Amplification of GC-rich DNA for High-Throughput Family-Based Genetic Studies

Researchers face a significant problem in PCR amplification of DNA fragments with high GC contents. Analysis of these regions is of importance since many regulatory regions of different genes and their first exons are GC-rich. There are a large number of protocols for amplification of GC-rich DNA, some of which perform well but are costly. Most of the economical protocols fail to perform consistently, especially on products with >80 % GC contents and a size of >300 bp. One of these protocols requires multiple additions of DNA polymerase during thermal cycling which therefore rules out its utility if a large number of samples have to be amplified. We have established a method for simultaneous amplification of specific PCR products from a large number of human DNA samples using general laboratory reagents. These amplicons have GC contents ranging from 65–85 % and sizes up to 870 bp. The protocol uses a PCR buffer containing co-solvents including 2-mercaptoethanol and bovine serum albumin for amplification of DNA. A specific thermal cycling profile is also used which incorporates a high annealing temperature in the first 7 cycles of the reactions. The PCR products are suitable for different molecular biology applications including sequencing.     

Exponential quadruplex priming amplification for DNA‐based isothermal diagnostics

Polymerase chain reaction (PCR) is a method of choice for molecular diagnostics. However, PCR relies on thermal cycling, which is not compatible with the goals of point‐of‐care diagnostics. A simple strategy to turn PCR into an isothermal method would be to use specific primers, which upon polymerase elongation can self‐dissociate from the primer‐binding sites. We recently demonstrated that a monomolecular DNA quadruplex, GGGTGGGTGGGTGGG, meets these requirements, which led to the development of the linear versions of quadruplex priming amplification (QPA). Here we demonstrate exponential version of isothermal QPA, which allows an unprecedented 10¹⁰‐fold amplification of DNA signal in less than 40 min. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 88–95, 2015.     

Clinical evaluation of micro-scale chip-based PCR system for rapid detection of hepatitis B virus

The polymerase chain reaction (PCR) is widely used to amplify a small amount of DNA in samples for genetic analysis. Rapid and accurate amplification is prerequisite for broad applications including molecular diagnostics of diseases, food safety, and biological warfare tests. We have developed a rapid real-time micro-scale chip-based PCR system, which consists of six individual thermal cycling modules capable of independent control of PCR protocols. The PCR volume is 1 microl and it takes less than 20 min to complete 40 thermal cycles. To test utility of a chip-based PCR system as a molecular diagnostic device, we have conducted the first large-scale clinical evaluation study. Three independent clinical evaluation studies (n = 563) for screening the hepatitis B virus (HBV) infection, the most popular social epidemic disease in Asia, showed an excellent sensitivity, e.g. 94%, and specificity, e.g. 93%, demonstrating micro-scale chip-based PCR can be applied in molecular diagnostics.     

PCR-based sexing in conservation biology: Wrong answers from an accurate methodology?

Molecular tests of sex based on the polymerase chain reaction (PCR) are now commonplace in conservation biology, routinely guiding management decisions. While molecular approaches to sexing can be highly reliable, current practices may leave an undesirable level of uncertainty in the sexes identified, because researchers focus on determining the sex-specific nature of a test, largely ignoring the accuracy of the test to correctly sex individuals. This latter step requires considerably more known-sex individuals. We argue that, due to the well-known technical problems associated with PCR amplification, the demonstrated potential for sexing errors and few known-sex individuals being available from threatened species, conservationists should place greater emphasis on verifying the sexes identified with PCR tests. We propose that all individuals of the sex indistinguishable from an amplification failure (e.g., females in mammals XX, males in birds ZZ) should be verified with a second independent sex test. Such a consensus approach to molecular sexing would reduce errors that could arise due to technical failure and PCR anomalies, but may also reduce field and laboratory bookkeeping errors.     

Direct and efficient cloning of full-length genes from environmental DNA by RT-qPCR and modified TAIL-PCR

Environmental DNA (eDNA) is defined as the total DNA that can be isolated from environmental samples. In total, therefore, eDNA includes a vast functional genes, and various approaches have been developed to retrieve full-length functional genes from eDNA. The efficiency of PCR amplification of eDNA is limited, however, because in truth, the net content of actual target functional genes is rather low in eDNA. To address this technical challenge, we developed a fast and effective approach to cloning full-length functional genes from eDNA. Two important modifications were made to existing PCR-based methods: first, a real-time quantitative PCR step was added to assess the difficulty of obtaining full-length genes; second, we improved the thermal asymmetric interlaced PCR program to make it more effective for cloning the flanking regions of known fragments that are present at low abundance in eDNA. Using this approach, five novel full-length functional genes with very low identity to known genes were cloned from environmental samples. This approach has great potential for allowing discovery of functional genes from environmental sources and may be broadly applicable to molecular biology research.     

Pre-PCR processing

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.     

Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates

ABSTRACT: BACKGROUND: While being a standard powerful molecular biology technique, applications of the PCR to the amplification of high GC-rich DNA samples still present challenges which include limited yield and poor specificity of the reaction. Organic solvents, including DMSO and formamide, have been often employed as additives to increase the efficiency of amplification of high GC content (GC > 60%) DNA sequences. Bovine serum albumin (BSA) has been used as an additive in several applications, including restriction enzyme digestions as well as in PCR amplification of templates from environmental samples that contain potential inhibitors such as phenolic compounds. FINDINGS: Significant increase in PCR amplification yields of GC-rich DNA targets ranging in sizes from 0.4 kb to 7.1 kb were achieved by using BSA as a co-additive along with DMSO and formamide. Notably, enhancing effects of BSA occurs in the initial PCR cycles with BSA additions having no detrimental impact on PCR yield or specificity. When a PCR was set up such that the cycling parameters paused after every ten cycles to allow for supplementation of BSA, combining BSA and organic solvent produced significantly higher yields relative to conditions using the solvent alone. The co-enhancing effects of BSA in presence of organic solvents were also obtained in other PCR applications, including site-directed mutagenesis and overlap extension PCR. CONCLUSIONS: BSA significantly enhances PCR amplification yield when used in combination with organic solvents, DMSO or formamide. BSA enhancing effects were obtained in several PCR applications, with DNA templates of high GC content and spanning a broad size range. When added to the reaction buffer, promoting effects of BSA were seen in the first cycles of the PCR, regardless of the size of the DNA to amplify. The strategy outlined here provides a cost-effective alternative for increasing the efficiency of PCR amplification of GC-rich DNA targets over a broad size range.     

Rapid cycle DNA amplification: time and temperature optimization

Rapid temperature cycling with hot air allows rigorous optimization of the times and temperatures required for each stage of the polymerase chain reaction. A thermal cycler based on recirculating hot air was used for rapid temperature control of 10-microliters samples in thin glass capillary tubes with the sample temperature monitored by a miniature thermocouple probe. The temperatures and times of denaturation, annealing and elongation were individually optimized for the amplification of a 536-base pair beta-globin fragment from human genomic DNA. Optimal denaturation at 92 degrees-94 degrees C occurred in less than one second; yield decreased with denaturation times greater than 30 seconds. Annealing for one second or less at 54 degrees-56 degrees C gave the best product specificity and yield. Non-specific amplification was minimized with a rapid denaturation to annealing temperature transition (9 seconds) as compared to a longer transition (25 seconds). An elongation temperature of 75 degrees-79 degrees C gave the greatest yield and increased yields were obtained with longer elongation times. Product specificity was improved with rapid air cycling when compared to slower conventional heat block cycling. Rapid thermal control of the temperature-dependent reactions in DNA amplification can improve product specificity significantly while decreasing the required amplification time by an order of magnitude.     

Strand Invasion Based Amplification (SIBA?): A Novel Isothermal DNA Amplification Technology Demonstrating High Specificity and Sensitivity for a Single Molecule of Target Analyte

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2′-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.     

Selection of deletion mutants by polymerase chain reaction

Polymerase chain reaction (PCR) based DNA amplification has replaced many time-consuming protocols in molecular biology. Here we describe a simple strategy to quickly select deletion mutants based on PCR methodology which then can be confirmed by nucleotide sequencing. A forward PCR primer is designed in such a way to recognize only the wild type sequences in the amplification reaction and thus a negative selection identifies the deletion in the samples.     

Development of a temperature sensor array chip and a chip-based real-time PCR machine for DNA amplification efficiency-based quantification

A temperature sensor array chip was developed to monitor the thermal cycling profiles of a polymerase chain reaction (PCR). DNA amplification efficiency of each cycle was estimated through temperature data to fit the stochastic model. A fluorescence detector system was constructed to detect the PCR amplifications of latter cycles, at which the fluorescence intensity passed the optical detection threshold. Through monitoring of both temperature and fluorescence, DNA amplification efficiency curve was completed for quantification. The F?rster resonance energy transfer (FRET) was employed to detect the measurements of the PCR product amount at the reaction endpoint. The chip-based, real-time PCR machine was constructed to perform the amplification efficiency curve-based quantification method. This novel method achieved the absolute quantification of the Hepatitis B virus (HBV) DNA using a single sample without the construction of the standard curve. The coefficient of variation (CV) of the 15 replicates inter assay experiments was less than 5.87%. Compared with the CV values obtained from the commercial machine in the range of 4.33-14.56%, it is noted that CV values of the prototype with respect to the samples of different initial concentration ranging from 10(7) to 10(3)copies/ml are almost equable.     

Ready-to-use thermally stable mastermix pills for molecular biology applications

Rolling circle amplification (RCA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP), are powerful tools that can be used for gene manipulation, pathogen detection, and infectious disease diagnostics. However, these techniques require trained personnel, as the pipetting steps involved can lead to contamination and, consequently, erroneous results. Furthermore, many of the reagents used in molecular biology are thermally labile and must be kept within a cold-chain. In this article, we present a simple and cost-effective method that allows molecular biology reagents to be thermally stabilized into ready-to-use mastermixes via drying in pullulan and trehalose films. Our experimental results demonstrate that this method is capable of preserving the activity of RCA, PCR, LAMP, ligase, polynucleotide kinase, and Klenow fragment mastermixes for at least 3 months at ambient conditions. Thus, stabilizing reagents via drying in pullulan and trehalose film may allow for a drastic reduction in the number of pipetting steps and the elimination of the need for a cold chain. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2764, 2019.     

Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek''s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: ● Set up reactions and thermal cycling conditions for a conventional PCR experiment ● Understand the function of various reaction components and their overall effect on a PCR experiment ● Design and optimize a PCR experiment for any DNA template ● Troubleshoot failed PCR experiments     

Parallel DNA amplification by convective polymerase chain reaction with various annealing temperatures on a thermal gradient device

We present a thermal gradient convective polymerase chain reaction (PCR) for parallel DNA amplification with different annealing temperatures. The thermal gradient for microfluidic gradient PCR is produced by an innovative fin design whose formation principle is given. Without the need for a pump, the buoyancy forces continuously circulate reagents in a closed loop through different thermal zones, which brings self-actuated convective-flow PCR. In our prototype, we measured a temperature difference of about 45 °C along the gradient direction on the copper flake (45 × 40 × 4 mm). When the temperature of the hot zone is 90-97 °C and the temperature of the cold zone is 60-70 °C, the convection triggered two-temperature amplification of 112-bp fragment of Escherichia coli DNA. The time for amplification is less than 45 min. Interestingly, parallel DNA amplification with different annealing temperatures ranging from 60 to 70 °C was performed by this method. The PCR thermocycler demonstrated herein can be further scaled down and the loop length can be further reduced, and therefore the PCR times can be further reduced. These devices are suited as a platform for a new generation of low-power, portable DNA analysis systems.     

Microarrays for the detection of HBV and HDV

The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.     

Plastic microfluidic chip for continuous-flow polymerase chain reaction: simulations and experiments

A continuous flow polymerase chain reaction (CF-PCR) device comprises a single fluidic channel that is heated differentially to create spatial temperature variations such that a sample flowing through it experiences the thermal cycling required to induce amplification. This type of device can provide an effective means to detect the presence of a small amount of nucleic acid in very small sample volumes. CF-PCR is attractive for global health applications due to its less stringent requirements for temperature control than for other designs. For mass production of inexpensive CF-PCR devices, fabrication via thermoplastic molding will likely be necessary. Here we study the optimization of a PCR assay in a polymeric CF-PCR device. Three channel designs, with varying residence time ratios for the three PCR steps (denaturation, annealing, and extension), were modeled, built, and tested. A standardized assay was run on the three different chips, and the PCR yields were compared. The temperature gradient profiles of the three designs and the residence times of simulated DNA molecules flowing through each temperature zone were predicted using computational methods. PCR performance predicted by simulation corresponded to experimental results. The effects of DNA template size and cycle time on PCR yield were also studied. The experiments and simulations presented here guided the CF-PCR chip design and provide a model for predicting the performance of new CF-PCR designs prior to actual chip manufacture, resulting in faster turn around time for new device and assay design. Taken together, this framework of combined simulation and experimental development has greatly reduced assay development time for CF-PCR in our lab.     

Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.

A Hot Start Polymerase Chain Reaction (PCR) entails the withholding of at least one reagent from the reaction mixture until the reaction tube temperature has reached 60-80 degrees C. Hot Start amplification with an AmpliWax vapor barrier uses a layer of solid wax to separate the retained reagent(s) and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated Hot Start PCR greatly increases the specificity, yield, and precision of amplifying low copy numbers of three HIV targets. In the presence of 1 microgram of human placental DNA (1.6 x 10(5) diploid genomes) the specificity improvement entails considerable to complete reduction in the amplification of mis-primed sequences and putative primer oligomers. When mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization. Hot Start PCR with an AmpliWax vapor barrier permits routine amplification of a single target molecule with detection by ethidium stained gel electrophoresis; nonisotopically visualized probing suffices for confirmation. The improved amplification performance is evident for target copy numbers below approximately 10(3).     

Micromachined polymerase chain reaction system for multiple DNA amplification of upper respiratory tract infectious diseases

This paper presents a micro polymerase chain reaction (PCR) chip for the DNA-based diagnosis of microorganism genes and the detection of their corresponding antibiotic-resistant genes. The micro PCR chip comprises cheap biocompatible soda-lime glass substrates with integrated thin-film platinum resistors as heating/sensing elements, and is fabricated using micro-electro-mechanical-system (MEMS) techniques in a reliable batch-fabrication process. The heating and temperature sensing elements are made of the same material and are located inside the reaction chamber in order to ensure a uniform temperature distribution. This study performs the detection of several genes associated with upper respiratory tract infection microorganisms, i.e. Streptococcus pneumoniae, Haemopilus influenze, Staphylococcu aureus, Streptococcus pyogenes, and Neisseria meningitides, together with their corresponding antibiotic-resistant genes. The lower thermal inertia of the proposed micro PCR chip relative to conventional bench-top PCR systems enables a more rapid detection operation with reduced sample and reagent consumption. The experimental data reveal that the high heating and cooling rates of the system (20 and 10 degrees C/s, respectively) permit successful DNA amplification within 15 min. The micro PCR chip is also capable of performing multiple DNA amplification, i.e. the simultaneous duplication of multiple genes under different conditions in separate reaction wells. Compared with the large-scale PCR system, it is greatly advantageous for fast diagnosis of multiple infectious diseases. Multiplex PCR amplification of two DNA segments in the same well is also feasible using the proposed micro device. The developed micro PCR chip provides a crucial tool for genetic analysis, molecular biology, infectious disease detection, and many other biomedical applications.     

PCR and sequencing from a single pollen grain

In order to eliminate the laborious step of DNA extraction preceding all studies within the field of plant molecular biology we attempted to do PCR amplifications directly on pollen grains. Successful PCR amplification was obtained in reactions including a single pollen grain from Hordeum vulgare or Secale strictum. Both the plastid gene encoding ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) and the nuclear-encoded internal transcribed spacer regions (ITS) and the 5.8S rDNA region were amplified and sequenced to verify PCR amplification.     

An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

Background

PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents.

Methodology/Principal Findings

Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein.

Conclusions/Significance

There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA.