Showing papers on "Multiple displacement amplification published in 2004"

Helicase-dependent isothermal DNA amplification

Abstract: Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.

Methods of amplifying and sequencing nucleic acids

Abstract: An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step

Isothermal reactions for the amplification of oligonucleotides

Abstract: Described is a new class of isothermal reactions for amplifying DNA. These homogeneous reactions rapidly synthesize short oligonucleotides (e.g., 8-16 bases) specified by the sequence of an amplification template. Versions of the reactions can proceed in either a linear or an exponential amplification mode. Both of these reactions utilize simple, stable conditions. The rate of amplification depends entirely on the molecular parameters governing the interactions of the molecules in the reaction. The exponential version of the method is a molecular chain reaction that uses the oligonucleotide products of each linear reaction to create a producer of more of the same oligonucleotide. It is a highly sensitive chain reaction that can be triggered by specific DNA sequences and can achieve amplifications of more than 106 fold. Several similar reactions in this class are described here. The robustness, speed and sensitivity of the exponential reaction render it useful in rapidly detecting the presence of small amounts of a specific DNA sequence in a sample, and a range of other applications including many currently making use of the polymerase chain reaction (PCR).

Pre-PCR processing : Strategies to generate PCR-compatible samples

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

Genome coverage and sequence fidelity of ϕ29 polymerase‐based multiple strand displacement whole genome amplification

Abstract: Major efforts are underway to systematically define the somatic and germline genetic variations causally associated with disease. Genome-wide genetic analysis of actual clinical samples is, however, limited by the paucity of genomic DNA available. Here we have tested the fidelity and genome representation of f29 polymerase-based genome amplification (f29MDA) using direct sequencing and high density oligonucleotide arrays probing >10 000 SNP alleles. Genome representation was comprehensive and estimated to be 99.82% complete, although six regions encompassing a maximum of 5.62 Mb failed to amplify. There was no degradation in the accuracy of SNP genotyping and, in direct sequencing experiments sampling 500 000 bp, the estimated error rate (9.5 3 10 ‐6 ) was the same as in paired unamplified samples. The detection of cancer-associated loss of heterozygosity and copy number changes, including homozygous deletion and gene amplification, were similarly robust. These results suggest that f29MDA yields high fidelity, near-complete genome representation suitable for high resolution genetic analysis.

A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences

Abstract: Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are approximately 500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5-7 days) and suitable for synthesizing long segments of DNA (5-6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.

Two Methods of Whole-Genome Amplification Enable Accurate Genotyping Across a 2320-SNP Linkage Panel

Abstract: Comprehensive genome scans involving many thousands of SNP assays will require significant amounts of genomic DNA from each sample. We report two successful methods for amplifying whole-genomic DNA prior to SNP analysis, multiple displacement amplification, and OmniPlex technology. We determined the coverage of amplification by analyzing a SNP linkage marker set that contained 2320 SNP markers spread across the genome at an average distance of 2.5 cM. We observed a concordance of >99.8% in genotyping results from genomic DNA and amplified DNA, strongly indicating the ability of both methods used to amplify genomic DNA in a highly representative manner. Furthermore, we were able to achieve a SNP call rate of >98% in both genomic and amplified DNA. The combination of whole-genome amplification and comprehensive SNP linkage analysis offers new opportunities for genetic analysis in clinical trials, disease association studies, and archiving of DNA samples.

Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease

Abstract: Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.

A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

Abstract: The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with phi29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 x 10(4)-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.

Group-specific polymerase chain reaction for DNA-based analysis of species diversity and identity in dietary samples.

Abstract: Unique DNA sequences are present in all species and can be used as biomarkers for the detection of cells from that species. These DNA sequences can most easily be detected using the polymerase chain reaction (PCR), which allows very small quantities of target DNA sequence to be amplified even when the target is mixed with large amounts of nontarget DNA. PCR amplification of DNA markers that are present in a wide range of species has proven very useful for studies of species diversity in environmental samples. The taxonomic range of species to be identified from environmental samples may often need to be restricted to simplify downstream analyses and to ensure that less abundant sequences are amplified. Group-specific PCR primer sets are one means of specifying the range of taxa that produce an amplicon in a PCR. We have developed a range of group-specific PCR primers for studying the prey diversity found in predator stomach contents and scats. These primers, their design and their application to studying prey diversity and identity in predator diet are described.

Strategies to Generate PCR-Compatible Samples

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

Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR.

Abstract: PCR amplification of limited amounts of DNA template carries an increased risk of product redundancy and contamination. We use molecular barcoding to label each genomic DNA template with an individual sequence tag prior to PCR amplification. In addition, we include molecular ‘batch-stamps’ that effectively label each genomic template with a sample ID and analysis date. This highly sensitive method identifies redundant and contaminant sequences and serves as a reliable method for positive identification of desired sequences; we can therefore capture accurately the genomic template diversity in the sample analyzed. Although our application described here involves the use of hairpin-bisulfite PCR for amplification of double-stranded DNA, the method can readily be adapted to single-strand PCR. Useful applications will include analyses of limited template DNA for biomedical, ancient DNA and forensic purposes.

Nucleic acid amplification strategies for DNA microarray-based pathogen detection.

Abstract: DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, phi29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and phi29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.

In vitro dna immortalization and whole genome amplification using libraries generated from randomly fragmented dna

Abstract: The present invention regards a variety of methods and compositions for whole genome amplification. In a particular aspect of the present invention, there is a method of amplifying a genome in a non-biased manner utilizing adaptor-attached randomly generated fragments following modification of the DNA ends prior to the adaptor attachment. In an additional aspect of the present invention, there are methods and compositions for whole genome amplification regarding a one-step endonuclease cleavage and linker ligation reaction.

Multiple displacement amplification on single cell and possible PGD applications

Abstract: Multiple displacement amplification (MDA) is a technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high-quality DNA. We used MDA to amplify the whole genome directly from a single cell. The most common techniques used in PGD are PCR and fluorescent in-situ hybridization (FISH). There are many limitations to these techniques including, the number of chromosomes diagnosed for FISH or the quality of DNA issued from a single cell PCR. This report shows, for the first time, use of MDA for single cell whole genome amplification. A total of 16 short tandem repeats (STRs) were amplified successfully with a similar pattern to the genomic DNA. Furthermore, allelic drop out (ADO) derived from MDA was assessed in 40 single cells by analysing (i) heterozygosity for a known beta globin mutation (IVSI-5 C-G) and by studying (ii) the heterozygous loci present in the STRs. ADO turned out to be 10.25% for the beta globin gene sequencing and 5% for the fluorescent PCR analysis of STRs. Moreover, the amplification accuracy of MDA permitted the detection of trisomy 21 on a single cell using comparative genome hybridization-array. Altogether, these data suggest that MDA can be used for single cell molecular karyotyping and the diagnosis of any single gene disorder in PGD.

Low-fidelity Pyrococcus furiosus DNA polymerase mutants useful in error-prone PCR

Abstract: Random mutagenesis constitutes an important approach for identifying critical regions of proteins, studying structure-function relations and developing novel proteins with desired properties. Perhaps, the most popular method is the error-prone PCR, in which mistakes are introduced into a gene, and hence a protein, during DNA polymerase-catalysed amplification cycles. Unfortunately, the relatively high fidelities of the thermostable DNA polymerases commonly used for PCR result in too few mistakes in the amplified DNA for efficient mutagenesis. In this paper, we describe mutants of the family B DNA polymerase from Pyrococcus furiosus (Pfu-Pol), with superb performance in error-prone PCR. The key amino acid changes occur in a short loop linking two long alpha-helices that comprise the 'fingers' sub-domain of the protein. This region is responsible for binding the incoming dNTPs and ensuring that only correct bases are inserted opposite the complementary base in the template strand. Mutations in the short loop, when combined with an additional mutation that abolishes the 3'-5' proof-reading exonuclease activity, convert the extremely accurate wild-type polymerase into a variant with low fidelity. The mutant Pfu-Pols can be applied in error-prone PCR, under exactly the same conditions used for standard, high-fidelity PCR with the wild-type enzyme. Large quantities of amplified product, with a high frequency of nearly indiscriminate mutations, are produced. It is anticipated that the Pfu-Pol variants will be extremely useful for the randomization of gene, and hence protein, sequences.

Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process

Abstract: The present invention regards a variety of methods and compositions for whole genome amplification and whole transcriptome amplification. In a particular aspect of the present invention, there is a method of amplifying a genome comprising a library generation step followed by a library amplification step. In specific embodiments, the library generating step utilizes specific primer mixtures and a DNA polymerase, wherein the specific primer mixtures are designed to eliminate ability to self-hybridize and/or hybridize to other primers within a mixture but efficiently and frequently prime nucleic acid templates.

DNA amplification method tolerant to sample degradation

Abstract: Despite recent advances in linear whole genome amplification of intact DNA/RNA, amplification of degraded nucleic acids in an unbiased fashion remains a serious challenge for genetic diagnosis. We describe a new whole genome amplification procedure, RCA-RCA (Restriction and Circularization-Aided Rolling Circle Amplification), which retains the allelic differences among degraded amplified genomes while achieving almost complete genome coverage. RCA-RCA utilizes restriction digestion and whole genome circularization to generate genomic sequences amenable to rolling circle amplification. When intact genomic DNA is used, RCA-RCA retains gene-amplification differences (twofold or higher) between complex genomes on a genome-wide scale providing highly improved concordance with unamplified material as compared with other amplification methodologies including multiple displacement amplification. Using RCA-RCA, formalin-fixed samples of modest or substantial DNA degradation were successfully amplified and screened via array-CGH or Taqman PCR that displayed retention of the principal gene amplification features of the original material. Microsatellite analysis revealed that RCA-RCA amplified genomic DNA is representative of the original material at the nucleotide level. Amplification of cDNA is successfully performed via RCA-RCA and results to unbiased gene expression analysis (R(2) = 0.99). The simplicity and universal applicability of RCA-RCA make it a powerful new tool for genome analysis with unique advantages over previous amplification technologies.

Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping.

Abstract: Genome-wide screening of genetic alterations between normal and cancer cells, as well as among subgroups of tumors, is important for establishing molecular mechanism and classification of cancer. Gene silencing through loss of heterozygosity is widely observed in cancer cells and detectable by analyzing allelic loss of single nucleotide polymorphism and/or short tandem repeat markers. To use minute quantities of DNA that are available through laser capture microdissection (LCM) of cancer cells, a whole genome amplification method that maintains locus and allele balance is essential. We have successfully used a o29 polymerase-based isothermal whole genome amplification method to amplify LCM DNA using a proteinase K lysis procedure coupled with a pooling strategy. Through single nucleotide polymorphism and short tandem repeat genotype analysis we demonstrate that using pooled DNA from two or three separate amplification reactions significantly reduces any allele bias introduced during amplification. This strategy is especially effective when using small quantities of source DNA. Although a convenient alkaline lysis DNA extraction procedure provided satisfactory results from using 1500 to 3000 LCM cells, proteinase K digestion was superior for lower cell numbers. Accurate genotyping is achieved with as few as 100 cells when both proteinase K extraction and pooling are applied.

Balanced‐PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples

Abstract: Analysis of genomic DNA derived from cells and fresh or fixed tissues often requires whole genome amplification prior to microarray screening. Technical hurdles to this process are the introduction of amplification bias and/or the inhibitory effects of formalin fixation on DNA amplification. Here we demonstrate a balanced-PCR procedure that allows unbiased amplification of genomic DNA from fresh or modestly degraded paraffin-embedded DNA samples. Following digestion and ligation of a target and a control genome with distinct linkers, the two are mixed and amplified in a single PCR, thereby avoiding biases associated with PCR saturation and impurities. We demonstrate genome-wide retention of allelic differences following balanced-PCR amplification of DNA from breast cancer and normal human cells and genomic profiling by array-CGH (cDNA arrays, 100 kb resolution) and by real-time PCR (single gene resolution). Comparison of balanced-PCR with multiple displacement amplification (MDA) demonstrates equivalent performance between the two when intact genomic DNA is used. When DNA from paraffin-embedded samples is used, balanced PCR overcomes problems associated with modest DNA degradation and produces unbiased amplification whereas MDA does not. Balanced-PCR allows amplification and recovery of modestly degraded genomic DNA for subsequent retrospective analysis of human tumors with known outcomes.

Improving specificity of DNA hybridization-based methods

Abstract: Methods based on DNA reassociation in solution with the subsequent PCR amplification of certain hybrid molecules, such as coincidence cloning and subtractive hybridization, all suffer from a common imperfection: cross-hybridization between various types of paralogous repetitive DNA fragments. Although the situation can be slightly improved by the addition of repeat-specific competitor DNA into the hybridization mixture, the cross-hybridization outcome is a significant number of background chimeric clones in resulting DNA libraries. In order to overcome this challenge, we developed a technique called mispaired DNA rejection (MDR), which utilizes a treatment of resulting reassociated DNA with mismatch-specific nucleases. We examined the MDR efficiency using cross-hybridization of complex, whole genomic mixtures derived from human and chimpanzee genomes, digested with frequent-cutter restriction enzyme. We show here that both single-stranded DNA-specific and mismatched double-stranded DNA-specific nucleases can be used for MDR separately or in combination, reducing the background level from 60 to 4% or lower. The technique presented here is of universal usefulness and can be applied to both cDNA and genomic DNA subtractions of very complex DNA mixtures. MDR is also useful for the genome-wide recovery of highly conserved DNA sequences, as we demonstrate by comparing human and pygmy marmoset genomes.

Integrated method for single-cell DNA extraction, PCR amplification, and sequencing of ribosomal DNA from harmful dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella.

Abstract: A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and Alexandrium catenella successfully amplified the target ribosomal DNA regions. DNA sequencing of the unpurified PCR products showed that DNA sequences corresponded to the expected locus of ribosomal DNA regions of both A. catenella and C. polykrikoides (each zero genetic distance and 100% sequence similarity). Using the protocol described in this article, there was little DNA loss during the purification step, and the technique was found to be rapid and inexpensive. This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.

DNA polymerase fusions and uses thereof

Abstract: The present invention discloses methods of using DNA polymerase fusions at high pH in PCR, DNA sequencing and mutagenesis protocols.

Genotyping of three single nucleotide polymorphisms following whole genome preamplification of DNA collected from buccal cells.

Abstract: Collection of genomic DNA from buccal cells is a simple and convenient procedure for genotyping individuals. One disadvantage is that the amount of genomic DNA may be inadequate for genotyping projects that require a large number of determinations per sample. Primer Extension Preamplification (PEP) methods that can amplify the entire genome 100-fold or more offer a potential solution to this problem. To test whether the preamplified DNA product retains the original allelic distribution of the genomic DNA sample from which it was derived, we compared PEP buccal DNA with genomic buccal DNA from 94 pairs of monozygotic twins. Using the 5′ nuclease (Taqman®) method, we genotyped three SNPs that are of wide interest in behavioral and psychiatric genetic studies. The percent agreement between allele calls using PEP DNA samples and those using genomic DNA samples ranged between 99% and 100% depending on SNP assay. These data complement our earlier findings with respect to microsatellite markers and variable number tandem repeat (VNTR) polymorphisms and indicate that whole-genome preamplification is an appropriate method for providing DNA for SNP genotyping these and possibly other loci.

Presence of Bacterial Phage-Like DNA Sequences in Commercial Taq DNA Polymerase Reagents

Abstract: Many studies have reported the presence of bacterial DNA contamination in commercial Taq DNA polymerase reagents. This is the first report of the presence of phage-like DNA sequences in certain commercial Taq DNA polymerase reagents. Precautions are needed when using amplification reagents with exogenous DNAs.

Strand displacement amplification: a versatile tool for molecular diagnostics.

Abstract: Strand displacement amplification is an isothermal process that permits 10(10)-fold amplification of a DNA target sequence in as little as 15 min. In the form of the BD ProbeTec ET System, strand displacement amplification was the first nucleic acid amplification technology to be coupled with real-time homogeneous fluorescence-based detection for routine application in the clinical laboratory. The isothermal nature of the reaction process offers distinct advantages with regard to the cost and simplicity of instrumentation, while a universal detection format permits the use of the same fluorescent detector probes across multiple analytes. This has important potential in the field of genetic analysis, in which disease predisposition and therapeutic efficacy are frequently determined by multiple nucleic acid markers.