Successful PCR Amplification Research Articles

Quantitative and qualitative analysis of three DNA extraction methods from soybean, maize, and canola oils and investigation of the presence of genetically modified organisms (GMOs)

The objective of this study was to develop a DNA-based method for the identification and tracking of edible oils, which is important for health management. Three different DNA extraction methods (CTAB, MBST kit, and manual hexane-based method) were used to obtain high-purity DNA from crude and refined soybean, maize, and canola oils. PCR was then conducted using specific primers to identify the presence of genes related to each oil type and to assess transgenicity. The results showed that DNA was present in crude and refined oils, but in very low amounts. However, using method 3 for DNA extraction provided sufficient quantity and quality of DNA for successful PCR amplification. The study concluded that the main challenge in DNA extraction from oils is the presence of PCR inhibitors, which can be overcome using the manual hexane-based method. Also, the examination of protein presence in the oils using SDS-PAGE did not indicate any protein bands.

The quality of DNA isolated from autopsy formalin-fixed and formalin-fixed paraffin-embedded tissues: study of 1662 samples.

There are enormous formalin-fixed paraffin-embedded tissue archives and a constantly growing number of methods for molecular analyses but, the isolation of DNA from this tissue is still challenging due to the damaging effect of formalin on DNA. To determine the extent to which DNA purity, yield and integrity depend on the process of fixation in formalin, and to what extent on the process of tissue paraffin embedding, we compared the quality of DNA isolated from fixed tissues and DNA isolated from tissues embedded in paraffin blocks after fixation. Heart, liver and brain tissues obtained from healthy people who suddenly died a violent death were fixed in 10% buffered formalin as well as in 4% unbuffered formalin for 6h, 1-7days (every 24h), 10, 14, 28days and 2months. Additionally, the same tissues were fixed in 4% unbuffered formalin embedded in a paraffin block and stored from a few months to 30years. The yield and purity of the DNA samples isolated from these tissues were measured using spectrophotometry. PCR amplification of the hTERT gene was performed to evaluate the degree of DNA fragmentation. Although the purity of the DNA isolated from almost all tissue samples was satisfactory, the DNA yields changed significantly. There was a decrease in successful PCR amplification of the hTERT gene in DNA samples isolated from tissue fixed in buffered and unbuffered formalin for up to 2months from 100% to 8.3%. Archiving the tissue in paraffin blocks for up to 30years also impacts the integrity of DNA, so there was a decrease in PCR amplification of the hTERT gene from 91% success to 3%. The largest decrease in DNA yield was observed after tissue formalin fixation after 14days of fixation in buffered and unbuffered formalin. DNA integrity depends on the time of tissue formalin fixation, especially after 6days for tissue fixed in unbuffered formalin, while for tissue fixed in buffered formalin the time is prolonged up to 28days. The age of paraffin blocks also impacted DNA integrity, after 1year and 16years of archiving the paraffin blocks of tissues, there was a decrease in the success of PCR amplification.

Sex-typing of ingested human blood meal in Anopheles stephensi mosquito based on the amelogenin gene.

Identifying the sex of human hosts of insect disease vectors, using PCR amplification of the amelogenin gene (AMEL) from the ingested blood meal is an increasingly useful technique for epidemiological studies of vector-borne diseases, as well as within the criminal justice system. Detection of DNA from ingested blood is influenced by the choice of DNA extraction method, genomic target region, type and length of PCR, and rate of degradation in the DNA samples over time. Here, we have tested two types of PCR (i.e. conventional and nested), producing differently-sized PCR products, in time-course assays targeting the human AMEL gene in Anopheles stephensi mosquitoes that were fed on human male and female blood. The fed female mosquitoes were allowed to digest at 28°C for times ranging from 0 to 120h. Three AMEL primer pairs were used to amplify three sequences that were 977, 539, and 106 bp for the X chromosome and 790, 355, and 112 bp for Y. We found that time since feeding had a significant negative effect on the success of PCR amplification. The shortest fragments (106 and 112 bp) were amplified for the longest time after blood feeding (up to 60h), whereas the medium and longest loci were not amplified by conventional PCR even at 0h. However, the nested PCR protocol, targeting the medium sequence, could detect small amounts of human DNA up to 36h (1.5 days) after the blood meal. The shortest PCR assay standardized herein successfully detected small amounts of human DNA in female mosquitoes up to 60h after the blood meal. This assay represents a promising tool for identifying the sex of the human host from the blood meal in field-collected female mosquitoes.

A simple alkali lysis method for Plasmodium falciparum DNA extraction from filter paper blood samples

A fast, simple, easy, efficient, and inexpensive method for DNA extraction from malaria parasites collected on filter paper would be very useful for molecular surveillance. The quality and quantity of DNA are critical to molecular diagnosis and analysis. Here, we developed a simple alkali lysis method for DNA extraction from blood samples on filter paper. The results showed that 10–50 mM NaOH and deionized water all effectively isolated parasite DNA at higher parasitemia, as witnessed by successful PCR amplification, while at a parasitemia of 0.01%, the 10 mM NaOH lysis condition generated the best results. Furthermore, DNA extracted by this method was successfully used to amplify a fragment of > 2000 bp. This method successfully extracted DNA from 1 µl of blood at a parasitemia as low as 0.0001% (equivalent to 5 parasites /µl). The DNA isolated by the 10 mM NaOH lysis method was stable to yield PCR products after storage at 4 °C or − 20 °C for 12 months. These results indicate that this alkali lysis method is simple, effective, sensitive, and inexpensive for isolating stable Plasmodium DNA from dried blood spots on filter paper.

Effects of storage conditions and digestion time on DNA amplification of biting midge (Culicoides) blood meals

BackgroundMolecular analysis of blood meals is increasingly used to identify the hosts of biting insects such as midges and mosquitoes. Successful host identification depends on the availability of sufficient host DNA template for PCR amplification, making it important to understand how amplification success changes under different storage conditions and with different durations of blood meal digestion within the insect gut before being placed into the storage medium.MethodWe characterised and compared the digestion profile of two species of Culicoides over a 96-h period using a novel set of general vertebrate primers targeting the 16S rRNA gene. A set number of individuals from each species were killed over 13 time points post-blood feeding and preserved in 95% ethanol. Samples were stored either at ambient room temperature or in a − 20 °C freezer to examine the effect of storage condition on the PCR amplification success of host DNA.ResultsWe found that amplification success across the 96-h sampling period post-feeding was reduced from 96 to 6% and 96% to 14% for Culicoides nubeculosus and Culicoides sonorensis, respectively. We found no effect of storage condition on PCR amplification success, and storage in 95% ethanol was sufficient to maintain high rates of amplifiable host DNA for at least 9 months, even at room temperature.ConclusionsThese findings highlight the limited time frame during which an individual may contain amplifiable host DNA and demonstrate the importance of timely sample capture and processing post-blood feeding. Moreover, storage in 95% ethanol alone is sufficient to limit host DNA degradation. These results are relevant to the design of studies investigating the biting behaviour and disease transmission potential of Culicoides and other biting Diptera.Graphical

Rapid and inexpensive method of PCR ready DNA isolation from human peripheral blood and saliva

BackgroundThe isolation of nucleic acids is a frequently performed procedure in the molecular biology area. Although several rapid DNA isolation techniques from human peripheral blood and saliva have been developed, there are still some disadvantages – volume, time, cost, and yield are a few notable ones. ObjectiveWe aim to develop a rapid and inexpensive method to isolate high-molecular-weight genomic DNA from human peripheral blood and saliva that can be used for molecular biology experiments. MethodsFive DNA isolation methods with slightly varying protocols were used. High-quality DNA obtained from one specific method was further amplified by PCR and the template with good amplification was further used for performing RFLP and sequencing. ResultsOut of 5 different isolation methods (R1 to R5), DNA obtained from the R4 was of good quality (molecular weight is > 10 kb and 260/280 ratio is 1.89 ± 0.2), which allows successful PCR amplification and good separation in Restriction Fragment Length Polymorphism analysis. Sequencing by the Sanger Sequencing produced a good readable sequence of an amplified fragment from Method R4 DNA. ConclusionIn the present study we have developed a simple, rapid, and cost-effective DNA isolation method, which uses low sample volume and yields good quantity and high-quality product. The DNA obtained is highly fit for molecular genetics research applications.

Genome-wide mining of potentially-hypervariable microsatellites and validation of markers in Momordica charantia L.

Relatively large number of bitter melon microsatellite markers have been reported; however, only few resulted in successful PCR amplification and a small fraction shown polymorphisms. This limited chance of recovering polymorphic markers makes the primer screening a cost-demanding process. To test the hypothesis that microsatellites with longer motifs as well as shorter motifs repeated substantially shall have better prospects to be polymorphic, we performed a genome-wide microsatellite mining. We selected a sample of genome-wide microsatellites with prescribed motif lengths or satisfying a target repeat number, which were considered potentially-hyper variable, for primer designing and validation. Seventy five microsatellites satisfying these criteria were identified, of which 69 were validated through successful PCR amplification. Among them, 40 (53.33% of the markers identified) were polymorphic. This result showed a significantly higher success compared to our initial results of 51 (20.64%) polymorphic markers out of the 188 amplified when 247 previously reported markers were screened. The screening of two cultivars revealed that markers were efficient to identify up to three alleles. The characterization of these 69 new markers with 247 markers previously reported showed that di-nucleotide motifs were most abundant, followed by tri- and tetra-nucleotide motifs. TC motif markers were most polymorphic (12.08%) followed by AG and CT motifs (both 9.89%). Similarly, AGA (6.59%) and TATT (3.29%) were most polymorphic among the tri- and tetra-nucleotide motifs. These 69 hypervariable microsatellite markers along with 188 markers initially validated in this study shall be useful for phylogenetic analyses, studies of linkage, QTL, and association mapping in bitter melon.

Biodiversity of South Indian tea clones with detection of plant-based adulterants in tea dust using DNA barcoding

Tea is by and large a highly penetrated product in south India. Hence the adulteration risk in tea dust gets hiked in the markets. We constructed a standard database using plant plastid markers (rbcL, matK, trnH-psbA, rpoC, rpoB, ycf 1) and nuclear (ITS2) locus from prominent south Indian tea clones representing Assam, China, and Cambod varieties. These barcodes were used as reference algorithm to investigate the authenticity of 10 sampled commercial tea dust by recovering its DNA barcodes using rbcL, matK, and ITS2 loci. PCR amplification success, sequencing efficiency, genetic polymorphisms, BLAST search, and phylogenetic analysis were performed to enhance genotypic information on south tea cultivars and in authenticating the commercial samples of Camellia sinensis. Findings suggest that the chloroplast and nuclear loci can identify tea plant at the genus and varietal level respectively and rbcL as the potential marker for detecting plant-based admixtures coupled with TA cloning after DNA barcoding.

Optimized methodology for obtention of high-yield and -quality RNA from the mycelium of the bioluminescent fungus Neonothopanus gardneri

Neonothopanus gardneri, also known as coconut flower mushroom (flor-de-coco), is a Brazilian bioluminescent basidiomycete found in Palm Forest, a transitional biome between the Amazonian Forest and Caatinga (Savanna-like vegetation) in Northeast Brazil, especially in Piauí State. Recent advances toward the elucidation of fungal bioluminescence have contributed to the discovery of four genes (hisps, h3h, luz and cph) involved with the bioluminescence process, the so-called Caffeic Acid Cycle (CAC) and to develop biotechnological applications such autoluminescent tobacco plants and luciferase-based reporter genes. High-yield and -quality RNA-extraction methods are required for most of these purposes. Herein, four methods for RNA isolation from the mycelium of N. gardneri were evaluated: RNeasy® kit (QIAGEN), TRI+, TRI18G+, and TRI26G+. Highest RNA yield was observed for TRI18G+ and TRI26G+ methods, an increase of ~130% in comparison to the RNeasy® method and of ~40% to the TRI+ protocol. All the RNA samples showed good purity and integrity, except by gDNA contamination in RNA samples produced with the RNeasy® method. High quality of RNA samples was confirmed by successful cDNA synthesis and PCR amplification of the coding sequence of h3h gene, responsible for the hydroxylation of the precursor of fungal luciferin (3-hydroxyhispidin). Similarly, RT-qPCR amplification of ef-tu gene, related to the protein biosynthesis in the cell, was demonstrated from RNA samples. This is the first report of a reproducible, time-saving and low-cost optimized method for isolation of high-quality and -yield, DNA-free RNA from a bioluminescent fungus, but that can also be useful for other basidiomycetes.

Calcium-Alginate Tissue Gels (CATG): Proof-of-concept biomaterial development

Hydrogels are desirable materials to the field of forensic science and offer many advantages for use as tissue simulants in research and training scenarios. In this work, we demonstrate a proof-of-concept study for our biomaterial described as the Calcium-Alginate Tissue Gel (CATG). CATG biomaterials integrate functional DNA strands designed to amplify with known human primer sets for genetic profiling. Our range of CATG materials demonstrate successful DNA extraction, PCR amplification and genotyping when both fresh and aged for 21 days. The rheological properties of the CATGs were measured and the incorporation of DNA into the CATGs was assessed. Overall, the CATGs demonstrated increased viscoelastic behavior with the addition of DNA. In addition, two methods of sampling were considered, where it was found that cutting a sample of the dried CATG produced higher allele peak heights in the genotype compared to swabbing. Overall, our CATG biomaterials can be designed for multiple applications in forensic science with tunable properties for various training and research needs.

From Sampling to Sequencing: A Liquid Biopsy Pre-Analytic Workflow to Maximize Multi-Layer Genomic Information from a Single Tube.

Simple SummaryLiquid biopsies seek to isolate tumor derived genetic material that circulates in blood plasma or cerebrospinal fluid. The less-invasive character of liquid biopsies combined with the option for serial analyses bears enormous potential for treatment monitoring or surveillance. We aimed to establish robust sampling protocols and pre-analytical workflows to allow for site independent multi-layer liquid biopsy testing. For an optimal usage of precious material, we explored sample stabilization in various conservation tubes and describe a protocol for the parallel isolation of cell-free DNA and RNA. Quantification and quality control steps were optimized for minimal sample use with both high sensitivity and reproducibility. We provide detailed step-by-step information on how to i) choose the best-suited protocol and ii) implement this in the liquid biopsy workflow. We believe that our study has potential to increase comparability of liquid biopsy approaches to bring these one step closer to routine clinical application.Liquid biopsies hold great promise for the management of cancer. Reliable liquid biopsy data depend on stable and reproducible pre-analytical protocols that comply with quality measures, irrespective of the sampling and processing site. We established a workflow for plasma preservation, followed by processing, cell-free nucleic acid isolation, quantification, and enrichment of potentially tumor-derived cell-free DNA and RNA. Employing the same input material for a direct comparison of different kits and protocols allowed us to formulate unbiased recommendations for sample collection, storage, and processing. The presented workflow integrates the stabilization in Norgen, PAX, or Streck tubes and subsequent parallel isolation of cell-free DNA and RNA with NucleoSnap and NucleoSpin. Qubit, Bioanalyzer, and TapeStation quantification and quality control steps were optimized for minimal sample use and high sensitivity and reproducibility. We show the efficiency of the proposed workflow by successful droplet digital PCR amplification of both cell-free DNA and RNA and by detection of tumor-specific alterations in low-coverage whole-genome sequencing and DNA methylation profiling of plasma-derived cell-free DNA. For the first time, we demonstrated successful parallel extraction of cell-free DNA and RNA from plasma samples. This workflow paves the road towards multi-layer genomic analysis from one single liquid biopsy sample.

Assessment of DNA Integrity From Trap-Captured Boll Weevil (Coleoptera: Curculionidae) for Use in a New PCR-Based Diagnostic Tool.

The boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is a major pest of commercial cotton (Gossypium hirsutum) in the southern United States and throughout Central and South America. Efforts are underway to develop a PCR-based diagnostic tool that can be used to rapidly and accurately differentiate boll weevils from other weevil species that are commonly captured in pheromone traps. However, the quantity and integrity of weevil DNA must be sufficient for a successful PCR assay. Currently, active eradication programs service traps weekly, but post-eradication programs service traps at 2- or 3-wk intervals. Consequently, captured weevils may be dead, dismembered, and exposed to environmental conditions for prolonged periods which may adversely affect the quantity and quality of weevil DNA. We documented DNA quantity and integrity in boll weevils and weevil body parts aged in traps over a 3-wk period under field conditions. The quantity of DNA extracted from whole weevils, heads, abdomens, and legs generally remained sufficient (> 1 ng/μl) for successful PCR amplification throughout the 21-d period. The integrity (fragment length) of extracted DNA declined over time but generally was sufficient (> 700 bp) for successful amplification. PCR amplification of three marker genes validated that the quality and integrity of DNA extracted from dead weevils and individual weevil body parts aged in traps up to 21 d remained at sufficient levels for the PCR-based assay. However, our data also suggested that rain events may accelerate degradation of weevil DNA.

Prediction of PCR amplification from primer and template sequences using recurrent neural network

We have developed a novel method to predict the success of PCR amplification for a specific primer set and DNA template based on the relationship between the primer sequence and the template. To perform the prediction using a recurrent neural network, the usual double-stranded formation between the primer and template nucleotide sequences was herein expressed as a five-lettered word. The set of words (pseudo-sentences) was placed to indicate the success or failure of PCR targeted to learn recurrent neural network (RNN). After learning pseudo-sentences, RNN predicted PCR results from pseudo-sentences which were created by primer and template sequences with 70% accuracy. These results suggest that PCR results could be predicted using learned RNN and the trained RNN could be used as a replacement for preliminary PCR experimentation. This is the first report which utilized the application of neural network for primer design and prediction of PCR results.

Genotypic Detection of Fish-Borne Zoonotic Trematodes Using the Hotshot DNA Extraction Method

Efficient DNA extraction is a keystone in molecular assays. A wide range of methods is used to extract DNA from biological materials. These methods vary in their efficiency, economic costs, and availability. Therefore, the present study was conducted to assess the efficiency of a Hotshot method for DNA extraction from minute fish trematodes in comparison to a commercial kit. A total of 68 samples were examined. Samples included 29 encysted metacercariae from the African catfish(Clarias gariepinus) collected from the Nile tributaries in Ismailia Governorate, and 39 adult trematodes collected from rats after being experimentally infected with encysted metacercariae from fish samples. Helminths were morphologically identified where they belonged to family Cyathocotylidae, species Mesostephanus burmanicus, Prohemistomum vivax, and Mesostephanus appendiculatus. The DNA yield by the Hotshot method was compared to the commercial kit. The hotshot method produced 28% higher DNA yield per sample (mean ± SE: 2.65 ± 0.83 µg) than the commercial kit (2.07 ± 0.16 µg), but the difference was not significant (Mann-Whitney U test, P>0.05). PCR amplification success of the internal transcribed spacer 2 (ITS-2) was also higher for trematode samples extracted by the Hotshot method than the commercial kit. Although the differences were not statistically significant, the Hotshot method is still rapid, cheap, and does not require special reagents or equipment for DNA extraction from minute helminths.

PCR procedures to amplify GC-rich DNA sequences of Mycobacterium bovis

Amplification of high GC content genes by PCR is a major challenge during the creation of recombinant GC-rich DNA constructs. This may be due to the difficulty in DNA denaturation or the possibility of forming secondary structures from DNA templates. Tools have been described to address the technical problems associated with the amplification of shorter sequences (<1000 bp). However, obstacles of synthesizing larger-sized GC-rich sequences by PCR continue to exist. This study aims to investigate the amplification of long and high GC content genes by PCR from the Mycobacterium bovis, a genome with GC content >60%, in comparison to amplifying a gene from the Listeria monocytogenes genome, a genome with a 37.8% GC content. Three PCR protocols were designed and experimented at various conditions with two M. bovis genes, Mb0129, a large gene of 1794 bp with 77.5% GC content, mpb83, a smaller gene of 663 bp in length with moderate GC content of 63%, together with LMHCC_RS00060, a large L. monocytogenes gene of 1617 bp with a lower GC content of 41.5%. The result demonstrated the superiority of the 2-step PCR protocol over other protocols in PCR amplification of Mb0129 when specific high fidelity DNA polymerases were used in the presence of an enhancer. The study highlighted the importance of manipulating the cycling conditions to perform the annealing and extension steps at higher temperatures while adjusting the ramp speed at a lower speed for a successful PCR amplification of a large GC-rich DNA template. A final PCR protocol was developed and enabled the amplification of 51 GC-rich targets. This can be a valuable tool for the amplification of long GC-rich DNA sequences for various downstream applications.

At the crossroads of botanical collections and molecular genetics laboratory: a preliminary study of obtaining amplifiable DNA from moss herbarium material.

BackgroundResearch focused on extreme environments is often associated with difficulties in obtaining fresh plant material. Herbaria may provide great support as they house large collections of specimens from different parts of the world. Accordingly, there is also a growing interest in methods using herbarium specimens in molecular studies. Much of the literature on herbarium DNA is aimed to improve extraction and PCR amplification and is focused mostly on vascular plants. Here, I provide a brief study of DNA extraction efficiency from moss herbarium specimens, emphasizing the importance of herbaria as an invaluable source of material from hard-to-access geographical areas, such as the Antarctic region.MethodsThe presented study is based on herbarium collections of 25 moss species collected in the austral polar regions between 1979 and 2013. The majority of samples were obtained using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The remaining, smaller part was extracted using an adapted CTAB-based approach. The performance of DNA extraction methods in terms of PCR amplification success was measured by testing several DNA fragments of various size. Furthermore, in order to estimate of DNA fragmentation level, an automated on-chip electrophoresis system was used.ResultsResults reveal that DNA purity and the length of the target genetic region are the fundamental agents which drive the successful PCR reaction. Conversely, the DNA yield and specimen age seem to be less relevant. With this study, I present also an optimized CTAB-based approach which may effectively suppress inhibitors in the herbarium DNA. This method can be considered a cheaper alternative to column-based technology, particularly useful for dealing with a large number of samples. Results of this study confirmed previous reports and contribute to filling the existing gap in molecular analyses which involve the use of herbarium collections of mosses.

Molecular identification of dried squid products sold in China using DNA barcoding and SYBR green real time PCR

ABSTRACT Dried squid products are popular in China as a snack food, side dishes, or refreshments, and the market appeal can be reflected by the high price that occasionally reaches 497 RMB per kg. However, the absence of harmonisation around the definition of squid, as well as the problems with visual inspection for processed seafood products, make alternative species substitution for dried squid products a frequent occurrence. The aim of the present study was to apply a DNA barcoding approach for species identification of 48 dried squid products collected from the largest online shopping platform in China. Moreover, we also developed a novel SYBR green real-time PCR assay (simplex and duplex followed by a melting curve analysis) specific for Illex argentinus and Todarodes pacificus based on cytochrome C oxidase subunit I (COI) gene. Results highlighted the successful DNA extraction and PCR amplification of a 655 bp COI gene fragment from all products. A maximum similarity value in the range of 98-100% was obtained for all readable sequences using the BOLD and BLAST public databases and four species (Dosidicus gigas, Uroteuthis edulis, I. argentinus, and T. pacificus) were identified. The specificity of the designed primer sets was confirmed against 23 non-target species, and the newly developed methods were successfully applied to screen I. argentinus and T. pacificus in dried squid products. Overall, DNA barcoding is a robust tool for seafood species identification and the novel method is effective in screening I. argentinus and T. pacificus in food products.

Efficient Method for Molecular Characterization of the 5' and 3' Ends of the Dengue Virus Genome.

Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 5′ and 3′ ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 5′ and 3′ ends of dengue virus types 1 to 4. The 5′ and 3′ ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 5′ end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 3′ end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 5′ and 3′ ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods.

RNA isolation efficacy of commercial and modified conventional methods for Citrus tristeza virus and mRNA internal control amplification

High quality RNA is critical for molecular and viral diagnostic applications. In case of citrus, polyphenol and polysaccharide rich content hinders many RNA isolation methods causing low quality RNA. These shortcomings can affect the Citrus tristeza virus (CTV) detection which is responsible to cause worldwide destruction of citrus orchards. Thus, in this study we analysed the RNA quality, quantity and integrity of different commercial and conventional methods. Commercial methods include TRIzol and kit-based method (PureLink RNA kit), while Cetyl trimethylammonium bromide (CTAB) and SDS/ Phenol (TENS-PCI) based method were optimized as conventional methods. For further validation, amplification potential was evaluated by performing Reverse transcriptase polymerase chain reaction (RT PCR) of CTV coat protein (p25) and mRNA internal control (nad5) genes to affirm RNA quality. Results indicated poor RNA recovery from TRIzol method with no successful amplification at RT PCR. Contrastingly, the modified conventional methods indicated RNA quality and purity for nucleic acids at Absorbance 260/280 (1.8–2.1) and Absorbance 260/230 (1.8–2.2) proving drastic improvement in RNA quality with reproducible amplification results. These results were further demonstrated with stored samples of different citrus varieties which indicated poor RNA integrity but successful RT PCR amplification. Consequently, our results may provide a brief comparison between isolation procedures by providing reference values to determine RNA purity requirements prior to RT PCR and help to determine rapidity, reproducibility and yield for each isolation method.

DNA Yields and PCR Amplification Success Using Degraded Animal Corpses

A recurring problem with ancient specimens from wildlife animals is that their preserved tissues contain small amounts of DNA in a degraded state. The specific objectives of the present study were: (1) To determine DNA yields from different animal tissues; (2) to compare traditional (manual) DNA extraction protocols with commercial procedures and (3) to assess the success of PCR amplification of Inter-Simple Sequence Repeats (ISSR) loci in degraded animal samples. Liver, stomach and muscle samples were extracted from coyote (Canis latrans) and long-tailed weasel (Mustela frenata) for this research. Manual protocols for DNA extraction were compared to a commercial kit procedure (Qiagen DNeasy kit). Genomic DNA in different states (intact, apoptotic and degraded) were amplified using a panel of ISSR primers. No DNA was recovered from coyote stomach samples using the manual extraction protocol. DNA concentrations in stomach and liver samples from coyote were 10.31 ng/μL and 15.8 ng/μL, respectively using the Qiagen extraction kit. In general, the kit extraction method yielded more DNA than the manual extraction procedure but it is more expensive. Intact and apoptotic genomic DNA were successfully amplified by PCR resulting in a similar profile. Artificially degraded DNA showed partial amplification. Thus, the ISSR marker system is suitable for animal population genetics when only limited and/or degraded animal DNA is available.