Amplification Yield Research Articles

Direct Fluorescence Detection of Allele-Specific PCR Products Using Novel Energy-Transfer Labeled Primers.

Background: Currently analysis of point mutations can be done by allele-specific polymerase chain reaction (PCR) followed by gel analysis or by gene-specific PCR followed by hybridization with an allele-specific probe. Both of these mutation detection methods require post-PCR laboratory time and run the risk of contaminating subsequent experiments with the PCR product liberated during the detection step. The author has combined the PCR amplification and detection steps into a single procedure suitable for closed-tube analysis. Methods and Results: Allele-specific PCR primers were designed as Sunrise energy-transfer primers and contained a 3' terminal mismatch to distinguish between normal and mutant DNA. Cloned normal (W64) and mutant (R64) templates of the beta3-adrenergic receptor gene were tested to verify amplification specificity and yield. A no-target negative control was also run with each reaction. After PCR, each reaction was tested for fluorescence yield by measuring fluorescence on a spectrofluorimeter or fluorescent microtitreplate reader. The cloned controls and 24 patient samples were tested for the W64R mutation by two methods. The direct fluorescence results with the Sunrise allele-specific PCR method gave comparable genotypes to those obtained with the PCR/ restriction digest/gel electrophoresis control method. No PCR artifacts were observed in the negative controls or in the PCR reactions run with the mismatched target. Conclusions: The results of this pilot study indicate good PCR product and fluorescence yield from allele-specific energy-transfer labeled primers, and the capability of distinguishing between normal and mutant alleles based on fluorescence alone, without the need for restriction digestion, gel electrophoresis, or hybridization with an allele-specific probe.

Enhancing the efficiency of a PCR using gold nanoparticles

We found that the PCR could be dramatically enhanced by Au nanoparticles. With the addition of 0.7 nM of 13 nm Au nanoparticles into the PCR reagent, the PCR efficiency was increased. Especially when maintaining the same or higher amplification yields, the reaction time could be shortened, and the heating/cooling rates could be increased. The excellent heat transfer property of the nanoparticles should be the major factor in improving the PCR efficiency. Different PCR systems, DNA polymerases, DNA sizes and complex samples were compared in this study. Our results demonstrated that Au nanoparticles increase the sensitivity of PCR detection 5- to 10-fold in a slower PCR system (i.e. conventional PCR) and at least 104-fold in a quicker PCR system (i.e. real-time PCR). After the PCR time was shortened by half, the 100 copies/µl DNA were detectable in real-time PCR with gold colloid added, however, at least 106 copies/µl of DNA were needed to reach a detectable signal level using the PCR reagent without gold colloid. This innovation could improve the PCR efficiency using non-expensive polymerases, and general PCR reagent. It is a new viewpoint in PCR, that nanoparticles can be used to enhance PCR efficiency and shorten reaction times.

Méthode de dénombrement des leucocytes résiduels dans les plasmas par amplification génomique en temps réel

Background. – Systematic plasma leukoreduction, which was introduced in France in April 1st 2001, has given rise to more sensitive methods for residual leukocytes counting. The technologies in application at this moment (Nageotte hemocytometers and flow cytometry methods) have been modified by a thirty fold sample concentration prior to analysis, inducing frequent downgrading of the plasma unit. So, in order to improve the detection threshold, we developed a more sensitive assay using “Real-Time Polymerase Chain Reaction” technology. Materials and methods. – Real-time polymerase chain reaction was performed on a highly conserved HLADQα1 gene sequence. In order to determine the analytical performances of the method (accuracy, sensitivity, linearity and specificity) serial dilutions series ranging from 10 4 to 1 cells/ml were performed. A total of 18 series were prepared from three leukocyte stock solutions, by 1 in 10 serial dilutions in three plasmas completely devoided of white cells (called negative plasmas). To examine the specificity of the assay two negative controls were analyzed in each run. Results. – a sensitivity of 10 cells/ml (10 4 leukocytes/l) was achieved and the assay was linear between 10 and 10 4 cells/ml. The slope (–3.72) of the average standard curve calculated from all series, showed an amplification yield of 92.85%. Conclusion. – we developed a quantitative assay for residual leukocytes in leukodepleted plasma, that agreed with the quality control requirement specifications.

Optimal Storage Conditions for Highly Dilute DNA Samples: A Role for Trehalose as a Preserving Agent

DNA extraction from trace samples or noninvasively collected samples often results in the recovery of low concentration solutions of DNA that are prone to DNA degradation or other loss. Because of the difficulty in obtaining such samples, and their potentially high value in wildlife and forensic studies, it is critical that optimal methods are employed for their long-term storage. We assessed the amplification yield of samples kept under different storage conditions with the addition of potential preserving agents. We stored dilutions of known concentration human placental DNA, and gorilla fecal DNA, under four conditions (+4 degrees C, -20 degrees C, -80 degrees C, dry at room temperature), and with three additives (Tris EDTA (TE) buffer, Hind III digested Lambda DNA, trehalose). The effectiveness of the treatment methods was tested at regular intervals using qPCR to assess the quantity of amplifiable DNA, and a PCR assay of a larger 757 bp fragment to evaluate the quality of that remaining DNA. The highest quantity of DNA remained in samples stored at -80 degrees C, regardless of storage additives, and those dried at room temperature in the presence of trehalose. Surprisingly, DNA quality was best preserved in the presence of trehalose, either dried or at -80 degrees C; significant quality loss occurred with -20 degrees C and +4 degrees C storage.

Comparison of yield and genotyping performance of multiple displacement amplification and OmniPlex™ whole genome amplified DNA generated from multiple DNA sources

The promise of whole genome amplification (WGA) is that genomic DNA (gDNA) quantity will not limit molecular genetic analyses. Multiple displacement amplification (MDA) and the OmniPlex PCR-based WGA protocols were evaluated using 4 and 5 ng of input gDNA from 60 gDNA samples from three tissue sources (mouthwash, buffy coat, and lymphoblast). WGA DNA (wgaDNA) yield and genotyping performance were evaluated using genotypes determined from gDNA and wgaDNA using the AmpFlSTR Identifiler assay and N = 49 TaqMan SNP assays. Short tandem repeat (STR) and SNP genotyping completion and concordance rates were significantly reduced with wgaDNA from all WGA methods compared with gDNA. OmniPlex wgaDNA exhibited a greater reduction in genotyping performance than MDA wgaDNA. Reduced wgaDNA genotyping performance was due to allelic (all protocols) and locus (OmniPlex) amplification bias leading to heterozygote and locus dropout, respectively, and %GC sequence content (%GC) was significantly correlated with TaqMan assay performance. Lymphoblast wgaDNA exhibited higher yield (OmniPlex), buffy coat wgaDNA exhibited higher STR genotyping completion (MDA), whereas mouthwash wgaDNA exhibited higher SNP genotyping discordance (MDA). Genotyping of wgaDNA generated from < or = 5 ng gDNA, e.g., from archaeological, forensic, prenatal diagnostic, or pathology samples, may require additional genotyping validation with gDNA and/or more sophisticated analysis of genotypes incorporating observed reductions in genotyping performance.

Utilization of polymerase chain reaction on archival cytologic material: a comparison with fresh material with special emphasis on cerebrospinal fluids

Use of the polymerase chain reaction (PCR) for the detection of B- and T-cell clonality, Epstein–Barr virus (EBV) and Human Herpes Virus 8 (HHV 8) infection is gaining increasing importance as a diagnostic modality. These tests are usually performed on fresh specimens. There are instances when fresh material is not available and there is a clinical utility for the performance of PCR on archival material via slide scrape lysates (SSL). However, the suitability of archival material may be questioned. Records were searched for all archival cytology cases submitted for SSL molecular diagnostics tests since 1998. Results for each case were analyzed for PCR amplification status and individual test results. A randomly chosen control group of equivalent cytologic samples submitted fresh was evaluated for comparison of amplification status. In all, 241 PCR runs were performed on SSL of archival material from 112 cytologic samples (89 cerebrospinal fluids (CSFs), 13 fine-needle aspirates (FNAs), 10 effusions). Out of these samples, 95 (85%) had amplifiable DNA, as assessed by a positive reaction for glyceraldehyde phosphate dehydrogenase (GAPDH). For the control group, 320 PCR runs were performed on 112 fresh cytologic samples (89 CSFs, 13 FNAs, 10 effusions). In total, 102 samples (91%) had amplifiable DNA. There was no statistical difference in the amplification yield between the two groups (P=0.2177). A morphologic review of 16 of the 17 SSL archival cytologic cases that did not show amplification revealed 11/16 to be of sparse cellularity. Molecular diagnostic tests are performed routinely on fresh cytologic samples with excellent results. At times critical decisions on patient care may need to be made when fresh tissue is not available for molecular diagnostic tests. SSL of archival cytologic material can be used with excellent results for molecular diagnostic tests when fresh material is not available or when the cytologic diagnosis needs further clarification.

Rapid preparative purification of West Nile and Sindbis virus PCR products utilizing a microbore anion-exchange column

Analysis and purification of specific PCR products from PCR reactions can be problematic due to several issues relating to amplification and low product yield. The use of HPLC as a preparative tool in PCR product analysis is common but has not replaced traditional electrophoretic techniques for purifying DNA to be used in subsequent experiments. Gel purification of PCR products can result in a net loss greater than 50% of the starting DNA amount. Thus, this method of recovery can become the limiting factor in the overall cloning protocol. This paper describes a simple and relatively inexpensive micro-preparative HPLC method to purify and analyze nM quantities of DNA. A microbore polyethyleneimine-based anion-exchange column fractionates PCR mixtures in less than 40 min with a recovery of the purified specific product as high as 80%, thus eliminating the need for gel purification. Using this method, nested PCR products from Sindbis virus differing by 18 bp in some cases and a 277 bp fragment from West Nile virus were resolved and quantified. This method differs from existing methodologies because separation is based on size and charge as well as the overall G + C content of the PCR product.

Multiplex real-time quantitative RT-PCR assay for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1

A multiplex real-time quantitative reverse transcription (RT)-PCR assay was developed for simultaneous detection, identification and quantification of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) in plasma or serum samples. Genomic amplification of one virus was unaffected by the simultaneous amplification of the other two. Competition between HCV and HIV-1 amplifications slightly affected the yield of HIV-1 amplification. However, quantitation was possible when a single virus was present. The 95% detection limits were 30, 167 and 680 IU/ml for HBV DNA, HCV RNA and HIV-1 RNA, respectively. The multiplex assay detected with similar efficiency strains of HBV genotypes A–F, HCV genotypes 1–6, and HIV-1 subtypes A–G. Applied to 267 pools of 10 plasmas from blood donors, multiplex screening indicated that the assay was reproducible, sensitive, and specific. This assay has the potential to be used for large-scale nucleic acid testing (NAT) of blood donations.

Influence of histochemical stains on quantitative gene expression analysis after laser-assisted microdissection

Laser-assisted microdissection (LAM) allows isolation of specific cell populations for molecular studies. The combination of LAM and of real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) enables generation of quantitative cell-specific gene expression data. Histochemical stains used to identify cells desired for LAM should provide acceptable morphology and not interfere with RNA or with subsequent molecular analysis techniques. To determine a reliable stain for analysing RNA, using the housekeeping gene, RPL13A, we performed quantitative gene expression analysis of laser microdissected cells from prostatic frozen tissues. The frozen sections were histochemically stained with hematoxylin, methyl green, toluidine blue O and May-Grunwald. After laser microdissection real-time quantitative RT-PCR was performed. Methyl green yielded more RT-PCR product than did the other dyes. The lowest yield of amplification was obtained after May-Grunwald staining. Therefore we recommend methyl green for general use in gene expression analysis, especially when handling small amounts of RNA.

Mitochondrial DNA analysis of ancient human teeth from a XVIth century archeological excavation

Our laboratories are involved in a large-scale study investigating the different hypotheses about the founding Canary Islands populations—making use of physical–anthropological, genetic and archeological approaches. The main problems associated with ancient DNA studies are low yield, high fragmentation and contamination with exogenous DNA. In this study, we tried to evaluate the viability of our laboratory set-up to accomplish mitochondrial DNA (mtDNA) isolation and analysis from ancient samples. We present preliminary results of HVI region mtDNA sequencing of several teeth from the Monastery of Los Silos (North of Tenerife Island), an archeological excavation site dating back to the XVIth century, used as a graveyard for some time. We also make some considerations about the amplification yield by comparing two different PCR strategies.

Variations in primer sequences are the origin of allele drop-out at loci D13S317 and CD4.

STRs have become almost the exclusive tool of genetic scientists in forensic typing work. Consequently, large numbers of samples are genotyped and the detection of rare abnormalities is to be expected. We found rare losses of alleles, also known as drop-out, at the two STR loci D13S317 and CD4. Drop-out at D13S317 was accidentally found in typing of suspects in a murder case and three other examples of drop-out were found at locus CD4 during paternity testing. The lost alleles reappeared when alternative PCR primer pairs were used. Sequences of lost alleles were characterised at the molecular level after cloning. Variations were found in the primer sequences and these are believed to prevent amplification or to reduce amplification yield and to be the origin of the allele drop-out.

Linked Linear Amplification: A New Method for the Amplification of DNA

Linked Linear Amplification (LLA) is a new nucleic acid amplification method that uses multiple cycles of primer extension reactions. The presence of nonreplicable elements in LLA primers renders primer extension products unusable as templates for further amplification, leading to linear accumulation of products. Through the use of nested primers, linear reactions can be "linked", providing total amplification yields comparable to those obtained by PCR. The LLA model predicts (a) that amplification yield will approach that of PCR as the number of primers increases and (b) that the unique composition of LLA products will give lower carryover amplification efficiency compared with PCR. To test these hypotheses, the human ss-globin gene was amplified by 10-, 14-, or 18-primer LLA and the yield was compared with PCR. Carryover contamination was simulated by reamplifying a dilution series of LLA or PCR products. To demonstrate the clinical utility of the method, LLA coupled with allele-specific oligonucleotide (ASO) capture was used to detect the factor V Leiden mutation in a panel of 111 DNA samples. Fourteen- and 18-primer LLA gave amplification yields comparable to PCR. However, LLA carryover amplification efficiency was four orders of magnitude lower than that of PCR. The LLA-ASO assay detected the correct factor V Leiden genotype in all 111 samples. LLA is a robust target amplification method that is comparable to PCR in yield. However, LLA is more resistant to false results caused by carryover amplicon contamination.

New specificity and yield enhancer of polymerase chain reactions.

Tetramethylammonium (TMA) chloride, dimethyl sulfoxide and formamide are known to increase, under certain conditions, the specificity and efficiency of the polymerase chain reaction (PCR). We compared the ability of several TMA derivatives and some other reagents to increase the specificity of PCR and to improve the yield of amplification. A novel combination of the enhancer TMA and oxalate as anion is demonstrated to be a powerful enhancer of PCR. Addition of 2 mM TMA oxalate to the PCR mixture decreases the formation of non-specific DNA fragments and increases the yield of specific PCR products.

Preparation of a Sol-Gel Broadband Antireflective and Scratch-Resistant Coating for Amplifier Blastshields of the French Laser LIL

A broadband antireflective and scratch resistant coating made from tantalum and silicon oxide-based layers has been developed using the sol-gel route for amplifier blastshields of the french laser LIL. Sol-gel synthesis have been carried out starting from cheap precursors in order to produce metallic-based solutions, each one suitable for liquid-deposition technique use such as dip-coating. Baking step temperature does not exceed 150°C during 30 minutes allowing to coat various substrates and to offer scratch-resistance properties in compliance with US-MIL-C-0675C severe test. The antireflective properties of the coating (layer thickness) were optimized to maximize the amplification yield (in comparaison to uncoated blastshield), taking into account the optical and geometrical parameters of the multipass amplification laser cavity. Experimental antireflective coating deposition process onto large-area glass plates sized to the LIL amplifier blastshields (1811 × 635 × 6 mm) is described.

Optimization of an automated DNA purification protocol for neonatal screening.

Collection of blood from newborns is a standard clinical procedure used for genetic screening. Typically, blood from a heel prick is absorbed onto standard collection paper and dried before analysis of metabolites, proteins, hormones, and more recently DNA. To evaluate strategies to purify DNA for use with automated workstations. Two factors were used to evaluate several DNA purification protocols: residual heme contamination and amplification yield. The protocol that produced DNA with the lowest heme content and the highest amplification yield was selected. In combination with those two performance factors, the protocol with the fewest number of steps was chosen to reduce reagent use and processing time. Industrial research and development laboratory. Robust amplification of DNA isolated from dried blood spots was demonstrated using both fluorescence and agarose gel-based detection methods. In addition, the samples had consistent DNA volumes and had no detectable cross-contamination. Suggested instrument settings, equipment, and supplies were included for automated processing of DNA from dried blood spots. A 4-step DNA processing protocol was developed for dried blood spots. The protocol could be performed in either a manual or automated format, making it possible to process hundreds of samples in 1 day.

Endonuclease-Mediated Long PCR and Its Application to Restriction Mapping

The polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for PCR have been extended significantly by the development of "long" PCR, a technique that makes it possible to amplify DNA fragments up to 40 kb in length. This article describes two novel applications of the long PCR technique, one which simplifies restriction mapping and another which enhances amplification specificity and yield. The same primers used to perform the long PCR amplification can be used as probes to perform restriction mapping of the DNA fragment amplified. Restriction digestion performed prior to long PCR amplification can be used to selectively suppress the amplification of members of families of closely related DNA sequences, thereby making it possible to selectively amplify one of a group of highly homologous sequences. These two complimentary techniques, both involving use of the long PCR paired with restriction digestion, have potential application in any laboratory in which PCR is performed.

DAF optimization using Taguchi methods and the effect of thermal cycling parameters on DNA amplification.

Taguchi methods, which are widely applied in industrial process design, were used to optimize DNA amplification finger-printing (DAF). Quadratic loss functions that penalize deviations from prediction values and L9 (3(4)) and L18 (3(8)) orthogonal arrays revealed effects and interactions of amplification reaction components and thermal cycling parameters. Analysis of variance (ANOVA) decomposed the contribution of individual factors to the experimental response (amplification yield and product number), while verification experiments established that optimum conditions were predictable, verifiable and reproducible. While several amplification components (primer, magnesium and enzyme) conditioned the amplification reaction, annealing temperature and time were the only important thermal cycling contributing factors. The Taguchi strategy defined a robust and transportable amplification protocol based on high annealing temperatures (typically 48 degrees C) and primer concentrations (typically 8 microM), which can be applied to the fingerprinting of a wide range of DNA templates of plant and fungal origin. The general strategy of robust experimental design holds potential as an optimization tool for other methods in molecular biology.

Improved method facilitates reliable APOE genotyping of genomic DNA extracted from formaldehyde-fixed pathology specimens

Apolipoprotein E (APOE) genotyping of genomic DNA extracted from formaldehyde-fixed specimens is cumbersome: there is not only a low yield or failure of PCR amplification (presumably due to degradation of DNA in the formaldehyde-fixed and paraffin-embedded tissue), but the standard method also involves the separation of DNA fragments as small as 48, 72, 81 and 91 bp requiring high-yield PCR products. Here we report about a semi-nested PCR method suitable for providing specific high-yield PCR products from DNA that has been extracted from formaldehyde-fixed specimens which initially generate low-quality templates. This method facilitates reliable APOE genotyping of DNA from difficult templates.

Theoretical Description of the Polymerase Chain Reaction

Taking into account expressions for the efficiency of the polymerase chain reaction (PCR) recently deduced from enzymological considerations, and making use of a continuous model based on the law of mass action, closed form solutions are derived that enable a complete description of the standard and quantitative competitive PCR methods. The resulting behaviour is in reasonable agreement with that from a previous, empirical, discrete approach; but the latter is nonetheless shown to overestimate the amplification yield by as much as 30% due to the weak assumption of a constant efficiency during the cycle duration. The present formalism will facilitate the implementation of accurate fitting procedures of experimental data to manage quantification.

Chromosome-specific panels of tri- and tetranucleotide microsatellite markers for multiplex fluorescent detection and automated genotyping: evaluation of their utility in pathology and forensics.

A set of 391 microsatellite markers (Weber set 6), 85% of which consist of tri- and tetranucleotide repeat markers, were used to design chromosome-specific panels that allowed for a high degree of multiplexing with respect to the fragment size range and fluorophore (FAM, HEX, TET). This marker set has an average coverage of 10.5 cM, with the largest gap being 28.1 cM. The markers were divided into 49 panels, with a maximum degree of multiplexing of 15 markers per panel. The utility of the markers for analysis of DNA from blood, hair, and formalin-fixed archival tissue biopsies was evaluated with respect to amplification efficiency, product yield, and degree of preferential amplification of the shorter allele in heterozygotes. The amplification efficiency was inversely related to repeat length and amplicon length. Based on the analysis of DNA from formalin-fixed biopsies, 51 markers suitable for loss of heterozygosity (LOH) studies were identified. The utility of the marker set for genome scanning, LOH, and forensic analyses is discussed.