Precipitation Of Nucleic Acids Research Articles

One-pot method for preparing DNA, RNA, and protein for multiomics analysis

Typical multiomics studies employ separate methods for DNA, RNA, and protein sample preparation, which is labor intensive, costly, and prone to sampling bias. We describe a method for preparing high-quality, sequencing-ready DNA and RNA, and either intact proteins or mass-spectrometry-ready peptides for whole proteome analysis from a single sample. This method utilizes a reversible protein tagging scheme to covalently link all proteins in a lysate to a bead-based matrix and nucleic acid precipitation and selective solubilization to yield separate pools of protein and nucleic acids. We demonstrate the utility of this method to compare the genomes, transcriptomes, and proteomes of four triple-negative breast cancer cell lines with different degrees of malignancy. These data show the involvement of both RNA and associated proteins, and protein-only dependent pathways that distinguish these cell lines. We also demonstrate the utility of this multiomics workflow for tissue analysis using mouse brain, liver, and lung tissue.

Combined biolistic and cell penetrating peptide delivery for the development of scalable intradermal DNA vaccines

Physical-based gene delivery via biolistic methods (such as the Helios gene gun) involve precipitation of nucleic acids onto microparticles and direct transfection through cell membranes of exposed tissue (e.g. skin) by high velocity acceleration. The glycosaminoglycan (GAG)-binding enhanced transduction (GET) system exploits novel fusion peptides consisting of cell-binding, nucleic acid condensing, and cell-penetrating domains, which enable enhanced transfection across multiple cell types. In this study, we combined chemical (GET) and physical (gene gun) DNA delivery systems, and hypothesized the combination would generate enhanced distribution and effective uptake in cells not initially transfected by biolistic penetration. Physicochemical characterization, optimization of bullet contents and transfection experiments in vitro in cell monolayers and engineered tissue demonstrated these formulations transfected efficiently, including DC2.4 dendritic cells. We incorporated these formulations into a biolistic format for gene gun by forming fireable dry bullets obtained via lyophilization (freeze drying). This system is simple and with enhanced scalability compared to conventional methods to generate bullets. Flushed GET bullet contents retained their ability to mediate transfection (17-fold greater and 13-fold greater reporter gene expression than standard spermidine bullets in the absence and presence of serum, respectively). Fired GET bullets in vitro (in cells and collagen gels) and in vivo (mice) showed increased reporter gene transfection compared to untreated controls, whilst maintaining cell viability in vitro and having no obvious toxicity in vivo. Lastly, a SARS-CoV-2 plasmid DNA vaccine with spike (S) protein-receptor binding domain (S-RBD) was delivered by gene gun using GET bullets. Specific T cell and antibody responses comparable to the conventional system were generated. The non-physical and physical combination of GET‑gold-DNA carriers using gene gun shows potential as an alternative DNA delivery method that is scalable for mass deployable vaccination and intradermal gene delivery.

Soil Metatranscriptomics: An Improved RNA Extraction Method Toward Functional Analysis Using Nanopore Direct RNA Sequencing

Soil microbes play an undeniable role in sustainable agriculture, plant health, and soil management. A deeper understanding of soil microbial composition and function has been gained through next-generation sequencing. Although soil metagenomics has provided valuable information about microbial diversity, issues stemming from RNA extraction, low RNA abundance in some microbial populations (e.g., viruses), and messenger RNA enrichment have slowed the progress of soil metatranscriptomics. A variety of soil RNA extraction methods have been developed thus far yet none of the available protocols can obtain RNA with high quality, purity, and yield for third-generation sequencing. The latter requires RNA with high quality and large quantities (with no or low contamination such as humic acids). Also, use of commercial kits for in-batch soil RNA extraction is quite expensive, and these commercial kits lack buffer composition details, which prevents the optimization of protocols for different soil types. An improved and cost-effective method for extracting RNAs from mineral and organic soils is presented in this article. An acidic sodium acetate buffer and phosphate buffer with modifications to bead beating and nucleic acid precipitation lead to higher RNA yields and quality. Using this method, we obtained almost DNA-free RNA. By using nanopore's direct RNA sequencing, the extracted contamination-free RNAs were successfully sequenced. Finally, taxonomic groups such as bacteria, fungi, archaea, and viruses were classified and profiled, and functional annotation of the datasets was carried out using an in-house customized bioinformatics workflow.

Monitoring COVID-19 spread in Prague local neighborhoods based on the presence of SARS-CoV-2 RNA in wastewater collected throughout the sewer network

Many reports have documented that the presence of SARS-CoV-2 RNA in the influents of municipal wastewater treatment plants (WWTP) correlates with the actual epidemic situation in a given city. However, few data have been reported thus far on measurements upstream of WWTPs, i.e. throughout the sewer network. In this study, the monitoring of the presence of SARS-CoV-2 RNA in Prague wastewater was carried out at selected locations of the Prague sewer network from August 2020 through May 2021. Various locations such as residential areas of various sizes, hospitals, city center areas, student dormitories, transportation hubs (airport, bus terminal), and commercial areas were monitored together with four of the main Prague sewers. The presence of SARS-CoV-2 RNA was determined by reverse transcription – multiplex quantitative polymerase chain reaction (RT-mqPCR) after the precipitation of nucleic acids with PEG 8,000 and RNA isolation with TRIzol™ Reagent. The number of copies of the gene encoding SARS-CoV-2 nucleocapsid (N1) per liter of wastewater was compared with the number of officially registered COVID-19 cases in Prague. Although the data obtained by sampling wastewater from the major Prague sewers were more consistent than those obtained from the small sewers, the correlation between wastewater-based and clinical-testing data was also good for the residential areas with more than 7,000 registered inhabitants. It was shown that monitoring SARS-CoV-2 RNA in wastewater sampled from small sewers could identify isolated occurrences of COVID-19-positive cases in local neighborhoods. This can be very valuable while tracking COVID-19 hotspots within large cities.

A systematic investigation of key factors of nucleic acid precipitation toward optimized DNA/RNA isolation.

Nucleic acid precipitation is important for virtually all molecular biology investigations. However, despite its crucial role, a systematic study of the influence factors of nucleic acid precipitation has not been reported. In the present work, via rational experimental design, key factors of nucleic acid precipitation, including the type of nucleic acid, temperature and time of incubation, speed and time of centrifugation, volume ratio of ethanol/isopropanol to nucleic acid solution, type of cation-containing salt solution and type of coprecipitator, were comprehensively evaluated in an attempt to maximize the efficiency of nucleic acid precipitation. Our results indicate that the optimal conditions of each influence factor of nucleic acid precipitation may vary in accordance with the chemistry, structure and length of nucleic acids.

AZF-deletion – one of the genetic causes of male infertility

The possibility of investigating the causes of infertility in men increased significantly thanks to the human genome, the sequencing of the Y-chromosome, and the introduction of modern molecular techniques in medical practice. We conducted a survey of 80 men with idiopathic infertility, which included clinical and urological examination: complete blood count, urinalysis, Wassermann, blood sugar, blood biochemical examination, hormonal examination (testosterone, LH, FSH, prolactin), karyotyping, ultrasound examination of the scrotum and prostate, microscopic and bacteriological examination of ejaculate, a study on sexually transmitted infections. 68 patients with the concentration of sperm in the ejaculate <5*106/ml conducted a genetic blood test for the presence of deletions in the AZF locus Y-chromosome. Isolation and purification of DNA from whole venous blood passes through a standard method of precipitation of nucleic acids. For the analysis of the variations of the gene regions of AZF deletions specific DNA amplification was carried out in vitro. In the study group (68 patients) in the locus AZF deletions were detected in 6 patients (8.8%). According to the world of literature and the results of his observations is obvious need for research azoospermia factor for all men with azoospermia/oligozoospermia severe.

Isolation of high quality RNA from pistachio (Pistacia vera L.) and other woody plants high in secondary metabolites.

The quality and quantity of RNA are critical for successful downstream transcriptome-based studies such as microarrays and RNA sequencing (RNA-Seq). RNA isolation from woody plants, such as Pistacia vera, with very high amounts of polyphenols and polysaccharides is an enormous challenge. Here, we describe a highly efficient protocol that overcomes the limitations posed by poor quality and low yield of isolated RNA from pistachio and various recalcitrant woody plants. The key factors that resulted in a yield of 150μg of high quality RNA per 200mg of plant tissue include the elimination of phenol from the extraction buffer, raising the concentration of β-mercaptoethanol, long time incubation at 65°C, and nucleic acid precipitation with optimized volume of NaCl and isopropyl alcohol. Also, the A260/A280 and A260/A230 of extracted RNA were about 1.9-2.1and 2.2-2.3, respectively, revealing the high purity. Since the isolated RNA passed highly stringent quality control standards for sensitive reactions, including RNA sequencing and real-time PCR, it can be considered as a reliable and cost-effective method for RNA extraction from woody plants.

Changes in nucleic acid and protein levels during in vitro germ¬nation and elongation of Lilium longifforum cv. "Ace" polleni

While changes in nucleic acid and protein levels during germination and subsequent tube elongation have been determined for a number of pollens, they have not been extensively examined for &lt;em&gt;in vitro&lt;/em&gt; grown &lt;em&gt;Lilium longiflorum&lt;/em&gt;, cv. `Ace' pollen. Nucleic acids and proteins were extracted with cold trichloroacetic acrid (TCA), cold-hot TCA or cold TCA and potassium hydroxide-perchloric acid (KOH-HClO&lt;sub&gt;4&lt;/sub&gt;). Following extraction, RNA, DNA and total protein were assayed colorimetrically with orcinol, diphenylamine and Folin-Phenol reagents, respectively. Extraction of 500 x g supernatants with KOH-HClO&lt;sub&gt;4&lt;/sub&gt;, yielded less RNA than either of the TCA-extraction procedures which gave similar nucleic acids and protein recoveries. Whereas total protein levels decreased initially and then increased during 36 h, RNA and DNA levels rose throughout the time-course. Precipitation and quaritiation of nucleic acids and protein from homogenized and soaicated 500 x g pellets resulted in time-dependent alterations in levels of macromolecules which differed from those for 500 x g supernatants. Whereas DNA and RNA levels increased and then decreased over 36 h, total protein levels remained constant for 12 h and then declined during the : next 24 h. Addition of the data obtained for 500 x g supernatants to those for 500 x g pellets revealed that total protein levels increased 2.4 times for the first 12 h and thereafter remained constant, that RNA levels increased 9.8 times for the first 12 h and then levelled off and that the DNA content rose more than 5 times over 36 h.

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Introduction: Tumor lysis syndrome (TLS) is an oncologic emergency that is caused by massive tumor cell lysis with the release of large amount of potassium, phosphate, nucleic acids. Uric acid, a catabolic product of nucleic acid precipitation in renal tubules leads to acute kidney injury. TLS occurs mostly after initiation of cytotoxic therapy in patients with high-grade lymphomas and acute leukemia. TLS can also occur spontaneously with malignancies with a high proliferative rate and a large tumor burden. There are few reported cases of spontaneous TLS with solid organ malignancy. We present a case of spontaneous TLS associated with endometrial/ovarian carcinoma. A 45 years old female presented to Upstate University Hospital with a 5 day history of lower abdominal pain and abdominal distension. Her past medical/surgical history was uneventful but had limited preventative and gynecological care. On examination, she was noted to have ascites and 12 weeks size of uterus. Her initial work up revealed leukocytosis (white blood cell count 36000/µL with 90% polymorphonuclear cells), Hyperkalemia (5.6 mmol/L), Hyperphosphatemia (11.9 mg/dl), Hyperuricemia (22 mg/dl), Hypocalcemia (Ionized calcium 0.89 mmol/L), Uremia (Blood Urea Nitrogen 94 mg/dl and Creatinine 4.2 mg/dl) and anion gap metabolic acidosis (PH 7.26, Serum bicarbonate 16 mmol/L. A 13 cm enlarged uterus, 4x3x3 cm mass over the left side of uterine fundus, left adnexal mass, grossly thickened endometrium, thickened omentum/peritoneum, bilateral pleural effusion and ascites was noted on radiological examination. Cytopathological examination from pleural and ascitic fluid and tumor marker studies revealed poorly differentiated carcinoma of gynecologic origin. She was treated with intravenous fluids, Rasburic acid, urinary alkalinization and hemodialysis for spontaneous tumor lysis syndrome. Peripheral blood flow-cytometry was negative for hematological malignancy. She was deemed unfit for cyto-reductive surgery. Palliative chemotherapy with carboplatin and Paclitaxel was initiated but she later opted for hospice care and subsequently died in hospital. Although many cases of TLS have been described after treatment of non-hematologic solid tumors; there are very few reported cases of spontaneous TLS associated with solid tumors. The identification of patients at risk for TLS and determination of the degree of the risk are the cornerstone of management of TLS. Successful management of TLS include a high index of suspicion, aggressive hydration, allopurinol, urate oxidase, urinary alkalinization and dialysis.

Optimization of the Separation Method for Bioleaching Bacteria DNA and RNA Extracted Simultaneously

DNA and RNA based analysis was a useful way to characterize microbial communities during biohydrometallurgical processes. However, feasible, affordable and efficient DNA and RNA separation methods are rarely reported although several simultaneous DNA and RNA extraction methods have been developed recently. In this study, various salts including NaCl, CaCl2 and LiCl were tested for separation of DNA and RNA. Salt concentration, nucleic acid concentration, precipitation temperature and time were optimized. The results showed that LiCl was more efficient to separate DNA and RNA than the other two salts. The optimized conditions were as follows: 1/4 volume saturated LiCl was used for precipitating RNA first at -20°C for 30 min for total nucleic acid concentration of approximately 200-400 ng/μL, and then centrifuged at 12,000×g at room temperature for 20 min to collect RNA. DNA in the supernatant was precipitated using 0.6 volume isopropanol and then collected by centrifugation at 12,000×g at room temperature for 20 min. The results indicated that DNA and RNA could be extracted from not only pure culture of Acidithiobacillus ferrooxidans(A.ferrooxidans), Acidithiobacillus caldus(A. caldus), Acidithiobacillus albertensis(A. albertensis), Leptospirillum ferrooxidans(L. ferrooxidans), Ferroplasma thermophilium(F. thermophilium), but also the acid mine drainage(AMD) water samples from Daye copper mine, Xiangxi gold mine, Axi gold mine. The resulted DNA and RNA could be amplified and A260/280 was 1.8-2.0, A260/230 was 1.8-2.2, which indicated high quality of DNA and RNA. This method could be widely used for separation of bioleaching bacteria DNA and RNA extracted simultaneously.

Improving Griffith's protocol for co-extraction of microbial DNA and RNA in adsorptive soils

Quantification of microbial gene expression is increasingly being used to study key functions in soil microbial communities, yet major limitations still exist for efficient extraction of nucleic acids, especially RNA for transcript analysis, from this complex matrix. We present an improved extraction protocol that was optimized by: i) including an adsorption-site competitor prior to cell lysis to decrease adsorption of nucleic acids to soil particles, and ii) optimizing the PEG concentration used for nucleic acid precipitation. The extraction efficiency was determined using quantitative real-time PCR on both the RNA (after conversion to cDNA) and the DNA fraction of the extracts. Non-adsorptive soils were characterized by low clay content and/or high phosphate content, whereas adsorptive soils had clay contents above 20% and/or a strong presence of divalent Ca2+ in combination with high pH. Modifications to the co-extraction protocol improved nucleic acid extraction efficiency from all adsorptive soils and were successfully validated by DGGE analysis of the indigenous community based on 16S rRNA gene and transcripts in soils representing low biomass and/or high clay content. This new approach reveals a robust co-extraction protocol for a range of molecular analysis of diverse soil environments.

Prevalence of latent alpha-herpesviruses in Thoroughbred racing horses

The objective of this study was to detect and characterize latent equine herpes virus (EHV)-1 and -4 from the submandibular (SMLN) and bronchial lymph (BLN) nodes, as well as from the trigeminal ganglia (TG) of 70 racing Thoroughbred horses submitted for necropsy following sustaining serious musculoskeletal injuries while racing. A combination of nucleic acid precipitation and pre-amplification steps was used to increase analytical sensitivity. Tissues were deemed positive for latent EHV-1 and/or -4 infection when found PCR positive for the corresponding glycoprotein B (gB) gene in the absence of detectable late structural protein gene (gB gene) mRNA. The EHV-1 genotype was also determined using a discriminatory real-time PCR assay targeting the DNA polymerase gene (ORF 30). Eighteen (25.7%) and 58 (82.8%) horses were PCR positive for the gB gene of EHV-1 and -4, respectively, in at least one of the three tissues sampled. Twelve horses were dually infected with EHV-1 and -4, two carried a latent neurotropic strain of EHV-1, six carried a non-neurotropic genotype of EHV-1 and 10 were dually infected with neurotropic and non-neurotropic EHV-1. The distribution of latent EHV-1 and -4 infection varied in the samples, with the TG found to be most commonly infected. Overall, non-neurotropic strains were more frequently detected than neurotropic strains, supporting the general consensus that non-neurotropic strains are more prevalent in horse populations, and hence the uncommon occurrence of equine herpes myeloencephalopathy.

Reducing the effects of environmental inhibition in quantitative real-time PCR detection of adenovirus and norovirus in recreational seawaters

Quantitative real-time PCR (qPCR) is used commonly to detect adenovirus (Ads) and norovirus (Nvs) in recreational waters. However, qPCR detection may be limited by interference from inhibitory substances found in recreational waters. In previous work, viruses in Avalon and Doheny Beach water samples were concentrated by electropositive cartridge filtration and PEG precipitation, and high inhibition was found in the samples when using qPCR for detection of Ads and Nvs. Therefore, different approaches were evaluated for removal or blocking of inhibitory compounds that affect qPCR. Avalon and Doheny concentrates were spiked with known amounts of Ads 41 and Nvs GII, and spiked deionized water was used as a positive control. Modifications included gel chromatography with columns of Sephadex G-200/Chelex 100, different sample volumes for nucleic acid extraction, organic solvent extraction, and nucleic acid precipitation. The efficiency of each treatment varied according to sampling location and virus type. The best option for improved Nvs detection by reverse transcription-qPCR was to reduce the sample volume for nucleic acid purification. The best option for improving Ads detection in both beach samples was Sephadex/Chelex spin column chromatography. Chloroform extraction only improved virus detection in Doheny Beach samples but not in Avalon Beach samples. Observed differences in effective treatments between viruses may be related to the different PCR targets, amplification conditions, and enzymes used in each assay, and differences between beaches may be related to differences in PCR inhibitory environmental compounds at each location. The results suggest that methods for detecting viruses from marine beaches, including treatments for the removal of PCR inhibitory compounds, should be optimized for each sampling site and probably for each virus of interest.

Characterization of a Thermophilic l-Rhamnose Isomerase from Caldicellulosiruptor saccharolyticus ATCC 43494

L-Rhamnose isomerase (EC 5.3.1.14, l-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-rhi gene encoding L-RhI was PCR-cloned from Caldicellulosiruptor saccharolyticus ATCC 43494 and then expressed in Escherichia coli. A high yield of active L-RhI, 3010 U/g of wet cells, was obtained after 20 °C induction for 20 h. The enzyme was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 90 °C. The enzyme was stable at pH values ranging from 4 to 11 and retained >90% activity after a 6 h incubation at 80 °C and pH 7-8. Compared with other previously characterized L-RhIs, the L-RhI from C. saccharolyticus ATCC 43494 has a good thermostability, the widest pH-stable range, and the highest catalytic efficiencies (k(cat)/K(M)) against L-rhamnose, L-lyxose, L-mannose, D-allose, and D-ribose, suggesting that this enzyme has the potential to be applied in rare sugar production.

The recovery of nucleic acid from biomining and acid mine drainage microorganisms

The cornerstone of biological molecular techniques is the extraction of the intra- or extra-cellular component of interest. However, inefficiencies in the extraction method can lead to results that are not representative of the microbial population under investigation. It is particularly difficult to extract clean and pure samples of the cellular component of interest from the microbial inhabitants of low-pH, sulfidic environments, such as those found in biomining or acid mine drainage (AMD). A number of nucleic acid (NA) extraction methods were tested using microorganisms commonly found in biomining and AMD environments, including archaea and Gram-negative and Gram-positive bacteria. The different stages in the methods of NA extraction were investigated separately, including the: (i) removal of cells from pyrite, (ii) cell lysis and nucleic acid extraction, (iii) nucleic acid precipitation and (iv) RNA purification using, as appropriate, microscopy, spectrophotometry, agarose gel electrophoresis of NA, PCR, quantitative-PCR and quantitative reverse transcriptase-PCR to assess the quality and quantity of the DNA and RNA. The relative percentage of NA recovered from each microorganism using the optimised method discussed in this paper returned the following percentage of NA per cell: At. ferrooxidans 91% ± 1.4%; At. caldus 91% ± 2.7%; L. ferriphilum 98% ± 1.2%; F. acidiphilum 83% ± 2.9%; and S. thermosulfidooxidans 79% ± 0.7%. Differences in lysis methods and NA precipitation greatly impacted the quality and quantity of the extracted NA. A method for the reliable, representative and reproducible extraction of NA from five strains of biomining and AMD microorganisms from pyrite and from liquid culture is described.

Characterization of a Thermophilic l-Rhamnose Isomerase from Thermoanaerobacterium saccharolyticum NTOU1

L-rhamnose isomerase (EC 5.3.1.14, L-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-Rhi gene encoding L-Rhi was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. A high yield of the active L-RhI, 9780 U/g of wet cells, was obtained in the presence of 0.2 mM IPTG induction. L-RhI was purified sequentially using heat treatment, nucleic acid precipitation, and anion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 75 °C. The enzyme was stable at pH values ranging from 5 to 9, and the activity was fully retained after a 2 h incubation at 40-70 °C. L-RhI from T. saccharolyticum NTOU1 is the most thermostable L-RhI to date, and it has a high specific activity (163 U/mg) and an acceptable purity after heat treatment, suggesting that this enzyme has the potential to be used in rare sugar production.

Prevalence of equine herpesvirus type 1 in trigeminal ganglia and submandibular lymph nodes of equids examined postmortem

The objective of this study was to detect and characterise the biovar of equine herpesvirus type 1 (EHV-1) from submandibular lymph nodes (SMLNs) and trigeminal ganglia from 153 equids undergoing...

Development and Application of a Rapid and Simple Method for Extracting Nucleic Acids from Microbes.

Rapid, efficient, and easy-to-use procedures for isolating high-quality genomic DNA or RNA of pathogenic bacteria or viruses prior to gene amplification procedures are essential for the molecular identification of biowarfare agents. In this study, we developed a rapid and simple nucleic acid extraction method for bacteria and its spore. The method is based on cell disruption using zirconia/silica beads followed by precipitation of nucleic acids with isopropanol by using a personal microcentrifuge. In spectrometric and agarose gel electrophoretic analysis, we confirmed that bacterial genomic DNA was efficiently purified from gram-negative, gram-positive bacteria and spores. We evaluated this extraction method for the application for real-time PCR-based biological detection system, R.A.P.I.D.TM. The detection sensitivities of the R.A.P.I.D.TM system to the samples prepared from Bacillus anthracis vegetative cells and spores by our extraction method were 100-fold higher than those by using the IT1-2-3TM extraction kit, which is recommended by the instruction of R.A.P.I.D.TM. In the loop-mediated isothermal amplification (LAMP) detection, which we previously developed, B. anthracis DNA in the extracts of 2×102 cfu/mL of spore suspensions could be detected. In addition, B. anthracis in 0.5% (w/v) white powder (e.g. sugar) suspension was also detected by R.A.P.I.D.TM and LAMP. The DNA extraction method we developed here is highly efficient and applicable for nucleic acid amplification techniques. In addition, this method was able to be performed with simple devices. Therefore our method was considered to be suitable for the on-site use for detection of microbiological agents.

Nucleic acid contamination of glycogen used in nucleic acid precipitation and assessment of linear polyacrylamide as an alternative co-precipitant

Molecular-grade glycogen is widely used to recover nanogram or picogram quantities of DNA and RNA across molecular biology applications in the life sciences. As a result, its purity is critical to obtain reliable results. Using agarose gel electrophoresis, we detected pg/microL (DNA) to ng/microL (RNA) concentrations of nucleic acid in two of the nine glycogen samples obtained from commercial suppliers. Denaturing gradient gel electrophoresis of 16S rRNA gene PCR-amplified products indicated that an additional two samples contained detectable contamination. We also tested a synthetic polymer co-precipitant, linear polyacrylamide (LPA); none of the four samples tested with LPA were detectably contaminated. The partial 16S rRNA gene sequence associated with the contaminated samples of the shellfish-derived glycogen was nearly identical to the sequence of Actinobacteria lwoffii, which has been isolated from mussels previously. By testing the recovery of low-nanogram amounts of DNA with multiple precipitants and simulated experimental conditions, we demonstrated that LPA was a preferable co-precipitant for sensitive protocols.

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