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TaqMan probe (dual-labeled DNA probe)–based real-time detection, one of the most sensitive and specific fluorescent detection methods, has been widely utilized in conjunction with polymerase chain reaction (PCR). Helicase-dependent amplification (HDA) is an isothermal amplification technology that has a similar reaction scheme to PCR, but replaces thermocycling with a helicase capable of unwinding a DNA duplex. Here we describe a novel isothermal real-time detection method (HDA-TaqMan) that combines the advantages of both HDA and a TaqMan assay. In this assay, the reactions of DNA unwinding, primer annealing, polymerization, probe hybridization, and subsequent hydrolysis by the polymerase are coordinated and synchronized to perform at a single temperature. It not only provides a useful tool for real-time detection of HDA, but also provides an isothermal format for the TaqMan system. With this platform, we have successfully developed rapid real-time isothermal assays for biodefense targets that include Vibrio cholerae and Bacillus anthracis.

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

Single nucleotide polymorphisms (SNPs) are increasingly recognized as important diagnostic markers for the detection and differentiation ofBacillus anthracis. The use ofSNP markers for identifying B. anthracis DNA in environmental samples containing genetically similar bacteria requires the ability to amplify and detect DNA with single nucleotide specificity. We designed a TaqMan® mismatch amplification mutation assay (TaqMAMA) around a SNP in the plcR gene ofB. anthracis. The assay permits specific, low-level detection (25fg DNA) of this B. anthracis-specific SNP, even in the presence of environmental DNA extracts containing a 20,000-fold excess of the alternate allele. We anticipate that the ability to selectively amplify and detect low copy number DNAs with single nucleotide specificity will represent a valuable tool in the arena ofbiodefense and microbialforensics.

Real-time PCR methods have become widely used within the past few years. However, real-time PCR is rarely used to study chronic diseases with low pathogen loads, presumably because of insufficient sensitivity. In this report, we developed an integrated nucleic acid isolation and real-time PCR platform that vastly improved the sensitivity of the quantitative detection of the intracellular bacterium, Chlamydia spp., by fluorescence resonance energy transfer realtime PCR. Determinants of the overall detection sensitivity were analyzed by extracting nucleic acids from bovine milk specimens spiked with low amounts of chlamydial organisms. Nucleic acids were optimally preserved and recovered by collection in guanidinium stabilization buffer, binding to a matrix of glass fiber fleece, and elution in low volume. Step-down thermal cycling and an excess of hot-start Taq polymerase vastly improved the robustness and sensitivity of the real-time PCR while essentially maintaining 100% specificity. The amplification of Chlamydia 23S rRNA allowed for the differentiation of chlamydial species and was more robust at low target numbers than amplification of the omp1 gene. The best combined method detected single targets per a 100-μL specimen equivalent in a 5-μL real-time PCR input. In an initial application, this high-sensitivity realtime PCR platform demonstrated a high prevalence of chlamydial infection in cattle.

The commonly used DNA polymerase assay is based on the detection of incorporated radiolabeled nucleotides in a DNA elongation reaction. It is laborious, radioactive, and can be highly variable. Here we report a nonradioactive fluorescence-based assay. The method consists of CydyeTM-labeled nucleotides, biotinylated primer, and a streptavidin-coated microplate. The assay is found to have sensitivity and dynamic range comparable to the classical radioactive method. Moreover, it has the advantages of being simple, stable, nonradioactive, and suitable for high-throughput applications. We have also found that, to ensure efficient measurement of the enzyme activity, the template DNA used in this method should have a sequence that avoids the incorporation of the fluorescence-labeled nucleotide in a consecutive way.

The performance of a novel thermal cycler has been characterized in a 30-cycle PCR. The device consists of a microcapillary equipped with bidirectional pressure-driven flow and in situ optical position sensors. A 1-microL droplet of reaction mixture moves between three heat zones in a 1-mm i.d., oil-filled capillary using a multi-element scattered light detector and active feedback. The design permits time and number of cycles to be changed without hardware modification, unlike other flow-in-capillary PCR systems. Temperature optimization has been performed on the three PCR heat steps. The optimal denaturation temperature is 94 degrees C-96 degrees C, which is identical to commercial machines. The optimal extension temperature of 62 degrees C-66 degrees C is lower than reported for Taq DNA polymerase (70 degrees C-80 degrees C) because of the high enzyme concentration and/or the absence of detergent in the PCR mixture. The optimal annealing temperature seems to be the same as the optimal extension temperature. This is because extension occurs when the sample is inside of the annealing heat zone. Annealing takes place as the sample travels between heat zones. Device speed (23 minfor 30 cycles without time optimization) is competitive with other rapid PCR designs for efficiencies comparable to a commercial machine.

The rapid development of molecular genetic analysis tools has made it possible to analyze most biological material found at the scene of a crime. Evidence materials containing DNA quantities too low to be analyzed using nuclear markers can be analyzed using the highly abundant mtDNA. However, there is a shortage of sensitive nDNA and mtDNA quantification assays. In this study, an assay for the quantification of very small amounts of DNA, based on the real-time Taq-Man® assay, has been developed. This analysis will provide an estimate of the total number of nDNA copies and the total number of mtDNA molecules in a particular evidence material. The quantification is easy to perform, fast, and requires a minimum of the valuable DNA extracted from the evidence material. The results will aid in the evaluation of whether the specific sample is suitable for nDNA or mtDNA analysis. Furthermore, the optimal amount of DNA to be used in further analysis can be estimated, ensuring that the analysis is successful and that the DNA is retained for future independent analysis. This assay has significant advantages over existing techniques because of its high sensitivity, accuracy, and the combined analysis of nDNA and mtDNA. Moreover, it has the potential to provide additional information about the presence of inhibitors in forensic samples. Subsequent mitochondrial and nuclear analysis of quantified samples illustrated the potential to predict the number of DNA copies required for a successful analysis in a certain typing assay.

The error rate of conventional PCR is problematic when amplifying from single cells or amplifying segments for protein functional analysis by in vitro translation. We describe truncated amplification, a method for high-fidelity amplification in which DNA polymerase errors are not propagated efficiently and original DNA templates exert greater influence on the amplification process. Truncated amplification utilizes pairs of oligonucleotides and thermal cycling, but it differs from PCR. Truncated amplification amplifies non-exponentially with one or two chimeric oligonucleotides and produces truncated terminal products that are no more than three rounds of replication from the original template. Exon 6 of the p53 gene was utilized as a model system to demonstrate proof of principle. Chimeric oligonucleotides containing three 3′→5′ reversed-deoxynucleotides or 2′-OMe-ribonucleotides at 6-8 nucleotides from the 3′ terminus retained sequence specificity and primer extension activity. With PfuTurbo™, but not with Taq or Vent (exo-) DNA polymerases, the modified nucleotides completely truncated the DNA polymerase elongation. The resulting truncated terminal products are not templates for further amplification because of the short length of the 3′ complementary region. Truncated amplification can amplify quadratically or geometrically, depending on whether two or one chimeric oligonucleotides are used. Truncated amplification is a promising approach when template-driven amplification is desired to increase the frequency of error-free products.

Recently, we reported the isolation and characterization of a new human DING protein, extracellular DING from CD4 T cells (X-DING-CD4) (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"HQ586056","term_id":"507527165"}}HQ586056), which is involved in cellular resistance to HIV infection (1). Our attempt to amplify the X-DING-CD4 cDNA sequence using standard PCR methods was unsuccessful. Supplementation of PCR reaction buffer with 1.5–3 M betaine, or 2–5% DMSO, or 2–5% formamide, or nonionic detergents such as 0.1–1% Tween-20 or NP40 also did not improve amplification of X-DING-CD4 cDNA. We hypothesized that the X-DING-CD4 gene might be located in the unassembled stretches of the human genome characterized by high GC content and that the difficulty in amplifying this gene is caused by the unavailability of a linear cDNA template due to a high level of secondary structures and possibly its rapid renaturation into a highly folded form. We reasoned that coprecipitation of denatured cDNA with a neutral nucleic acid carrier might immobilize the linear form of the DNA and help us to overcome this difficulty. Linear polyacrylamide (LPA) is commonly used for the coprecipitation of minute quantities of DNA, and we speculated that it might enforce the linear state of a coprecipitated, denatured cDNA template. We employed a touchdown PCR protocol (2) where the availability of denatured X-DING-CD4 cDNA template for primers and polymerase was enforced by this LPA coprecipitation of the denatured cDNA. To show the advantage of LPA immobilization for amplification of our “difficult” DNA template, we set up several PCR reactions for the X-DING-CD4 and control β-globin cDNAs. X-DING-CD4 and β-globin cDNA was prepared using Transcriptor First Strand cDNA Synthesis kit (Roche, Mannheim, Germany) from 4 µg mRNA harvested from X-DING-CD4 (+) cells (1). RNA was pretreated with DNase I (Invitrogen, Carlsbad, CA, USA) and reverse transcribed using random hexamers (Invitrogen) and gene-specific primers (RD8 5′-CGTTCAGCTTGGTCATCACGCCTTGGC-3′) according to manufacturer’s protocol with the modification that the annealing temperature was set at 65°C. The reverse transcriptase reaction buffer was supplemented 0.2% (v/v) Triton X-100, 0.25 mM DTT, and 2.5% (v/v) glycerol. The PCR primers for X-DING-CD4—5′-GCCATGGCAGACATCAACGGCGGC GGCGCCA-3′ (sense) and 5′-GAATCAGCTTGCCCCAGGTCGCTTGTTTGGTGGAGGCGTAG-3′ (antisense)—were designed based on the X-DING-CD4 peptide amino acid back translation (1) and human Hsap_21_UR_H12 DNA sequence (GenBank no. CW626261). The Hsap_21_UR_H12 DNA is derived from an ultra-rapidly reannealing fragment of genomic DNA containing a portion of the X-DING-CD4 gene (unpublished data). Primers for the control β-globin gene were synthesized based on sequence published by Bauer et al. (3). The annealing temperature for X-DING-CD4 primers and template was optimized according to the empirical formulation developed by Rychlik et al. (4): TaOPT=0.3×Tmprimer+0.7×Tmproduct−14.9, [Eq. 1] where Tmprimer is the calculated Tm of the less stable primer-template pair and the Tmproduct is the Tm of the PCR product. Tmprimer was calculated based on Allawi’s thermodynamic parameters (5) and is 74.74°C for the sense primer and 76.7°C for the antisense primer. Tmproduct is 88.3°C and TaOPT was calculated as 69.3°C. Five PCR amplifications were each set up in a 50-µL volume containing 2.5 mM MgSO4, 50 mM each of the four dNTPs, 1 µM each forward and reverse primer, and 2 units GoTaq polymerase (Promega, Madison, WI, USA) supplemented with 5× GoTaq reaction buffer (for reactions 1 and 2); 2 units GoTaq polymerase supplemented with 5× GoTaq reaction buffer and 3M betaine (for reaction 3); 2 units GC-RICH polymerase mix composed of thermostable Taq polymerase and proofreading Tgo DNA polymerases and 10 µL 5× GC-RICH buffer supplemented with 1.5 mM DMSO and 30 µL 5 M GC-RICH resolution solution (Roche) (for reactions 4 and 5). Reactions 1–4 contained 100 ng soluble unprocessed cDNA, while reaction 5 contained 100 ng cDNA coprecipitated with LPA as follows: 5 µg/µL stock of LPA was prepared according to the method described by Gaillard et al. (6). One hundred nanograms of cDNA placed in a PCR tube was mixed with 4.5 µL 2N NaOH and 26 µL DNase and RNase-free water; following 3 min incubation at room temperature the denatured DNA sample was combined with 10 µg LPA carrier, 0.1 volume 4M ammonium acetate (pH 4.6), and 2.5 volumes of 100% ethanol. The DNA sample was incubated at −20°C for 1 h, followed by centrifugation at 4°C for 15 min. The DNA pellet was washed with 70% ethanol, dried briefly, and supplemented with PCR mix for amplification. The cycling conditions for reaction 1 were as follows: initial denaturation at 94°C for 5min; 35 cycles of 1 min denaturation at 94°C, 1 min annealing at 55°C, and 1 min extension at 72°C; then 7 min final extension at 72°C. Reactions 2–5 were amplified through a touchdown PCR as follows: initial denaturation at 94°C for 7min; then 20 cycles of denaturation at 94°C for 45 s, annealing for 1 min, and extension at 72°C for 1 min. The annealing temperature decreased 1°C every second cycle from 10°C above TaOPT to a touchdown at TaOPT, at which point 10–15 cycles were carried out followed by 7 min final extension at 72°C. PCR-amplified products were analyzed by electrophoresis on a 2% agarose gel. For DNA sequencing, amplified PCR products were gel-extracted and purified with QIAquick gel extraction kit (Qiagen, Valencia, CA, USA) as specified by the manufacturer. The amplified fragments of X-DING-CD4 cDNA were cloned in TOPO –TA vector (Invitrogen) and sequenced by GENEWIZ Inc. (South Plainfield, NJ, USA) using M13 primers. As shown in Figure 1A, PCR amplification of the control β-globin gene produced strong amplicons in all experimental conditions. Amplification of X-DING-CD4 cDNA template under the standard PCR conditions (Figure 1B, lane 1) produced a smear of nonspecific bands, including longer products, and the touchdown PCR cycling did not improve the amplification in this system (Figure 1B, lane 2). Addition of 3 M betaine into the touchdown PCR reaction also did not produce the expected amplicon (Figure 1B, lane 3). Touchdown cycling in the presence of GC-RICH polymerase mix, GC-RICH resolution solution, and DMSO produced a weak X-DING-CD4 amplicon and a high background of nonspecific bands (Figure 1B, lane 4); the same PCR system including LPA-coprecipitated, denatured X-DING-CD4 cDNA resulted in the amplification of a strong single DNA product, indicating that the linear denatured template was accessible to the primers and polymerase throughout the PCR reaction (Figure 1B, lane 5). Figure 1 Amplification of an ultra-rapidly renaturing cDNA under various PCR conditions In a separate experiment, we tested if denaturation of DNA prior to its coprecipitation with LPA was essential for the successful amplification of our “difficult” DNA template. Our data showed that while LPA coprecipitation of the X-DING-CD4 cDNA fragment without prior denaturation resulted in some specific amplification; denaturation of the DNA template prior to its coprecipitation with LPA increased both the quality and quantity of the X-DING-CD4 amplicon (Supplementary Figure S1). These data show that the combination of LPA coprecipitation of a denatured DNA template with touchdown PCR in the presence of GC-RICH polymerase mix, 3 M GC-RICH resolution solution, and DMSO enabled successful amplification of the rapidly renaturing X-DING-CD4 cDNA that is characterized by highly repetitive sequences and the formation of strong secondary structures (Figure 1C), and which has a GC content of 61% as determined by the sequencing of several amplicons (not shown). We believe that application of the LPA-PCR technique might help overcome difficulties in the amplification of “difficult” DNA templates in diverse fields, including plant and environmental biology.

The sensitivity and robustness of various DNA detection and amplification techniques are to a large extent determined by the properties of the DNA polymerase used. We have compared the performance of conventional Taq and Bst DNA polymerases to a novel Taq DNA polymerase mutant (SD DNA polymerase), which has a strong strand displacement activity, in PCR (including amplification of GC-rich and complex secondary structure templates), long-range PCR (LR PCR), loop-mediated amplification (LAMP), and polymerase chain displacement reaction (PCDR). Our results demonstrate that the strand displacement activity of SD DNA polymerase, in combination with the robust polymerase activity, provides a notable improvement in the sensitivity and efficiency of all these methods.

In quantitative real-time PCR assays, fluorophor-labeled oligonucleotide probes are employed to generate sequence-specific signals for the quantitative evaluation. Whereas TaqMan® probes have to be hydrolyzed during PCR by the endonucleolytic activity of Taq DNA polymerase to generate a signal, the hybridization probes in Light-Cycler® assays must not be hydrolyzed. In this study, we demonstrate for four different targets that the probes are degraded during PCR by Taq DNA polymerase. Signal yield, quality of amplification curves, and accuracy of quantitative measurements can be improved using the Stoffel fragment lacking an endonucleolytic activity and TaqStart® antibody suppressing the formation of nonspecific products, without laborious efforts to optimize the amplification protocol.

Homogeneous assays based on real-time fluorescence monitoring during PCR are relevant alternatives for large-scale genotyping of single-nucleotide polymorphisms (SNPs). We compared the performance of the homogeneous TaqMan® 5′-nuclease assay and the Molecular Beacon assay using three SNPs in the human estrogen receptor gene as targets. When analyzing a panel of 90 DNA samples, both assays yielded a comparable power of discrimination between the genotypes of a C-to-T transition in codon 10 and a G-to-A transition in codon 594 of the estrogen receptor gene. The Molecular Beacon probes distinguished better than the TaqMan probes between homozygous and heterozygous genotypes of a C-to-G transversion in codon 325. The sensitivity of detecting one allele, present as a minority in a mixed sample, varied between the SNPs and was similar for both assays. With the Molecular Beacon assay, the measured signal ratios were proportional to the amount of the minor allele over a wider range than with the TaqMan assay at all three SNPs.

The quantity of PCR products that are simultaneously amplified from two different loci in a duplex amplification (DA) are significantly lower for one of the loci, as compared to identical PCR amplification in separate single-band amplifications (SBA). This difference in amplification probably occurs already after the second cycle of amplification. To further analyze this phenomenon, we tested different reaction conditions, including annealing times, a wide range of temperatures, various quantities of the template, several nucleotide concentrations, different amounts of TaqI DNA Polymerase, number of amplification cycles and various amounts of primers and primers ratio. Changing the ratio between the sets of primers in DA had the most significant effect on the relative levels of amplification of the loci with an optimal ratio of 4:1 in favor of the set of primers used to amplify the underrepresented fragment. The optimal annealing temperatures for the tested sets of primers were identical in SBA and different in DA. Possible reasons for this phenomenon are discussed.

Previously we described a PCR protocol for detecting the inversion in the factor VIII gene, which is a common cause of Hemophilia A. This PCR assay is challenging due to the size of the amplification (10-12 kb), the varying GC content (30%-80%) and the multiplex PCR products involved (four for carrier female). Efficient amplification of the four segments depends on three unusual modifications to standard long-distance PCR protocols: (i) very high concentrations of dimethyl sulfoxide, (ii) addition of deaza-dGTP and (iii) high concentration of Taq and Pwo DNA polymerases. One of the segments was amplified much more efficiently than the others under standard three-temperature cycling conditions (12 s at 94 degrees C, 30 s at 65 degrees C and 14 min at 68 degrees C). To facilitate the uniform amplification of the multiple regions, subcycling-PCR (S-PCR) was developed. In S-PCR, the combined annealing/elongation step is composed of subcycles of shuttling between a low and a high temperature, e.g., shuttling four times between 60 degrees and 65 degrees C. S-PCR produces consistent robust amplification of the various segments produced by wild-type, mutant and carrier individuals. S-PCR is a simple generalization of PCR, which generally may be advantageous in three contexts: (i) amplification of long segments in which the GC content varies within the segment, (ii) multiplex amplification of long segments and (iii) multiplex amplification of short segments in which the GC content varies among the segments.

By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.

The precise quantification of rare mRNA copies from intronless genes by reverse transcription polymerase chain reaction (RT-PCR) requires the complete removal of genomic DNA because discrimination of cDNA and DNA amplification products by differing sizes of PCR products is not possible. Elimination of DNA is achieved by treating the RNA sample with RNase-free DNase I before RT-PCR. The lack of a PCR product from DNase-treated RNA samples before RT is usually accepted as a proof of efficient DNA destruction. However, this may vary depending on the metal cofactor used in the DNase I cleavage. Treating DNA-contaminated RNA samples with DNase I and magnesium as a cofactor creates a negative PCR control after digestion without further RT. Paradoxically, after additional RT-PCR, the original intron-containing DNA fragment size may be produced again. In the presence of manganese as cofactor, RT-created DNA fragments do not appear. This is because in the presence of manganese, DNase I cleaves both DNA strands at approximately the same site, yielding DNA fragments that are blunt-ended or that have protruding termini of only one or two nucleotides in length. However, overlapping fragments with the potential to recombine are created by DNase digestion with magnesium as cofactor. Because one cannot differentiate between a PCR signal produced by RNA and one produced by recombined DNA after DNase I digestion and RT, all such DNase I assays should be performed with manganese instead of magnesium.

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (>70°C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hotstart PCR with improved amplification performance.

The Applied Biosystems PRISMTM fluorescence-based genotyping system as well as the Invitrogen TA Cloning® vector system are influenced by the tendency of Taq DNA polymerase to add an adenine nucleotide to the 3′ end of PCR products after extension. Incomplete addition of adenine to a majority of PCR product strands creates problems in allele-calling during genotyping and potentially diminishes the cloning efficiency of such products. Experiments reported here show that certain terminal nucleotides can either inhibit or enhance adenine addition by Taq and that PCR primer design can be used to modulate this activity. The methods we propose can substantially improve allele-calling for problematic microsatellite markers when using GENOTYPERTM software.

Bisulfite treatment and PCR amplification of genomic DNA permits the methylation analysis of any cytosine residue in a target sequence. By cloning and sequencing the PCR product, the methylation of individual molecules can be determined, whereas direct sequencing of the PCR product can provide an average of the methylation status in the population of molecules. Reliable quantitation of cytosine methylation by direct sequencing, however, has not been possible with current methods. In this paper we describe an accurate and innovative protocol to directly quantitate the methylation of any cytosine residue in the target sequence by fluorescence-based automated genomic sequencing. Only the cytosine and thymine residues of bisulfite-treated and amplified genomic DNA are sequenced. The degree of methylation is obtained by direct comparison of the cytosine and thymine signals, which have been labeled with the same fluorescent dyes. GENESCANTM analysis is employed to achieve a fast and accurate estimate of methylation at every cytosine in the target sequence. Combining direct bisulfite genomic sequencing and GENESCAN analysis permits the rapid survey of detailed DNA methylation profiles. Using this approach we have found the unexpected result that multicopy plasmid DNA grown in a Dcm host is not always fully methylated as suggested by restriction enyzme data.

Taq DNA polymerase can catalyze nontemplated addition of a nucleotide (principally adenosine) to the 3′ end of PCR-amplified products. Recently, we showed that this activity, which is primer-specific, presents a potential source of error in genotyping studies based on the use of short tandem repeat (STR) markers. Furthermore, in reviewing our data, we found that non-templated nucleotide addition adjacent to a 3′ terminal C is favored and that addition adjacent to a 3′ terminal A is not. It was clear, however, that features of the template in addition to the 3′ terminal base also affect the fraction of product adenylated. To define consensus sequences that promote or inhibit product adenylation, we transplanted sequences between the 5′ ends of the reverse primers of markers that are adenylated and those of markers that are not adenylated. It proved difficult to identify a single sequence capable of protecting the products of all markers from non-templated addition of nucleotide. On the other hand, placing the sequence GTTTCTT on the 5′ end of reverse primers resulted in nearly 100% adenylation of the 3′ end of the forward strand. This modification or related ones (called “PIGtailing”) should facilitate accurate genotyping and efficient T/A cloning.

We investigated the use of Taq dye primer and Taq terminator sequencing chemistry to optimize the quality of sequence data obtained from templates containing homopolymer tracts and repetitive elements. In direct side-by-side comparisons using the Applied Biosystems Model 373A Fluorescent Sequencer, the Taqterminator sequencing chemistry gave much cleaner and more consistent results on long homopolymer tracts and dinucleotide repeats. We also investigated various thermal cycling conditions and determined that higher annealing temperatures and longer denaturation times improved the ability to sequence through these problem templates.

Stutter products are a common artifact in the PCR amplification of frequently used genetic markers that contain mononucleotide simple sequence repeats. Despite the importance of accurate determination of nucleotide sequence and allele size, there has been little progress toward decreasing the formation of stutter products during PCR. In this study, we tested the effects of lowered extension temperatures, inclusion of co-solutes in PCR, PCR cycle number, and the use of different polymerases on sequence quality for a set of sequences containing mononucleotide A/T repeats of 10–17 bp. Our analyses showed that sequence quality of mononucleotide repeats ≤15 bp is greatly improved with the use of proofreading DNA polymerases fused to nonspecific dsDNA binding domains. Our findings also suggest that the number of nucleotides with which the DNA polymerase interacts may be the most important factor in the reduction of slipped-strand mispairings in vitro.

In this study we present a quick and easy method for counting trinucleotide repeats by de-oxyadenosine overhang (A-overhang)–dependent repeat expansion determination (ADRED). During standard Taq DNA polymerase–based sequencing reactions, the unterminated sequencing products of short PCR fragments are tagged with a 3′-end A-overhang that is visible as an intense peak in an electropherogram; this allows for easy and precise determination of the fragment length and thus the extent of repeat expansions. ADRED has clear advantages over existing methods, because repeat numbers of both normal and pathogenic (expanded) alleles can be analyzed without using labeled primers or labeled DNA standards. Because ADRED includes a sequencing step, disease-relevant polymorphisms (e.g., CAA interruptions in spinocerebellar ataxia type 2) can simultaneously be detected.

Reorienting Directional Cloning The cloning of PCR-amplified DNA fragments is frequently the first step in expression vector construction. Commercial kits that exploit the tendency of DNA polymeras...

Since public and private efforts announced the first draft of the human genome last year, researchers have reported great numbers of single nucleotide polymorphisms (SNPs). We believe that the availability of well-mapped, quality SNP markers constitutes the gateway to a revolution in genetics and personalized medicine that will lead to better diagnosis and treatment of common complex disorders. A new generation of tools and public SNP resources for pharmacogenomic and genetic studies--specifically for candidate-gene, candidate-region, and whole-genome association studies--will form part of the new scientific landscape. This will only be possible through the greater accessibility of SNP resources and superior high-throughput instrumentation-assay systems that enable affordable, highly productive large-scale genetic studies. We are contributing to this effort by developing a high-quality linkage disequilibrium SNP marker map and an accompanying set of ready-to-use, validated SNP assays across every gene in the human genome. This effort incorporates both the public sequence and SNP data sources, and Celera Genomics' human genome assembly and enormous resource ofphysically mapped SNPs (approximately 4,000,000 unique records). This article discusses our approach and methodology for designing the map, choosing quality SNPs, designing and validating these assays, and obtaining population frequency ofthe polymorphisms. We also discuss an advanced, high-performance SNP assay chemisty--a new generation of the TaqMan probe-based, 5' nuclease assay-and high-throughput instrumentation-software system for large-scale genotyping. We provide the new SNP map and validation information, validated SNP assays and reagents, and instrumentation systems as a novel resource for genetic discoveries.

The development of transgenic events can be limited by many factors. These include expression levels, insert stability and inheritance, and the identification of simple insertion events. All of the factors can be related to the copy number of the transgene. Traditionally, copy number has been determined by laborious blotting techniques. We have developed an alternative approach that utilizes the fluorogenic 5′ nuclease (TaqMan®) assay to quantitatively determine transgene copy level in plants. Using this assay, hundreds of samples can be analyzed per day in contrast to the low throughput encountered with traditional methods. To develop the TaqMan copy number assay, we chose to utilize our highly efficient Agrobacterium-mediated transformation system of maize. This transformation procedure generates predominantly low copy number insertion events, which simplified assay development. We have also successful applied this assay to other crops and transformation systems.