potential users in addition to research institutes.

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS) is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

A Direct PCR Approach to Accelerate Analyses of Human-Associated Microbial Communities

Since the composition of the human microbiome is highly variable both within and between individuals, researchers are increasingly reliant on high-throughput molecular approaches to identify linkages between the composition of these communities and human health. While new sequencing technologies have made it increasingly feasible to analyze large numbers of human-associated samples, the extraction of DNA from samples often remains a bottleneck in the process. Here we tested a direct PCR approach using the Extract-N-Amp Plant PCR Kit to accelerate the 16S rRNA gene-based analyses of human-associated bacterial communities, directly comparing this method to a more commonly-used approach whereby DNA is first extracted and purified from samples using a series of steps prior to PCR amplification. We used both approaches on replicate samples collected from each of five body habitats (tongue surface, feces, forehead skin, underarm skin, and forearm skin) from four individuals. With the exception of the tongue samples, there were few significant differences in the estimates of taxon richness or phylogenetic diversity obtained using the two approaches. Perhaps more importantly, there were no significant differences between the methods in their ability resolve body habitat differences or inter-individual differences in bacterial community composition and the estimates of the relative abundances of individual taxa were nearly identical with the two methods. Overall, the two methods gave very similar results and the direct PCR approach is clearly advantageous for many studies exploring the diversity and composition of human-associated bacterial communities given that large numbers of samples can be processed far more quickly and efficiently.