Polymerase Chain Reaction Efficiency Research Articles

Market Analysis of Food and Feed Products for Detection of Horse DNA by a TaqMan Real-Time PCR

Food-labelling regulations require that meat species in food and feed are accurately declared to the consumer. Traceability systems based on species identification through DNA analysis are potent tools for the supervision of food adulteration. In this study, a TaqMan real-time polymerase chain reaction (PCR) assay targeting a short mitochondrial 12S ribosomal RNA (rRNA) gene fragment of 73 base pair (bp) was developed for detection of horse DNA in different commercial meat products for human and pet consumption. The method was found to be specific for horse and did not show any cross-reactivity with different species of mammals, birds, fish and plants. The assay complies with the acceptance criteria required for real-time PCR methods in terms of applicability, linear dynamic range, accuracy and PCR efficiency and showed to be sensitive allowing the detection of 1 pg of horse DNA. A range of food and feed products (n = 171) was screened with the horse-specific real-time PCR to determine whether a correct labelling had been employed at the market level. Results obtained when testing the meat products for human consumption were in agreement with the labelling description provided by the suppliers. In the case of the pet foods, undeclared horse meat was detected in 21 % of the samples at low levels, suggesting unintentional cross-contamination during processing. The reported real-time PCR methodology may represent a suitable tool for the detection of food mislabelling.

DNA Polymerase Binding to the Primer/template Duplex Affects the Efficiency of PCR

Polymerase chain reaction(PCR) for DNA and RNA amplifications in vitro has a profound impact on modern molecular biology. Since design of proper primers is crucial for the success in PCR,many parameters have been used for primer design. However,the effect of DNA polymerase binding to primer /template duplex on PCR efficiency was not taken into account in conventional primer design programs. To reveal whether or not the DNA polymerase-primer /template binding affects the PCR efficiency,here we built structural models for the Taq DNA polymerase in complex with different primer /template sequences,and designed PCR primers according to relative binding free energies calculated by MM /GBSA method. We verified our primer design approach using Human Serum Albumin(HSA) gene and Mycobacterium tuberculosis pyrF gene,and found that the PCR efficiencies of different designed primers for both tested genes correlated well with the calculated binding free energies. Our finding indicates clearly that the DNA polymerase-primer /template binding affects the PCR efficiency significantly. Thus,the calculated binding free energy could be used as a new parameter to design efficient PCR primers.

The Effect of DNA-Dispersed Single-Walled Carbon Nanotubes on the Polymerase Chain Reaction

The unique properties of single-wall carbon nanotubes (SWCNT) make them useful in many new technologies and applications. The interaction of DNA and SWCNT is of interest for many uses, including molecular sensors. This study determined polymerase chain reaction (PCR) efficiency in amplifying a 76 base pair DNA sequence in the presence of SWCNT, of heterogeneous “Mix” and (6,5)-enriched chiralities, associated with three DNA sequences. The dependence of PCR efficiency on the concentration of DNA:SWCNT preparations was measured, as well as their age and level of dispersion (less than one month or between four and ten months). Additionally, the ability to directly amplify the DNA sequence associated with the SWCNT scaffold was investigated. In PCRs with DNA:SWCNT preparations less than one month old, concentrations greater than or equal to 0.1 mg/mL inhibited the PCR reaction. In PCRs with older preparations, no inhibition was seen at 0.01 or 0.1 mg/mL, with amplification at 1 mg/mL in some samples. Additionally, our studies showed that the DNA directly associated with the SWCNT can be amplified using PCR. This work provides an inhibitory concentration of DNA-dispersed SWCNT in PCR reactions for different preparations as well as a basis for future DNA:SWCNT studies that require PCR amplification. This will be useful for future studies focused on the use of SWCNT in molecular sensing technologies.

Determination of real-time polymerase chain reaction uncertainty of measurement using replicate analysis and a graphical user interface with Fieller's theorem.

Disease monitoring of viruses using real-time polymerase chain reaction (PCR) requires knowledge of the precision of the test to determine what constitutes a significant change. Calculation of quantitative PCR confidence limits requires bivariate statistical methods. To develop a simple-to-use graphical user interface to determine the uncertainty of measurement (UOM) of BK virus, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) real-time PCR assays. Thirty positive clinical samples for each of the three viral assays were repeated once. A graphical user interface was developed using a spreadsheet (Excel, Microsoft Corporation, USA) to enable data entry and calculation of the UOM (according to Fieller's theorem) and PCR efficiency. The confidence limits for the BK virus, CMV and EBV tests were ∼0.5 log, 0.5 log to 1.0 log, and 0.5 log to 1.0 log, respectively. The efficiencies of these assays, in the same order were 105%, 119% and 90%. The confidence limits remained stable over the linear range of all three tests. A >5 fold (0.7 log) and a >3-fold (0.5 log) change in viral load were significant for CMV and EBV when the results were ≤1000 copies/mL and >1000 copies/mL, respectively. A >3-fold (0.5 log) change in viral load was significant for BK virus over its entire linear range. PCR efficiency was ideal for BK virus and EBV but not CMV. Standardized international reference materials and shared reporting of UOM among laboratories are required for the development of treatment guidelines for BK virus, CMV and EBV in the context of changes in viral load.

Droplet Microfluidic Chip Based Nucleic Acid Amplification and Real-Time Detection of Influenza Viruses

Miniaturized bio-diagnostic devices have the potential to allow for rapid pathogen screening in clinical patient samples, as a low cost and portable alternative to conventional bench-top equipment. Miniaturization of key bio-diagnostic techniques, such as: nucleic acid detection and quantification, polymerase chain reaction (PCR), DNA fingerprinting, enzyme linked immunosorbent assay (ELISA), results in substantial reduction of reaction volumes (expensive samples/reagents) and shorter reaction times. Droplet microfluidics (DMF) is one of several miniaturized bio-sample handling techniques available for manipulating clinical samples and reagents in microliter (10−6 L) to picoliter (10−12 L) volume regime. Electro-actuation of sample and reagent in the form of droplets in the aforementioned volume regime, using dielectrophoresis (DEP) and/or Electrowetting (EW) are achieved by means of patterned, insulated metal electrodes on one or more substrates. In this work, we have utilized electro-actuation based DMF technology, integrated with suitably tailored resistive micro-heaters and temperature sensors, to achieve chip based real-time, quantitative PCR (qRT-PCR). This qRT-PCR micro-device was utilized to detect and quantify the presence of influenza A and C virus nucleic acids, using in-vitro synthesized viral RNA segments. The experimental analysis of the DMF micro-device confirms its capabilities in qRT-PCR based detection and quantification of pathogen samples, with accuracy levels comparable to established commercial bench-top equipment (PCR efficiency ∼95%). The limit of detection (LOD) of the chip based qRT-PCR technique was estimated to be ∼5 copies of template RNA per PCR reaction.

Mechanism of gold nanoparticle induced simultaneously increased PCR efficiency and specificity

Gold nanoparticles (AuNPs) have been reported to induce faster heat transfer of polymerase chain reaction (PCR) solution to enhance PCR efficiency. AuNPs can also increase the specificity of PCR by functioning analogously to single-stranded DNA binding protein (SSB). However, the simple structure of AuNPs makes it difficult to determine how AuNPs affect PCR efficiency and specificity. This study aimed to elucidate the effect of AuNPs on PCR efficiency by altering the denaturation times and annealing temperatures. In addition, comparative study of the effect of AuNPs on PCR inhibition caused by two competitive primers allowed investigation of the mechanisms of AuNP-enhanced PCR accuracy. Finally, heat-treated salmon sperm DNA was used to evaluate whether AuNPs could eliminate the inhibitory PCR effect caused by DNA impurities. This study demonstrated that enhanced thermal conductivity by AuNPs was the main mechanism for increased PCR efficiency and specificity. AuNPs promoted efficient double-stranded DNA template unwinding and dissociation between mismatched primers, DNA fragments and template, and enhanced PCR efficiency and specificity simultaneously. Thus, this significant finding suggests the future use of AuNP-assisted PCR in different fields, especially in rapid clinical diagnosis and screening by PCR.

Collaborative Ring Trial of the Papaya Endogenous Reference Gene and Its Polymerase Chain Reaction Assays for Genetically Modified Organism Analysis

The papaya (Carica papaya L.) Chymopapain (CHY) gene has been reported as a suitable endogenous reference gene for genetically modified (GM) papaya detection in previous studies. Herein, we further validated the use of the CHY gene and its qualitative and quantitative polymerase chain reaction (PCR) assays through an interlaboratory collaborative ring trial. A total of 12 laboratories working on detection of genetically modified organisms participated in the ring trial and returned test results. Statistical analysis of the returned results confirmed the species specificity, low heterogeneity, and single-copy number of the CHY gene among different papaya varieties. The limit of detection of the CHY qualitative PCR assay was 0.1%, while the limit of quantification of the quantitative PCR assay was ∼25 copies of haploid papaya genome with acceptable PCR efficiency and linearity. The differences between the tested and true values of papaya content in 10 blind samples ranged from 0.84 to 6.58%. These results indicated that the CHY gene was suitable as an endogenous reference gene for the identification and quantification of GM papaya.

Improved thermal cycling durability and PCR compatibility of polymer coated quantum dot

Quantum dots have experienced rapid development in imaging, labeling and sensing in medicine and life science. To be suitable for polymerase chain reaction (PCR) assay, we have tested QD thermal cycling durability and compatibility, which have not been addressed in previous reports. In this study, we synthesized CdSe/ZnS QDs with a surface modification with high-MW amphiphilic copolymers and observed that Mg2+ ions in the PCR reaction could induce the QDs to precipitate and reduce their fluorescence signal significantly after thermal cycling. To overcome this problem, we used mPEG2000 to conjugate the QD surface for further protection, and found that this modification enables QDs to endure 40 thermal cycles in the presence of other components essential for PCR reactions. We have also identified that QDs have different effects on rTaq and Ex Taq polymerization systems. A high QD concentration could apparently reduce the PCR efficiency, but this inhibition was relieved significantly in the Ex PCR system as the concentration of Ex Taq polymerase was increased. Real-time PCR amplification results showed that QDs could provide a sufficiently measurable fluorescence signal without excessively inhibiting the DNA amplification. Based on this improved thermal cycling durability and compatibility with the PCR system, QDs have the potential to be developed as stable fluorescent sensors in PCR and real-time PCR amplification.

Analytical Validation of a Practical Molecular Assay Prognostic of Survival in Nonsquamous Non–Small Cell Lung Cancer

A molecular assay prognostic of survival in resected nonsquamous non-small cell lung cancer designed to meet the need for improved risk stratification in early-stage disease has recently been described. This assay measures the expression levels of 14 genes using RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. The assay underwent blinded clinical validation in 2 large international cohorts involving approximately 1500 patients; the analytical precision and reproducibility of this assay, however, have not yet been reported. For each of the 14 TaqMan quantitative polymerase chain reaction (PCR) primer and probe sets used in the molecular prognostic assay, the linear range, PCR efficiency, limits of blank, limits of quantitation, and quantitative bias were determined using serial dilutions of pooled RNA extracted from FFPE samples. The reproducibility of the entire molecular assay was determined by performing repeat testing of FFPE samples over multiple days. The linear range of individual quantitative TaqMan PCR primer and probe sets was between 2(10)- and 2(15)-fold input RNA. The median C(T) of the quantitative PCR primer and probe sets at 10 ng of input RNA was 24.3; the median efficiency was 91.2%. The median quantitative bias across all quantitative PCR primer and probe sets was 0.75% (range, 0.32% to 1.32%). In repeat testing, the mean SD of the risk score (scaled from 1 to 100) was 2.18, with a mean coefficient of variation of 0.08. The molecular prognostic assay presented in this study demonstrates high precision and reproducibility, validating its clinical utility as a reliable prognostic tool that can contribute to the management of patients with early-stage disease.

Error estimation in environmental DNA targets quantification due to PCR efficiencies differences between real samples and standards

IntroductionNowadays, as in the past, our knowledge of microbiology islimited because of the capacity of available technologytools. In recent decades, molecular techniques are playinga central role in the understanding of the microbial world. Inthis sense, a complete scientific evaluation of biologicalsamples is not possible without using molecular biology.The most influential technique has probably been the poly-merasechainreaction(PCR),whichallowsustoworkbeyondclassicalmicrobiology.WithPCR,insomecases,theneedforculture may be avoided. Nevertheless, until the developmentof real-time PCR, our evaluation of the microbial complexityhas been merely qualitative.Real-time PCR is an evolution of the conventional end-point PCR wherein the amplification of a target gene and itsfluorescence detection occurs simultaneously during eachcycle. Different strategies based on the use of nonspecificDNA intercalating dyes or specific fluorescent probes existfor linking target amplification to fluorescence detection.Regardless of the fluorescence emission mechanisms in-volved, real-time PCR enables both detection and quantifica-tion of a genetic target by a continuous fluorescencemonitoring of DNA amplification (Higuchi et al. 1993).An advantage in real-time PCR isthat target quantificationsensitivity is independent of the copy number within thesample (Freeman et al. 1999). Moreover, the key aspect thatdifferentiates real-time PCR from previous semi-quantitativemethodologies is that target DNA concentration can be deter-mined from the fractional cycle at which a threshold amountof target DNA is produced (i.e., measured signal exceeds abackground level), set at a point where amplicon DNA justbecomesdetectablebutisstillwithintheexponentialphaseoftheamplification(Higuchietal.1993;Rasmussen2001).Thisquantificationcycle(Cq)(orcyclethreshold,Ct)areinverselyproportionaltotheamountoftargetnucleicacidinthesample(i.e., the lower the Cq value, the greater the amount of targetnucleic acid in the sample). This approach ensures that inter-fering factors associated with later stages of the amplificationare minimized and considerably improves the measurementprecision (Rasmussen 2001).One of the most important points is that real-time PCRallows quantitative measurements. In order to estimate atarget concentration in environmental samples, absolutequantification can be performed using a standard curveconstructed by amplifying known amounts of target DNA(Rutledge and Cote 2003). Nowadays, this approach is themost commonly used in environmental microbiology.Real-time PCR has many advantages over conventionalendpoint PCR including speed, broad dynamic range ofquantification, reduced risk of contamination, and goodsensitivity. However, despite all these objective strengths,real-time PCR sensitivity is not better than conventionalPCR if a protocol validation is not performed (Bastien etal. 2008).

Rapid real-time PCR detection of transgenic cry1C rice using plasmid molecule as calibrator

Transgenic Bacillus thuringiensis (Bt) rice expressing cry1C gene showed a high level of resistance to leaffolders (Cnaphalocrocis medinalis) and stemborers. Till now, no detection method based on the plasmid molecule as the calibrator has been reported. In this study, one plasmid molecule containing the rice root-specific gene (gos9) endogenous sequence and the cry1C rice 5′ event-specific sequence was developed. Real-time polymerase chain reaction (PCR) method was established using the developed plasmid molecule as the calibrator. Two standard curves for gos9 and the cry1C rice 5′ event-specific sequence showed high PCR efficiency and good linear regression. Limit of quantification of the plasmid molecule in quantitative PCR assays was 40 copies. Biases for 5 and 0.25 % content samples’ quantification were −6.01 and −3.55 % with acceptable standard deviation and repeatability standard deviation, respectively. Comparing with genomic DNA, the plasmid molecule was suitable for cry1C rice quantification as the calibrator. Furthermore, the present study provided a reliable and stable identification and quantification system for monitoring cry1C rice.

Effect of surface charge of PDDA-protected gold nanoparticles on the specificity and efficiency of DNA polymerase chain reaction

The polymerase chain reaction (PCR) has become an indispensable technique in molecular biology, however, it suffers from low efficiency and specificity problems. Developing suitable additives to effectively avoid nonspecific PCR reactions and explore the mechanism for PCR enhancing is a significant challenge. In this paper, we report three different modified gold nanoparticles (AuNPs) with different surface charge polarities and poly (diallyl dimethylammonium) chloride (PDDA) for use as novel PCR enhancers to improve the efficiency and specificity. These AuNPs included the positively charged PDDA protected AuNPs (PDDA-AuNPs), the neutral PDDA-AuNPs modified with excess chloride ion (PDDA.C-AuNPs), and the negatively charged sodium citrate (Na(3)Ct) protected AuNPs (Na(3)Ct-AuNPs). Our data clearly suggests that the positively charged PDDA-AuNPs with an optimum concentration as low as 1.54 pM could significantly enhance the specificity and efficiency of PCR, however, the optimum concentration of the negatively charged Na(3)Ct-AuNPs (2.02 nM) was more than 3 orders of magnitude higher than that of positively charged PDDA-AuNPs. The PCR specificity and efficiency are also improved by the neutral PDDA.C-AuNPs with an optimum concentration, much more than that of the PDDA-AuNPs. This suggests that there should be an electrostatic interaction between the positively charged PDDA-AuNPs and the negatively charged PCR components, and the surface charge polarities of PDDA-AuNPs may play an important role in improving the PCR specificity and efficiency.

Development and validation of qualitative SYBR®Green real-time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes.

A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the “Definition of minimum performance requirements for analytical methods of GMO testing”. The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).

Enhancing the efficiency of polymerase chain reaction using graphene nanoflakes

The effect of the recently developed graphene nanoflakes (GNFs) on the polymerase chain reaction (PCR) has been investigated in this paper. The rationale behind the use of GNFs is their unique physical and thermal properties. Experiments show that GNFs can enhance the thermal conductivity of base fluids and results also revealed that GNFs are a potential enhancer of PCR efficiency; moreover, the PCR enhancements are strongly dependent on GNF concentration. It was found that GNFs yield DNA product equivalent to positive control with up to 65% reduction in the PCR cycles. It was also observed that the PCR yield is dependent on the GNF size, wherein the surface area increases and augments thermal conductivity. Computational fluid dynamics (CFD) simulations were performed to analyze the heat transfer through the PCR tube model in the presence and absence of GNFs. The results suggest that the superior thermal conductivity effect of GNFs may be the main cause of the PCR enhancement.

Evaluation of Usefulness of Polymerase Chain Reaction in the Diagnosis of Malaria in Nigeria

Microscopy has been the most common technique for the diagnosis of malaria in south western Nigeria. This study was undertaken to determine the efficiency of PCR for malaria diagnosis in south western Nigeria. A total of 450 samples submitted for malaria diagnosis at Obafemi Awolowo University Teaching Hospital Complex (OAUTHC), Ile-Ife between the months of January and December, 2009 were used. Methods used included Giemsa staining procedure for estimation of parasite densities and polymerase chain reaction (PCR) to detect the presence of malaria parasite in the whole blood. Using microscopy as reference gold standard, patients comprising 120 males and 330 females with age ranging between less than 1 and 60 samples were used. In all, about 255 (56.7 %) of the samples were positive for microscopy, while 75 (16.7 %) with high parasitaemia on microscopy were positive for PCR analysis. The study concluded that PCR for diagnosis of malaria has sensitivity of 29.4% and specificity of 100% using crude method of DNA extraction while the use of DNA extraction kit has sensitivity of 90.2% and specificity of 100%, hence effort should be geared towards increasing the sensitivity and reduce the cost of doing the test in low resource country like Nigeria.

Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

Analysis of Diverse Plant Species using Mass Spectrometric Cleaved Amplified Polymorphic Sequences (MS-CAPS) and Improvement of PCR Efficiency by Addition of Cysteine

The applicability of mass spectrometric cleaved amplified polymorphic sequences (MS-CAPS) was evaluated in several plant species. This method consists of genomic DNA extraction from plant tissues, polymerase chain reaction (PCR) amplification of a specific genetic region, enzymatic digestion of amplicons, and followed by rapid analysis of single nucleotide polymorphisms (SNPs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Crude extracts obtained by homogenizing plant tissues in water were used as templates for short PCR amplifications for MS-CAPS analysis. For most plant species tested, these crude extracts could be used directly as templates for PCR. However, extracts from lettuce leaves and stems showed enzymatic browning as a result of polyphenol oxidase (PPO) activity, and were not suitable PCR templates. The addition of cysteine to the homogenizing solution inhibited enzymatic browning and did not affect the other MS-CAPS procedures, including PCR amplification, uracil-DNA glycosylase treatments, or MALDI-TOF MS analysis. Thus, this method inhibits PPO in crude extracts, allowing them to be used directly for MS-CAPS analysis.

Effects of Superparamagnetic Nanoparticle Clusters on the Polymerase Chain Reaction

The polymerase chain reaction (PCR) method is widely used for the reproduction and amplification of specific DNA segments, and a novel PCR method using nanomaterials such as gold nanoparticles has recently been reported. This paper reports on the effects of superparamagnetic nanoparticles on PCR amplification without an external magnetic field, and clarifies the mechanism behind the effects of superparamagnetic particle clusters on PCR efficiency by estimating the structures of such clusters in PCR. It was found that superparamagnetic nanoparticles tend to inhibit PCR amplification depending on the structure of the magnetic nanoparticle clusters. The paper also clarifies that Taq polymerase is captured in the spaces formed among magnetic nanoparticle clusters, and that it is captured more efficiently as a result of their motion from heat treatment in PCR thermal cycles. Consequently, Taq polymerase that should be used in PCR is reduced in the PCR solution. These outcomes will be applied to novel PCR techniques using magnetic particles in an external magnetic field.

Microsatellite Markers Within the α-Globin Gene Cluster for Robust Preimplantation Genetic Diagnosis of Severe α-Thalassemia Syndromes in Mediterranean Populations

In this study we report the development of a generic protocol for preimplantation genetic diagnosis (PGD) of severe α-thalassemia (α-thal) syndromes in α-thal carrier couples of Mediterranean origin. The in silico identification and design of primers for multiplex analysis of short tandem repeats (STRs), was followed by the optimization of polymerase chain reaction (PCR) conditions for multiplexed STR analysis within the α-globin gene cluster (16p3.3) and subsequent optimization and validation of a single-cell multiplex reaction including the selected STRs. Three simple dinucleotide repeats were selected based on their rate of heterozygosity, multiplex PCR efficiency and product size, and location within the α-globin gene cluster. The multiplex PCR was optimized in single lymphocytes with PCR efficiency ranging from 92.5 to 98% and an allele drop-out (ADO) rate of 0 to 9.0% for the three loci. The optimized method was applied in two clinical PGD cycles and genotypes were achieved in 17 out of 18 blastomeres (94%). Transfer of unaffected embryos led to a singleton pregnancy in one of the two couples. The triplex PCR validated for Greek and Cypriot populations is a robust generic method for α-thal PGD.

Enhancing the specificity and efficiency of polymerase chain reaction using polyethyleneimine-based derivatives and hybrid nanocomposites

There is a general necessity to improve the specificity and efficiency of the polymerase chain reaction (PCR), and exploring the PCR-enhancing mechanism still remains a great challenge. In this paper we report the use of branched polyethyleneimine (PEI)-based derivatives and hybrid nanocomposites as a novel class of enhancers to improve the specificity and efficiency of a nonspecific PCR system. We show that the surface-charge polarity of PEI and PEI derivatives plays a major role in their effectiveness to enhance the PCR. Positively charged amine-terminated pristine PEI, partially (50%) acetylated PEI (PEI-Ac50), and completely acetylated PEI (PEI-Ac) are able to improve PCR efficiency and specificity with an optimum concentration order of PEI < PEI-Ac50 < PEI-Ac, whereas negatively charged carboxyl-terminated PEI (PEI-SAH; SAH denotes succinamic acid groups) and neutralized PEI modified with both polyethylene glycol (PEG) and acetyl (Ac) groups (PEI-PEG-Ac) are unable to improve PCR specificity and efficiency even at concentrations three orders of magnitude higher than that of PEI. Our data clearly suggests that the PCR-enhancing effect is primarily based on the interaction between the PCR components and the PEI derivatives, where electrostatic interaction plays a major role in concentrating the PCR components locally on the backbones of the branched PEI. In addition, multiwalled carbon nanotubes modified with PEI and PEI-stabilized gold nanoparticles are also able to improve the PCR specificity and efficiency with an optimum PEI concentration less than that of the PEI alone, indicating that the inorganic component of the nanocomposites may help improve the interaction between PEI and the PCR components. The developed PEI-based derivatives or nanocomposites may be used as efficient additives to enhance other PCR systems for different biomedical applications.