Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction

Six TaqMan real-time polymerase chain reaction (PCR) systems using minor groove binding (MGB) probes have been developed for the detection quantitation of bovine, porcine, lamb, chicken, turkey, and ostrich DNA in complex samples. Species-specific amplification was achieved by combining only two fluorogenic probes and 10 oligonucleotide primers targeting mitochondrial sequences, decreasing the cost of the assay significantly. The limits of detection ranged from 0.03 to 0.80 pg of template DNA. Analysis of experimental mixtures containing two to four different species showed the suitability of the assay for detection of more than 1% of pork, chicken, or turkey and of more than 5% of cattle or lamb. The quantitation accuracy in samples containing 10-100% of beef or pork DNA was close to 90%. The system is complemented with one additional TaqMan MGB detector based on consensus sequence segments of the nuclear 18S ribosomal RNA gene. A method to evaluate the presence of unknown eukaryotic DNA in a mixture, where data derived from the species-specific detection are compared with the experimental values obtained from the general 18S detector, is presented. This method allows the validation of the quantitative measurements, providing an internal control of the total content of PCR-amplifiable DNA in the sample. The system was tested on DNA mixtures containing different shares of up to four different species and on DNA extracted from processed commercial food samples.     

A rapid chemiluminescent method for quantitation of human DNA.

A sensitive and simple method for the quantitation of human DNA is described. This method is based on probe hybridization to a human alpha satellite locus, D17Z1. The biotinylated probe is hybridized to sample DNA immobilized on nylon membrane. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for chemiluminescent detection using a luminol-based reagent and X-ray film. Less than 150 pg of human DNA can easily be detected with a 15 minute exposure. The entire procedure can be performed in 1.5 hours. Microgram quantities of nonhuman DNA have been tested and the results indicate very high specificity for human DNA. The data on film can be scanned into a computer and a commercially available program can be used to create a standard curve where DNA quantity is plotted against the mean density of each slot blot signal. The methods described can also be applied to the very sensitive determination of quantity and quality (size) of DNA on Southern blots. The high sensitivity of this quantitation method requires the consumption of only a fraction of sample for analysis. Determination of DNA quantity is necessary for RFLP and many PCR-based tests where optimal results are obtained only with a relatively narrow range of DNA quantities. The specificity of this quantitation method for human DNA will be useful for the analysis of samples that may also contain bacterial or other non-human DNA, for example forensic evidence samples, ancient DNA samples, or clinical samples.     

Effects of humic substances on fluorometric DNA quantification and DNA hybridization

DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities. Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic substances and vascular plant extract interfered with the fluorometric measurement of DNA concentration using Hoechst dye H33258 and PicoGreen reagent. Quantification of DNA amended with humic substances (20-80 ng/microl) using the Hoechst dye assay was more reliable than with PicoGreen reagent. A simple procedure was developed to improve the accuracy for determining the DNA concentration in the presence of humic substances. In samples containing up to 80 ng/microl of humic acids, the fluorescence of the samples were measured twice: first without Hoechst dye to ascertain any fluorescence from impurities in the DNA sample, followed with Hoechst dye addition to obtain the total sample fluorescence. The fluorescence of the Hoechst dye-DNA complex was calculated by subtracting the fluorescence of the impurities from the fluorescence of the sample. Vascular plant extract and humic substances reduced the binding of DNA onto the nylon membrane. Low amounts (<2.0 microg) of humic substances derived from estuarine waters did not affect the binding of 100 ng of target DNA to nylon membranes. DNA samples containing 1.0 microg of humic substances performed well in DNA hybridizations with DIG-labeled oliogonucleotide and chromosomal probes. Therefore, we suggest that DNA samples containing low concentrations of humic substances (<20 ng/microl) could be used in quantitative membrane hybridization without further purification.     

Facile determination of DNA-binding nuclear factor-kappaB by chemiluminescence detection

A simple, rapid, and sensitive method for the assay of a sequence-specific DNA-binding protein, nuclear factor-kappaB (NF-kappaB), has been developed by using a DNA-detectable chemiluminogenic reagent and a centrifugal filter that distinguishes different molecular sizes. After the formation of a complex between NF-kappaB and DNA, the unbound DNA is separated from the complex by the centrifugal filter. The amount of the bound NF-kappaB is estimated by chemiluminescence detection of the bound DNA. This detection is performed within 2 min at room temperature by the use of a chemiluminogenic reagent, 3',4',5'-trimethoxyphenylglyoxal, which selectively recognizes guanine moiety in oligonucleotides or DNAs. This method does not require any labeled probes or antibodies and can determine a concentration as low as 5 nM of DNA-binding NF-kappaB. The sensitivity is nearly the same as that of other methods such as gel shift assay using fluorescence-labeled probes and enzyme-linked immunosorbent assay. Therefore, the current method provides a convenient tool for surveying various DNA-binding proteins.     

An electrophoretic capture method for efficient recovery of rare sequences from heterogeneous DNA

It is difficult to isolate rare, PCR-quality DNA from specimens containing large quantities of nonspecific DNA from multiple sources (heterogeneous DNA). Extracting human DNA from stool for colorectal cancer (CRC) screening tests exemplifies this technically challenging sample preparation problem. The stool matrix is complex, the DNA composition heterogeneous, and CRC-associated mutated DNA is rare. This report describes a novel solid phase DNA sequence-specific hybrid capture sample preparation method: the reversible electrophoretic capture affinity protocol (RECAP). We show that RECAP, compared with other methods, is capable of extracting linearly increasing amounts of human DNA from increasing amounts of total stool DNA in a manner that avoids co-purifying PCR inhibitors. RECAP thereby increases the yield of rare mutated DNA molecules and thus increases the detection sensitivity for CRC-associated mutations.     

Sequence specificity modification of double-stranded DNA with an alkylating derivative of oligodeoxyribonucleotide pT(CT)6

It is shown that in slightly acidic solution (pH approximately 5.3) reagent CIRCH2NHpT(CT)6 (RCl = -C6H4-N(CH3)CH2CH2Cl) modifies a double-stranded DNA fragment (120 b. p.) containing A(GA)6.T(CT)6 sequence at a single nucleotide residue, viz. G29 located near to this sequence in the DNA chain. The location of this modification point suggests formation of a triple-stranded reactive complex with parallel orientation of the pyrimidine oligonucleotide moiety of the reagent and pyrine sequence of the target DNA. Analysing the modification extent dependence of the reagent concentration the association constant Kx between the reagent and DNA was calculated (Kx = (0.95 +/- 0.03).10(5) M-1, 25 degrees C, pH = 5.3, [NaCl] = 0.1 M). The modification by the reagent ClRCH2NHpT(m5CT)6 has the same quantitative characteristics as in the case of ClRCH2NHpT(CT)6.     

The impact of endogenous content,replicates and pooling on genome capture from faecal samples

Target‐capture approach has improved over the past years, proving to be very efficient tool for selectively sequencing genetic regions of interest. These methods have also allowed the use of noninvasive samples such as faeces (characterized by their low quantity and quality of endogenous DNA) to be used in conservation genomic, evolution and population genetic studies. Here we aim to test different protocols and strategies for exome capture using the Roche SeqCap EZ Developer kit (57.5 Mb). First, we captured a complex pool of DNA libraries. Second, we assessed the influence of using more than one faecal sample, extract and/or library from the same individual, to evaluate its effect on the molecular complexity of the experiment. We validated our experiments with 18 chimpanzee faecal samples collected from two field sites as a part of the Pan African Programme: The Cultured Chimpanzee. Those two field sites are in Kibale National Park, Uganda (N = 9) and Loango National Park, Gabon (N = 9). We demonstrate that at least 16 libraries can be pooled, target enriched through hybridization, and sequenced allowing for the genotyping of 951,949 exome markers for population genetic analyses. Further, we observe that molecule richness, and thus, data acquisition, increase when using multiple libraries from the same extract or multiple extracts from the same sample. Finally, repeated captures significantly decrease the proportion of off‐target reads from 34.15% after one capture round to 7.83% after two capture rounds, supporting our conclusion that two rounds of target enrichment are advisable when using complex faecal samples.     

A roadmap for high‐throughput sequencing studies of wild animal populations using noninvasive samples and hybridization capture

Large‐scale genomic studies of wild animal populations are often limited by access to high‐quality DNA. Although noninvasive samples, such as faeces, can be readily collected, DNA from the sample producers is usually present in low quantities, fragmented, and contaminated by microorganism and dietary DNAs. Hybridization capture can help to overcome these impediments by increasing the proportion of subject DNA prior to high‐throughput sequencing. Here we evaluate a key design variable for hybridization capture, the number of rounds of capture, by testing whether one or two rounds are most appropriate, given varying sample quality (as measured by the ratios of subject to total DNA). We used a set of 1,780 quality‐assessed wild chimpanzee (Pan troglodytes schweinfurthii) faecal samples and chose 110 samples of varying quality for exome capture and sequencing. We used multiple regression to assess the effects of the ratio of subject to total DNA (sample quality), rounds of capture and sequencing effort on the number of unique exome reads sequenced. We not only show that one round of capture is preferable when the proportion of subject DNA in a sample is above ~2%–3%, but also explore various types of bias introduced by capture, and develop a model that predicts the sequencing effort necessary for a desired data yield from samples of a given quality. Thus, our results provide a useful guide and pave a methodological way forward for researchers wishing to plan similar hybridization capture studies.     

Immunochromatographic assay for the detection of pseudojujubogenin glycosides

INTRODUCTION: Bacopa monnieri contains pseudojujubogenin glycosides as pharmacologically active compounds. In order to screen large numbers of plant samples for the presence of pseudojujubogenin glycosides, a rapid and simple assay system is required for application to small quantities of test materials. Immunoassays using monoclonal antibodies could be useful for the determination of small quantities of pseudojujubogenin glycosides in plant extracts. OBJECTIVE: The objective of this work was to develop a simple method for the detection of pseudojujubogenin glycosides by the immunochromatographic strip test using anti-bacopaside I monoclonal antibody. METHODOLOGY: The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of a colloidal gold particle coated with the respective anti-bacopaside I MAb. The capture reagent was a bacopaside I-human serum albumin conjugate immobilised onto a test strip membrane. RESULTS: The sample containing pseudojujubogenin glycosides and the detection reagent were incubated with the immobilised capture reagent. The glycosides in the sample competed in binding to the limited amount of antibodies in the detection reagent with the immobilised bacopaside I-HSA conjugates and, hence, positive samples showed no colour in the capture spot zone. The detection limit for the strip test was 125 ng/mL. CONCLUSION: The assay system was found to be useful as a rapid and simple screening method for the detection of pseudojujubogenin glycosides in plants.     

Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens

We developed a conceptually new subtraction strategy for the detection and isolation of target DNA and/or RNA from complex nucleic acid mixtures, called Primer Extension Enrichment Reaction (PEER). PEER uses adapters and class IIS restriction enzymes to generate tagged oligonucleotides from dsDNA fragments derived from specimens containing an unknown target ('tester'). Subtraction is achieved by selectively disabling these oligonucleotides by extension reaction using ddNTPs and a double stranded DNA template generated from a pool of normal specimens ('driver'). Primers that do not acquire ddNTP are used to capture and amplify the unique target DNA from the original tester dsDNA. We successfully applied PEER to specimens containing known infectious agents (Hepatitis B Virus and Walrus Calicivirus) and demonstrated that it has higher efficiency than the best comparable technique. The strategy used for PEER is versatile and can be adapted for the identification of known and unknown pathogens and mutations, differential expression studies and other applications that allow the use of subtractive strategies.     

Parallel sample preparation of proteins,from crude samples to crystals ready for MALDI-MS,in an integrated microfluidic system

A microfluidic structure is presented where selective capture of proteins in complex samples, followed by clean-up, enzymatic processing, and MALDI-MS sample preparation of peptides generated, can be performed. The structure uses an affinity column to capture the protein while all other components in the sample are disposed of. The protein of interest is then eluted from the affinity column and captured on a second column on which the enzymatic processing is performed. Salts and hydrophilic contaminants are then removed before the products from the enzymatic reaction are eluted together with a suitable MALDI matrix and the solvent evaporated in a designated MALDI target structure. All steps can be performed automatically in 54 parallel microstructures on a microfluidic compact disc. The process is demonstrated by the selective capture and tryptic digest of recombinant IgG molecules from samples containing other proteins: an excess of bovine serum albumin or spent cell culture media.     

Quantitation of supercoiled DNA cleavage in nonradioactive DNA: application to ionizing radiation and synthetic endonuclease cleavage.

Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to "normalize" the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a protein-based, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and normalizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays.     

A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion.

Free radicals produce a wide spectrum of damages; among these are DNA base damages and abasic (AP) sites. Although several methods have been used to detect and quantify AP sites, they either are relatively laborious or require the use of radioactivity. A novel reagent for detecting abasic sites in DNA was prepared by reacting O-(carboxymethyl)hydroxylamine with biotin hydrazide in the presence of carbodiimide. This reagent, called Aldehyde Reactive Probe (ARP), specifically tagged AP sites in DNA with biotin residues. The number of biotin-tagged AP sites was then determined colorimetrically by an ELISA-like assay using avidin/biotin complex conjugated to horseradish peroxidase as the indicator enzyme. With heat/acid-depurinated calf thymus or bacteriophage f1 DNA, ARP detected femtomoles of AP sites in DNA. Using this assay, DNA damages generated in calf thymus, phi X174 RF, and f1 single-stranded DNA, X-irradiated in phosphate buffer, were easily detectable at 10 rad (0.1 Gy). Furthermore, ARP sites were detectable in DNA isolated from heat-inactivated X-irradiated (10 Gy) and methyl methanesulfonate (MMS)-treated (5 microM) Escherichia coli cells. The rate of production of ARP sites was proportional to the X-ray dose as well as to the concentration of MMS. Thus, the sensitivity and simplicity of the ARP assay should provide a potentially powerful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.     

Comparison of Manual and Automatic (MagNA Pure) Isolation Methods of Total RNA from Adipose Tissue

Aim Comparison of manual and automatic (MagNA Pure) isolation methods of total RNA from adipose tissue with respect to its quality and recovery factor. Material 120 human subcutaneous adipose tissue samples (about 100 mg/sample) were collected from patients during surgical operations. The tissue sample was stabilized in RNAlater (QIAGEN GmbH, Germany). Methods Total RNA was extracted by the following kits: Rneasy Protect Mini, Rneasy Lipid Tissue (QIAGEN GmbH, Germany) and MagNA Pure Compact RNA Isolation (Tissue) for MagNA Pure Compact Instrument (Roche Diagnostics GmbH, Germany). Results The average RNA yields with Rneasy Lipid Tissue kits were about two-fold higher in comparison with the Rneasy Protect Mini kit. When the MagNA Pure Compact System was used, RNA yields from the same sample were more uniform compared with manual systems. It was also more convenient and less time-consuming than the manual approach. No DNA contamination of total RNA samples was detected except for samples isolated by Rneasy Protect Mini Kit. Conclusion Rneasy Lipid Tissue Kit and MagNA Pure Compact RNA Isolation Kit (Tissue) provide RNA samples of high quantity, purity and PCR amplificability. RNA samples are suitable for further processing using methods of molecular biology.     

Characterization and direct quantitation of cerebroside molecular species from lipid extracts by shotgun lipidomics

By using shotgun lipidomics based on the separation of lipid classes in the electrospray ion source (intrasource separation) and two-dimensional (2D) MS techniques (Han, X., and R. W. Gross. 2004. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom. Rev. First published on June 18, 2004; doi: 10.1002/mas.20023, In press), individual molecular species of most major and many minor lipid classes can be quantitated directly from biological lipid extracts. Herein, we extended shotgun lipidomics to the characterization and quantitation of cerebroside molecular species in biological samples. By exploiting the differential fragmentation patterns of chlorine adducts using electrospray ionization (ESI) tandem mass spectrometry, hydroxy and nonhydroxy cerebroside species are readily identified. The hexose (either galactose or glucose) moiety of a cerebroside species can be distinguished by examination of the peak intensity ratio of its product ions at m/z 179 and 89 (i.e., 0.74 +/- 0.10 and 4.8 +/- 0.7 for galactose- and glucose-containing cerebroside species, respectively). Quantitation of cerebroside molecular species (as little as 10 fmol) from chloroform extracts of brain tissue samples was directly conducted by 2D ESI/MS after correction for differences in (13)C-isotopomer intensities. This method was demonstrated to have a greater than 1,000-fold linear dynamic range in the low concentration region; therefore, it should have a wide range of applications in studies of the cellular sphingolipid lipidome.     

A Multi-sample Microdialysis Apparatus for Proteins and Nucleic Acids

Abstract

A multiposition microdialysis system suitable for simultaneous microsample applications (between 10 μL and 500 μL), has been developed. Each sample, contained in a specially designed microfuge dialysis tube (mDT), is dialysed independently from the other samples. Each mDT has its own membrane, and this feature allows the use of different membranes and dialysis times for different samples.

The microdialysis apparatus is kept at constant temperature by an external thermostat, avoiding the use of a cold box. The dialysis release time for small ions, a parameter used for quantitation of microdialysis efficiency, decreases from 22.9 min (for a 200 μL sample) to 7 min (for a 50 μL sample). The sample is efficiently recovered by centrifugation. Quantitative recoveries (90%) of different proteins and DNA were achieved after microdialysis by mDT.     

Nonlinear electrophoresis for purification of soil DNA for metagenomics

Purification of microbial DNA from soil is challenging due to the co-extraction of humic acids and associated phenolic compounds that inhibit subsequent cloning, amplification or sequencing. Removal of these contaminants is critical for the success of metagenomic library construction and high-throughput sequencing of extracted DNA. Using three different composite soil samples, we compared a novel DNA purification technique using nonlinear electrophoresis on the synchronous coefficient of drag alteration (SCODA) instrument with alternate purification methods such as direct current (DC) agarose gel electrophoresis followed by gel filtration or anion exchange chromatography, Wizard DNA Clean-Up System, and the PowerSoil DNA Isolation kit. Both nonlinear and DC electrophoresis were effective at retrieving high-molecular weight DNA with high purity, suitable for construction of large-insert libraries. The PowerSoil DNA Isolation kit and the nonlinear electrophoresis had high recovery of high purity DNA suitable for sequencing purposes. All methods demonstrated high consistency in the bacterial community profiles generated from the DNA extracts. Nonlinear electrophoresis using the SCODA instrument was the ideal methodology for the preparation of soil DNA samples suitable for both high-throughput sequencing and large-insert cloning applications.     

Reactions of coordinated molecules. 47. Preparation of rhena-β-ketoimine derivatives of iodoacetamide and of an N-hydroxysuccinimide ester: heavy-atom labeling reagents for sulfhydryl and amino groups

A rhena-β-ketoimine complex containing an iodoacetamide moiety has been prepared as a reagent for effecting heavy-atom labeling of molecules containing sulfhydryl groups. This complex reacts with benzyl mercaptan, ethanethiol, N-t-BOC-cystamine, N-acetyl-L-cysteine, and N-acetyl-DL-penicillamine to give the corresponding rhena-labeled derivatives. A rhena-β-ketoimine containing a free carboxylic acid moiety has been converted to its N-hydroxysuccinimide ester. This reagent should be useful in effecting the heavy-atom labeling of molecules containing primary and secondary amino groups.     

Sizing femtogram amounts of dsDNA by single-molecule counting

Modern molecular-biology applications raise renewed interest in sizing minute-amounts of DNA. In this work we utilize single-molecule imaging with in situ size calibration to accurately analyze the size and mass distribution of DNA samples. We exploit the correlation between DNA length and its fluorescence intensity after staining in order to assess the length of individual DNA fragments by fluorescence microscopy. Synthetic reference DNA standards are added to the investigated sample before staining and serve as internal size calibrators, supporting a robust assay for accurate DNA sizing. Our results demonstrate the ability to reconstruct the exact length distribution in a complex DNA sample by sizing a subset containing only femtogram amounts of DNA, thus, outperforming microfluidic gel electrophoresis which is the currently accepted gold standard. This assay may find useful applications for genetic analysis where the exact size distribution of DNA molecules is critical and the availability of genetic material is limited.     

Two-dimensional conformation-dependent electrophoresis (2D-CDE) to separate DNA fragments containing unmatched bulge from complex DNA samples

DNA fragments containing mispaired and modified bases, bulges, lesions and specific sequences have altered conformation. Methods for separating complex samples of DNA fragments based on conformation but independent of length have many applications, including (i) separation of mismatched or unmatched DNA fragments from those perfectly matched; (ii) simultaneous, diagnostic, mismatch scanning of multiple fragments; (iii) isolation of damaged DNA fragments from undamaged fragments; and (iv) estimation of reannealing efficiency of complex DNA samples. We developed a two-dimensional conformation-dependent electrophoresis (2D-CDE) method for separating DNA fragments based on length and conformation in the first dimension and only on length in the second dimension. Differences in migration velocity due to conformation were minimized during second dimension electrophoresis by introducing an intercalator. To test the method, we constructed 298 bp DNA fragments containing cytosine bulges ranging from 1 to 5 nt. Bulge-containing DNA fragments had reduced migration velocity in the first dimension due to altered conformation. After 2D-CDE, bulge-containing DNA fragments had migrated in front of an arc comprising heterogeneous fragments with regular conformation. This simple and robust method could be used in both analytical and preparative applications involving complex DNA samples.