Efficient PCR Amplification Research Articles

B-232 Versatile, Extraction-Free Nucleic Acid Sample Preparation in 15 Seconds Via Hyperbaric Heating

Abstract Nucleic acid extraction-based PCR sample prep is currently limited by long processing times, liquid transfers, kit cost, and lab storage requirements. Extraction-free approaches enable streamlined sample prep, but lack sensitivity compared to extraction-based methods. We studied several sample types to demonstrate the effectiveness of a novel 15-second Hyperbaric Heating (HBH) process to prepare nucleic acids for PCR. HBH exposes the sample to a short duration, high temperature environment to effectively lyse organisms and denature PCR inhibitors such as nucleases in seconds. Samples are introduced and sealed in a pressure-tight metallic vessel, which is then heated in seconds using magnetic induction. Within the sealed metallic vessel, the vaporization of the aqueous sample raises the internal pressure, enabling an internal temperature higher than 100°C to be achieved. Chelating agents and other proteins in HBH also protect the target nucleic acids from fragmentation. Post HBH, the sample is ready for direct PCR amplification without further dilution or extraction. Efficacy of HBH for efficient PCR amplification was tested against commercial kits. HBH was applied to viral, bacterial, eukaryotic, and fungal organisms. For SARS-CoV-2, HBH detected down to 250 viral copies/mL. Additionally, HBH outperformed commercial kits for PCR sample prep of Candida albicans and Bacillus subtilis. HBH is a generalizable sample prep technology with sensitivity comparable to that of current extraction kits. We have applied HBH to a multitude of organisms and have demonstrated that HBH can be employed as a rapid and comprehensive nucleic acid prep approach for applications that involve PCR amplification post sample prep. Future integration of HBH in devices suitable for point of care molecular testing are underway. We also envision HBH to be a useful, generalizable sample prep method for the molecular lab in applications where cost, simplicity, and speed are essential for preparing nucleic acids for amplification.

A Study of the Detection of SARS-CoV-2 by the Use of Electrochemiluminescent Biosensor Based on Asymmetric Polymerase Chain Reaction Amplification Strategy.

A new and reliable method has been constructed for detecting severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) open reading frames 1ab (ORF1ab) gene via highly sensitive electrochemiluminescence (ECL) biosensor technology based on highly efficient asymmetric polymerase chain reaction (asymmetric PCR) amplification strategy. This method uses magnetic particles coupled with biotin-labeled one complementary nucleic acid sequence of the SARS-CoV-2 ORF1ab gene as the magnetic capture probes, and [Formula: see text]-labeled amino-modified another complementary nucleic acid sequence as the luminescent probes, and then a detection model of magnetic capture probes-asymmetric PCR amplification nucleic acid products-[Formula: see text]-labeled luminescent probes is formed, which combines the advantages of highly efficient asymmetric PCR amplification strategy and highly sensitive ECL biosensor technology, enhancing the method sensitivity of detecting the SARS-CoV-2 ORF1ab gene. The method enables the rapid and sensitive detection of the ORF1ab gene and has a linear range of 1-[Formula: see text] copies/[Formula: see text], a regression equation of [Formula: see text] = [Formula: see text] + 2919.301 ([Formula: see text] = 0.9983, [Formula: see text] = 7), and a limit of detection (LOD) of 1 copy/[Formula: see text]. In summary, it can meet the analytical requirements for simulated saliva and urine samples and has the benefits of easy operation, reasonable reproducibility, high sensitivity, and anti-interference abilities, which can provide a reference for developing efficient field detection methods for SARS-CoV-2.

Quantitative and Single-Nucleotide Resolution Profiling of RNA 5-Methylcytosine.

RNA has coevolved with numerous posttranscriptional modifications to sculpt interactions with proteins and other molecules. One of these modifications is 5-methylcytosine (m5C) and mapping the position and quantifying the level in different types of cellular RNAs and tissues is an important objective in the field of epitranscriptomics. Both in plants and animals bisulfite conversion has long been the gold standard for detection of m5C in DNA but it can also be applied to RNA. Here, we detail methods for highly reproducible bisulfite treatment of RNA, efficient locus-specific PCR amplification, detection of candidate sites by sequencing on the Illumina MiSeq platform, and bioinformatic calling of non-converted sites.

Population genetic analysis suggests genetic recombination is responsible for increased zoonotic potential of Enterocytozoon bieneusi from ruminants in China

Enterocytozoon bieneusi is a zoonotic pathogen with worldwide distribution. Among the 11 established groups of E. bieneusi genotypes based on phylogenetic analysis of the ribosomal internal transcribed spacer (ITS), the human-infective potential and population genetics of the Group 1 genotypes from diverse hosts are well characterized. In contrast, Group 2 genotypes from ruminants have unclear population genetics, leading to poor understanding of their host range and zoonotic potential. In this study, we sequence-characterized 121 Group 2 isolates from dairy cattle, beef cattle, yaks, Tibetan sheep, golden takins, and deer from China at five genetic loci (ITS, MS1, MS3, MS4 and MS7), comparing with data from 113 Group 1 isolates from nonhuman primates. Except for MS7, most of the genetic loci produced efficient PCR amplification and high nucleotide identity between Groups 1 and 2 of E. bieneusi genotypes. In population genetic analyses of the sequence data, a strong linkage disequilibrium was observed among these genetic loci in the overall Group 2 population. The individual ITS genotypes (I, J and BEB4) within Group 2, however, had reduced linkage disequilibrium and increased genetic exchanges among isolates. There was only partial genetic differentiation between Group 1 and Group 2 genotypes, with some occurrence of genetic recombination between them. Genetic recombination was especially common between genotypes I and J within Group 2. The data presented indicate a high genetic identity between Group 1 and Group 2 genotypes of E. bieneusi, which could be responsible for the broad host range and high zoonotic potential of Group 2 genotypes in China. As there is no effective treatment against E. bieneusi, the One Health approach should be used in the control and prevention of zoonotic transmission of the pathogen.

Optimizing bacterial DNA extraction in urine.

Urine is an acceptable, non-invasive sample for investigating the human urogenital microbiota and for the diagnosis of sexually transmitted infections. However, low quantities of bacterial DNA and PCR inhibitors in urine may prevent efficient PCR amplification for molecular detection of bacteria. Furthermore, cold temperatures used to preserve DNA and bacteria in urine can promote precipitation of crystals that interfere with DNA extraction. Saline, Dulbecco’s Phosphate Buffered Saline, or Tris-EDTA buffer were added to urine from adult men to determine if crystal precipitation could be reversed without heating samples beyond ambient temperature. Total bacterial DNA concentrations and PCR inhibition were measured using quantitative PCR assays to compare DNA yields with and without buffer addition. Dissolution of crystals with Tris-EDTA prior to urine centrifugation was most effective in increasing bacterial DNA recovery and reducing PCR inhibition. DNA recovery using Tris-EDTA was further tested by spiking urine with DNA from bacterial isolates and median concentrations of Lactobacillus jensenii and Escherichia coli 16S rRNA gene copies were found to be higher in urine processed with Tris-EDTA. Maximizing bacterial DNA yield from urine may facilitate more accurate assessment of bacterial populations and increase detection of specific bacteria in the genital tract.

Comparison of Three Protocols to Preserve Leptospira spp. in Cat Urine for Efficient DNA Extraction and PCR Amplification

Background: The pathogenic leptospira infection in mammalian species can cause a range of acute or chronic manifestations and may result in a carrier state. Previous studies have suggested that cats were resistant to acute leptospirosis however, the description of some clinical cases suggests that Leptospira spp. may also be pathogenic to this species. Recent studies have shown that leptospires may be shed in the urine of infected cats. Endogenous substances present in urine may inhibit PCR and allow leptospires to evade detection. This study aims to compare three protocols for sample processing to optimize the detection of pathogenic leptospires in cat urine.Materials, Methods & Results: Three protocols to optimize the detection of pathogenic leptospires in cat urine were tested. Aliquots of standard concentration of L interrogans serovar Canicola culture were added to urine samples to achieve concentrations of 1×105 to 1×102 leptospires/mL for each protocol. In protocols A and B the urine was neutralized by the addition of phosphate-buffered saline (PBS), pH 7.4, in a proportion of 1 PBS: 2.5 urine (v/v). In protocol A, PBS was added to neutralize the urine pH for the leptospiral organisms immediately after addition of leptospires. In protocol B, PBS was added just before DNA extraction. In protocol C, no PBS was added. DNA extraction was performed at 4, 24 and 48 h after addition of the leptospires using a modified protocol. Samples were incubated at 37ºC for 10 min. Samples were then centrifuged (850 g) for 15 min, at 25ºC. The supernatants were transferred to another tube, and the pellets were discarded. The supernatants were centrifuged (16060 g) for 20 min at 4ºC. The supernatants were then discarded, and the pellets resuspended and washed with 1000 µL of PBS. All the samples were centrifuged at 16060 g for an additional 20 min at 25ºC. The supernatants were discarded and the pellets were resuspended in 100 µL of PBS and incubated at 94ºC for 10 min. DNA was stored at -20ºC until the molecular analysis. The PCR detection limit was evaluated. In samples from protocol A, leptospires were detected in concentrations up to 1×103 (4 h) and 1×104 (24 and 48 h). In protocol C, leptospires were detected in concentrations up to 1×104 (4 h) and 1×105 (24 and 48 h). No leptospiral DNA was detected in samples from protocol B.Discussion: Leptospires are sensitive to acid conditions, at pH 6.8 or lower and the urine pH of cats may vary from 5 to 7. In the present study, we found best results for DNA amplification with the addition of PBS immediately after urine collection (protocol A). Previous studies have shown the importance of neutralizing urine samples immediately after collection to avoid loss of bacterial DNA during the extraction process. However, protocols B and C may be an alternative in clinical practice, when PBS cannot be added immediately after collection. The delay after urine collection before DNA extraction is one more factor that may interfere with the PCR sensitivity. This was observed in the samples from protocol A, because although these samples were neutralized immediately, there was a 10-fold decrease in the detection limit of the test at 24 and 48 h. Leptospires rapidly lose their integrity in urine and the detection limit declines considerably over time, so prompt extraction is essential. These results show that the in-house method of preserving cat urine is useful to maintain the viability of leptospiral DNA extraction. In addition, this study highlights the importance of neutralizing urine samples immediately after collection and the need for prompt DNA extraction to improve PCR detection limit. However, if PBS cannot be added to the collected sample immediately, it is better to process the sample without PBS and extract DNA as soon as possible to minimize the risk of false-negative results.

Complete nucleotide sequence characterization of DRB5 alleles reveals a homogeneous allele group that is distinct from other DRB genes

Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. A better and comprehensive sequence assessment can be achieved by the inclusion of full gene sequences of all the common alleles at a given locus. The common alleles of DRB5 are under-characterized with the full exon-intron sequence of two alleles available. In the present study the DRB5 genes from 18 subjects alleles were cloned and sequenced; haplotype analysis showed that 17 of them had a single copy of DRB5 and one consanguineous subject was homozygous at all HLA loci. Methodological approaches including robust and efficient long-range PCR amplification, molecular cloning, nucleotide sequencing and de novo sequence assembly were combined to characterize DRB5 alleles. DRB5 sequences covering from 5′UTR to the end of intron 5 were obtained for DRB5*01:01, 01:02 and 02:02; partial coverage including a segment spanning exon 2 to exon 6 was obtained for DRB5*01:03, 01:08N and 02:03. Phylogenetic analysis of the generated sequences showed that the DRB5 alleles group together and have distinctive differences with other DRB loci. Novel intron variants of DRB5*01:01:01, 01:02 and 02:02 were identified. The newly characterized DRB5 intron variants of each DRB5 allele were found in subjects harboring distinct associations with alleles of DRB1, B and/or ethnicity. The new information provided by this study provides reference sequences for HLA typing methodologies. Extending sequence coverage may lead to identify the disease susceptibility factors of DRB5 containing haplotypes while the unexpected intron variations may shed light on understanding of the evolution of the DRB region.

Genotyping of the OATP1B1 c. 521 T>C Polymorphism from the Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens: an Optimized Protocol.

Organic anion transporting polypeptide (OATP) 1B1 is a liver-specific transport protein that plays an important role in hepatic drug disposition. It transports many drugs from the blood into the liver, including lipid-lowering statins. The c. 521 T>C polymorphism of OATP1B1 has reduced transport activity and is associated with statin-induced myopathy. Formalin-fixed paraffin-embedded (FFPE) liver tissues can be an enriched source for genotyping of this clinically significant OATP1B1 polymorphism in retrospective studies. The successfulness of genotyping using Sanger-sequencing of a PCR product from FFPE tissue relies on a successful PCR amplification using genomic DNA extracted from the FFPE tissues. Such PCR amplification is often limited by the quality of DNA extracted from the FFPE tissue. An optimized method for high-quality DNA extraction and efficient PCR amplification is highly needed in order to genotype polymorphisms such as the c. 521 T>C polymorphism using FFPE tissues. The current study established an optimized and reproducible method for a Sanger-sequencing-based genotyping method using FFPE human liver tissues that is applicable to even small FFPE tissues such as needle-core biopsy specimens.

PCR Amplification of Base-Modified DNA.

An efficient PCR amplification of various templates (short 57-mer, random 67- and 82-mer, and long DNA) with base-modified nucleoside triphosphates is presented here. Using 5-substituted pyrimidine and 7-substituted-7-deaza- or 8-substituted purine nucleoside triphosphates as substrates for thermostable DNA polymerases [Taq and Vent (exo- )], successful PCR amplification of partially or entirely modified DNA libraries and long DNA constructs (up to 1.5kb) is achieved. Visualization of double-stranded PCR product formation is improved through the use of primers with different fluorescent labels. This allows one to monitor the efficiency of modified substrate incorporation and the enzymatic recognition of the modified template during PCR. The redesigned fully base-modified DNA (denoted 'DZA') can be utilized for the straightforward production of diverse libraries for in vitro selection of aptamer and catalytic nucleic acids as well as for the synthesis of artificial genetic templates, replicons, or complex vectors. © 2018 by John Wiley & Sons, Inc.

Comparison of methods to preserve Rheum palmatum (Polygonaceae) for efficient DNA extraction and PCR amplification.

In this study, we compared the quality of DNA extracted using the modified CTAB method, from Rheum palmatum leaves preserved using fourteen different methods, including ones used commonly in other species: under ultra-cold (-80°C) temperatures, after drying with an absorbent paper, desiccating using a silica gel, drying at 60°C, in 70% ethanol, absolute ethanol, 70% ethanol supplemented with 50 mM EDTA, SDS-DNA extracting solution, nuclear separation buffer, improved NaCl-CTAB solution, TE-buffer, I-solution, or II-solution. DNA extracted from fresh leaves was used as the control. The quality of extracted DNA was evaluated based on the success of PCR amplification of the ITS2 region and a microsatellite marker. DNA was not extracted from samples preserved in the nuclear separation buffer and II-solution. The purities of DNA extracted from leaves preserved in ultra-cold temperatures, 70% ethanol, and 70% ethanol with 50 mM EDTA, and after desiccating using a silica gel and drying were higher, and comparable to the purity of DNA extracted from fresh leaves, than those of leaves preserved using other methods. In the present study, combined with the PCR amplifications, the preservation using ultra-cold temperatures, silica gel desiccation, or drying, and PCR amplification of the extracted DNA can be used for further molecular studies in R. palmatum.

A method suitable for DNA extraction from humus-rich soil.

A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils.

Challenges to design and develop of DNA aptamers for protein targets. I. Optimization of asymmetric PCR for generation of a single stranded DNA library.

Aptamers, or single stranded oligonucleotides, are produced by systematic evolution of ligands by exponential enrichment, abbreviated as SELEX. In the amplification and regeneration step of SELEX technique, dsDNA is conversed to ssDNA. Asymmetric PCR is one of the methods used for the generation of ssDNA. The purpose of this study was to design a random DNA library for selection of aptamers with high affinity for protein targets and develop an efficient asymmetric PCR amplification. Thus, the influence of factors including annealing temperature, number of amplification cycles, primer ratio, Mg(2+) concentration and the presence of a PCR enhancer on the amplification of the desired product were evaluated. Results obtained by agarose gel electrophoresis showed that the annealing temperature of 64 °C, Mg(2+) concentration of 0.25 mM, reverse to forward primer ratio of 15:1, amplification cycle of 25 and the presence L-ectoin as a PCR enhancer with the concentration of 0.4 M were the optimal conditions. Our results supported that the yield of this type of ssDNA production is sufficient for combinatorial screening of aptamers.

Improved Method for Bacterial Cell Capture after Flow Cytometry Cell Sorting

Fixed cells with different nucleic acid contents and scatter properties (low nucleic acid [LNA], high nucleic acid 1 [HNA1], and HNA2) were sorted by flow cytometry (FCM). For each sort, 10,000 cells were efficiently captured on poly-l-lysine-coated microplates, resulting in efficient and reproducible PCR amplification.

Comparison of dry- and wet-based fine bead homogenizations to extract DNA from fungal spores

The present study explored DNA extraction kinetics from fungal spores, i.e., Aspergillus niger, Penicillium chrysogenum and Cladosporium sphaerospermum, by fine bead mill homogenization. In particular, the study aimed to investigate basic differences between the dry- and wet-based methods. The results showed higher initial rates of the DNA extractions by the dry-based method than by the wet-based method, due to higher collision efficiency among fine beads and fungal spores. Based on the experimental results, we constructed kinetic models. While the results by the wet-based method were fitted well with an existing first-order release-degradation model, the results by the dry-based method were not fitted well. Meanwhile, a newly constructed first-order release-degradation model, assuming a proportion of the DNA remained inside the disrupted spore cells and protected from further sheer stress, showed good correlations. The real-time PCR assays showed the PCR efficiencies of the DNA obtained by the dry-based method were higher than those by the wet-based method likely due to increased moderate fragmentation of the DNA by the dry-based method. Thus, although wet-based methods have been commonly used, dry-based methods might also be applicable to achieve efficient extraction and PCR amplification of fungal DNA.

An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3′→5′ exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 107-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.

Identification of conserved core xylem gene sets: conifer cDNA microarray development, transcript profiling and computational analyses

One approach for investigating the molecular basis of wood formation is to integrate microarray profiling data sets and sequence analyses, comparing tree species with model plants such as Arabidopsis. Conifers may be included in comparative studies thanks to large-scale expressed sequence tag (EST) analyses, which enable the development of cDNA microarrays with very significant genome coverage. A microarray of 10,400 low-redundancy sequences was designed starting from white spruce (Picea glauca (Moench.) Voss) cDNAs. Computational procedures that were developed to ensure broad transcriptome coverage and efficient PCR amplification were used to select cDNA clones, which were re-sequenced in the microarray manufacture process. White spruce transcript profiling experiments that compared secondary xylem to phloem and needles identified 360 xylem-preferential gene sequences. The functional annotations of all differentially expressed sequences were highly consistent with the results of similar analyses carried out in angiosperm trees and herbaceous plants. Computational analyses comparing the spruce microarray sequences and core xylem gene sets from Arabidopsis identified 31 transcripts that were highly conserved in angiosperms and gymnosperms, in terms of both sequence and xylem expression. Several other spruce sequences have not previously been linked to xylem differentiation (including genes encoding TUBBY-like domain proteins (TLPs) and a gibberellin insensitive (gai) gene sequence) or were shown to encode proteins of unknown function encompassing diverse conserved domains of unknown function.

High-Throughput Quantitative Polymerase Chain Reaction in Picoliter Droplets

Limiting dilution PCR has become an increasingly useful technique for the detection and quantification of rare species in a population, but the limit of detection and accuracy of quantification are largely determined by the number of reactions that can be analyzed. Increased throughput may be achieved by reducing the reaction volume and increasing processivity. We have designed a high-throughput microfluidic chip that encapsulates PCR reagents in millions of picoliter droplets in a continuous oil flow. The oil stream conducts the droplets through alternating denaturation and annealing zones, resulting in rapid (55-s cycles) and efficient PCR amplification. Inclusion of fluorescent probes in the PCR reaction mix permits the amplification process to be monitored within individual droplets at specific locations within the microfluidic chip. We show that amplification of a 245-bp adenovirus product can be detected and quantified in 35 min at starting template concentrations as low as 1 template molecule/167 droplets (0.003 pg/microL). The frequencies of positive reactions over a range of template concentrations agree closely with the frequencies predicted by Poisson statistics, demonstrating both the accuracy and sensitivity of this platform for limiting dilution and digital PCR applications.

Efficient PCR amplification by an unnatural base pair system

Expansion of the genetic alphabet by an unnatural base pair system enables the site-specific incorporation of extra functional components into nucleic acids and proteins. In this system, PCR amplification of DNA templates containing unnatural base pairs is essential for modern biotechnology. We present a new unnatural base pair system, in which DNA duplexes containing the unnatural base pairs can be efficiently amplified by PCR. The system also provides a method for the site-specific incorporation of functional components into amplified DNA fragments by PCR, using unnatural base substrates linked with functional groups of interest.

A fundamental study of the PCR amplification of GC-rich DNA templates

A theoretical analysis is presented with experimental confirmation to conclusively demonstrate the critical role that annealing plays in efficient PCR amplification of GC-rich templates. The analysis is focused on the annealing of primers at alternative binding sites (competitive annealing) and the main result is a quantitative expression of the efficiency ( η) of annealing as a function of temperature ( T A), annealing period ( t A), and template composition. The optimal efficiency lies in a narrow region of T A and t A for GC-rich templates and a much broader region for normal GC templates. To confirm the theoretical findings, the following genes have been PCR amplified from human cDNA template: ARX and HBB (with 78.72% and 52.99% GC, respectively). Theoretical results are in excellent agreement with the experimental findings. Optimum annealing times for GC-rich genes lie in the range of 3–6 s and depend on annealing temperature. Annealing times greater than 10 s yield smeared PCR amplified products. The non-GC-rich gene did not exhibit this sensitivity to annealing times. Theory and experimental results show that shorter annealing times are not only sufficient but can actually aid in more efficient PCR amplification of GC-rich templates.

Development of an unnatural base pair for efficient PCR amplification

An unnatural base pair system could expand the genetic alphabet, enabling the site-specific incorporation of extra, functional components into nucleic acids and proteins. We developed an unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (denoted by Ds) and 2-nitropyrrole (denoted by Pn), which specifically and efficiently functions in DNA amplification by PCR. After 20 cycles of PCR, the mutation rate of the Ds-Pn pair in an amplified DNA fragment was approximately 1%. The Ds-Pn pair system in combination with other unnatural base pairs could be useful for DNA/RNA-based biotechnology.