Multiplex amplification of ancient DNA

This method is designed to assemble long, continuous DNA sequences using minimal amounts of fragmented ancient DNA as template. This is achieved by a two-step approach. In the first step, multiple fragments are simultaneously amplified in a single multiplex reaction. Subsequently, each of the generated fragments is amplified individually using a single primer pair, in a standard simplex (monoplex) PCR. The ability to amplify multiple fragments simultaneously in the first step allows the generation of large amounts of sequence from rare template DNA, whereas the second nested step increases specificity and decreases amplification of contaminating DNA. In contrast to current protocols using many template-consuming simplex PCRs, the method described allows amplification of several kilobases of sequence in just one reaction. It thus combines optimal template usage with a high specificity and can be performed within a day.     

Identification of a replication protein and repeats essential for DNA replication of the temperate lactococcal bacteriophage TP901-1

DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by orf13 and the repeats located within the gene. Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13). The putative origin of replication was identified as series of repeats within orf13 and was shown to confer a TP901-1 resistance phenotype when present in trans. Site-specific mutations were introduced into the replication protein and into the repeats. The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon. Subsequent analysis of induced phages showed that the protein encoded by orf13 and the repeats within orf13 were essential for phage TP901-1 amplification. In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo. These results show that orf13 encodes a replication protein and that the repeats within the gene are the origin of replication.     

TempliPhi,phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing

We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.     

Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering

DNA cloning and protein engineering are basic methodologies employed for various applications in all life-science disciplines. Manipulations of DNA however, could be a lengthy process that slows down subsequent experiments. To facilitate both DNA cloning and protein engineering, we present Transfer-PCR (TPCR), a novel approach that integrates in a single tube, PCR amplification of the target DNA from an origin vector and its subsequent integration into the destination vector. TPCR can be applied for incorporation of DNA fragments into any desired position within a circular plasmid without the need for purification of the intermediate PCR product and without the use of any commercial kit. Using several examples, we demonstrate the applicability of the TPCR platform for both DNA cloning and for multiple-site targeted mutagenesis. In both cases, we show that the TPCR reaction is most efficient within a narrow range of primer concentrations. In mutagenesis, TPCR is primarily advantageous for generation of combinatorial libraries of targeted mutants but could be also applied to generation of variants with specific multiple mutations throughout the target gene. Adaptation of the TPCR platform should facilitate, simplify and significantly reduce time and costs for diverse protein structure and functional studies.     

Closed-tube genotyping of apolipoprotein E by isolated-probe PCR with multiple unlabeled probes and high-resolution DNA melting analysis

Isolated-probe PCR (IP-PCR) is a method that combines asymmetric PCR, unlabeled probes, and high-resolution DNA melting while maintaining a closed tube system. A double-stranded DNA (dsDNA) dye LCGreen I was used to detect the unlabeled probes. LCGreen I is also used to detect the 277-base pair PCR product peak as an internal amplification control. To accomplish this, IP-PCR separates the asymmetric PCR amplification step and the detection step of the unlabeled probes. This prevents the probes from interfering with the amplification of the DNA target. The samples are then melted using a high-resolution DNA melting instrument: the HR-1. The closed tube system virtually eliminates PCR product contamination or sample carryover The target apolipoprotein E (APOE) was chosen to test the IP-PCR technique. APOE contains two single nucleotide polymorphisms (SNPs) located 139 base pairs apart in a GC-rich region of the human genome. The results from this study show that the IP-PCR technique was able to determine the correct APOE genotype for each of the 101 samples. The IP-PCR technique should also be useful in detecting SNPs in other high-GC regions of the human genome.     

Phage phi29 terminal protein residues Asn80 and Tyr82 are recognition elements of the replication origins.

Initiation of phage phi29 DNA replication starts with the recognition of the origin of replication, located at both ends of the linear DNA, by a heterodimer formed by the phi29 terminal protein (TP) and the phi29 DNA polymerase. The parental TP, covalently linked to the DNA ends, is one of the main components of the replication origin. Here we provide evidence that recognition of the origin is mediated through interactions between the TP of the TP/DNA polymerase heterodimer, called primer TP, and the parental TP. Based on amino acid sequence comparisons, various phi29 TP mutants were generated at conserved amino acid residues from positions 61 to 87. In vitro phi29 DNA amplification analysis revealed that residues Asn80 and Tyr82 are essential for functional interaction between primer and parental TP required for recognition of the origin of replication. Although these mutant TPs can form functional heterodimers with phi29 DNA polymerase that are able to recognize the origin of replication, these heterodimers are not able to recognize an origin containing a mutant TP.     

Cloning and direct sequencing of plant promoters using primer-adapter mediated PCR on DNA coupled to a magnetic solid phase.

A method that allows amplification and direct sequencing or cloning of an unknown DNA segment flanked by a known sequence is described using barley genomic DNA. The method avoids the step of circularization necessary for inverse PCR by ligation of primer-adapters to restricted genomic DNA. Specificity is achieved in the first amplification step; linear PCR with a biotinylated primer complementary to the known flanking sequence (primer 1-B) produces a single-stranded product that is purified employing streptavidin-coated magnetic beads. After this step, which removes genomic DNA, two rounds of exponential PCR are performed, first with the adapter-primer and primer 1 and second with primer 1 substituted by a nested primer 2. If the second primer is biotinylated, the product can be sequenced directly using solid-phase sequencing. We have employed this method to sequence directly and to clone the promoters of two late embryogenesis-abundant (Lea) genes (B19.4 and B19.3) from barley. Lea B19.4 and B19.3 encode putative desiccation-protective proteins that act in the final stages of embryogenesis and have previously been cloned as cDNAs. We demonstrate here that their proximal promoter regions are very similar (80% identity) and that both contain putative abscisic acid-responsive elements.     

Identification of a Replication Protein and Repeats Essential for DNA Replication of the Temperate Lactococcal Bacteriophage TP901-1

DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by orf13 and the repeats located within the gene. Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13). The putative origin of replication was identified as series of repeats within orf13 and was shown to confer a TP901-1 resistance phenotype when present in trans. Site-specific mutations were introduced into the replication protein and into the repeats. The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon. Subsequent analysis of induced phages showed that the protein encoded by orf13 and the repeats within orf13 were essential for phage TP901-1 amplification. In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo. These results show that orf13 encodes a replication protein and that the repeats within the gene are the origin of replication.     

Enhancement of PCR Amplification of Moderate GC-Containing and Highly GC-Rich DNA Sequences

PCR is a commonly used and highly efficient technique in biomolecular laboratories for specific amplification of DNA. However, successful DNA amplification can be very time consuming and troublesome because many factors influence PCR efficiency. Especially GC-rich DNA complicates amplification because of generation of secondary structures that hinder denaturation and primer annealing. We investigated the impact of previously recommended additives such as dimethylsulfoxide (DMSO), magnesium chloride (MgCl₂), bovine serum albumin (BSA), or formamide. Furthermore, we tested company-specific substances as Q-Solution, High GC Enhancer, and Hi-Spec; various actively promoted polymerases as well as different PCR conditions for their positive effects on DNA amplification of templates with moderate and extremely high CG-content. We found considerable differences of specificity and quantity of product between different terms. In this article, we introduce conditions for optimized PCR to help resolve problems amplifying moderate to high GC-rich templates.     

Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for microorganisms

We report the rapid generation of DNA probes for several Azospirillum strains. This method does not require any knowledge of the genetics and/or the molecular biology of the organism (genome) to be investigated. The procedure is based on the generation of random amplified polymorphic DNA (RAPD) fingerprints using primers with an embedded restriction site. The amplification product(s) peculiar to one strain or common to two or more strains can be purified, cloned, sequenced and used as molecular probes in hybridization experiments for the detection and identification of microorganisms. We have tested this methodology in the nitrogen-fixing bacterium Azospirillum by amplyfing the total DNA extracted from several Azospirillum strains. We have used amplification bands with different specificity as molecular probes in hybridization experiments performed on amplified DNA. Results obtained have demonstrated the usefulness of this methodology for Azospirillum. Its use in microbial ecology studies as a general strategy to generate specific DNA probes is also discussed.     

Sequencing PCR DNA amplified directly from a bacterial colony

We show that PCR product asymmetrically amplified directly from a bacterial colony can be sequenced to yield results as good as those obtained when purified template DNA is used for the PCR amplification step. With either template, greater than 300 nucleotides can be read from a typical sequencing reaction. Taq DNA polymerase was used for both the PCR amplification and sequencing reactions.     

Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method

Metagenomics generates and tests hypotheses about dynamics and mechanistic drivers in wild populations, yet commonly suffers from insufficient (相似文献   

Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA.

We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information.     

Preferential amplification is minimised in long-PCR systems

The advent of long PCR (XL-PCR) has proven to be a major advance in PCR technology and is currently being utilised to investigate numerous biological systems. The analysis of mixed DNA populations is a particularly useful application for XL-PCR. For example, XL-PCR has been used to investigate the occurrence of heterogeneous mitochondrial DNA (mtDNA) rearrangement mutations. With XL-PCR it became possible to amplify the entire length of the mtDNA chromosome and detect any mtDNA deletion or insertion mutations based on a measurable change in overall sequence length. In the present communication, XL-PCR and conventional short-length PCR were used to amplify mitochondrial DNA sequences from several human vastus lateralis skeletal muscle samples. The experiments demonstrated that there was minimal preferential amplification of shorter DNA sequences with XL-PCR and was significantly less than the preferential amplification of shorter sequences observed with conventional PCR. Also, XL-PCR amplification of the complete mtDNA sequence from control DNA containing a single mtDNA template (leucocyte extracts) showed that the generation of PCR artefacts was not a predisposed failing of the system but was dependant on the standard rules that govern the set up and optimisation of any PCR reaction. In optimised systems, XL-PCR artefacts were not generated and a single PCR product was always recovered.     

The ligation amplification reaction (LAR)--amplification of specific DNA sequences using sequential rounds of template-dependent ligation

A novel DNA sequence detection method that utilizes the ligation of oligonucleotide pairs that are complementary to adjacent sites on appropriate DNA templates is described. The product is increased by either linear or exponential amplification using sequential rounds of template-dependent ligation. In the case of linear amplification, a single pair of oligonucleotides is ligated, the reaction is heated to dissociate the ligation product, and an additional round of ligation is performed. After n rounds there is a (1 + x) X n-fold amplification of product, where x is the efficiency of the ligation reaction. Exponential amplification utilizes two pairs of oligonucleotides, one complementary to the upper strand and one to the lower strand of a target sequence. The products of the ligation reaction serve as templates for subsequent rounds of ligation. In this case there is (1 + x)(n-1)-fold amplification of product after n rounds. A single base-pair mismatch between the annealed oligonucleotides and the template prevents ligation, thus allowing the distinction of single base-pair differences between DNA templates. At high template concentrations, the ligation reaction has an efficiency approaching 100%. In this report, we demonstrate the use of the ligation amplification reaction (LAR) to distinguish the normal from the sickle cell allele of the human beta-globin gene. We also report the use of LAR as a detection system for polymerase chain reaction-enriched DNA sequences.     

Apolipoprotein B(Arg3500----Gln) allele specific polymerase chain reaction: large-scale screening of pooled blood samples.

A two-step polymerase chain reaction (PCR) method for the rapid detection of the apolipoprotein B(Arg3500----Gln) mutation in a mixture of pooled blood samples is described. In the first step PCR, a short gene fragment surrounding codon 3500 is amplified. Subsequently the reaction product is subjected to a second amplification in which a mutation-specific primer is used. A PCR product is generated only if the mutant sequence is present in the DNA pool. Individuals carrying the mutation can then be identified by PCR with mutagenic primers and MspI restriction typing, essentially as described by Hansen et al. (J. Lipid Res. 1991. 32: 1229-1233).