Ligation mediated PCR performed at low denaturation temperatures--PCR melting profiles

We show that using low denaturation temperatures (80-88 degrees C) during ligation mediated PCR (LM PCR) of bacterial DNA leads to the amplification of limited sets of the less stable DNA fragments. A set of electrophoretic patterns of such fragments obtained at different denaturation temperatures forms the PCR melting profile (PCR MP). A single pattern obtained for a given temperature and a set of patterns arising after application of several denaturation temperatures (PCR MP) are very specific for the given bacterial genome and may be used for strain characterisation and differentiation. The method may also be used for amplification and isolation of the less stable DNA fragments in a genome.     

PCR MP (PCR melting profile) technique--possibilities of application in epidemiological studies

The performance and convenience of a PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation mediated PCR (LM PCR) of bacterial DNA is shown. We found that PCR MP technique is a rapid method that offers good discriminatory power, excellent reproducibility and may be applied for epidemiological studies. Results from strain genotyping illustrate that PCR MP is useful for the study of intraspecific genetic relatedness of strains and is as effective in discriminating closely related strains as the PFGE method, which is currently considered to be the gold standard for epidemiological studies. The usefulness of the PCR MP for molecular typing was shown for clinical strains of Escherichia coli, Enterococcus faecium VRE and Stapylococcus aureus.     

Template-blocking PCR: An advanced PCR technique for genome walking

This article describes the development of an improved method for the isolation of genomic fragments adjacent to a known DNA sequence based on a cassette ligation-mediated polymerase chain reaction (PCR) technique. To reduce the nonspecific amplification of PCR-based genome walking, the 3′ ends of the restriction enzyme-digested genomic DNA fragments were blocked with dideoxynucleoside triphosphate (ddNTP) and ligated with properly designed cassettes. The modified genomic DNA fragments flanked with cassettes were used as a template for the amplification of a target gene with a gene-specific primer (GSP) and a cassette primer (CP). The ddNTP blocking of the genomic DNA ends significantly reduced the nonspecific amplification and resulted in a simple and rapid walking along the genome. The efficiency of the template-blocking PCR method was confirmed by a carefully designed control experiment. The method was successfully applied for the cloning of the PGK1 promoter from Pichia ciferrii and two novel cellulase genes from Penicillium sp.     

Effect of highly fragmented DNA on PCR.

We characterized the behavior of polymerase chain reactions (PCR) using degraded DNA as a template. We first demonstrated that fragments larger than the initial template fragments can be amplified if overlapping fragments are allowed to anneal and extend prior to routine PCR. Amplification products increase when degraded genomic DNA is pretreated by polymerization in the absence of specific primers. Secondly, we measured nucleotide uptake as a function of template DNA degradation. dNTP incorporation initially increases with increasing DNA fragmentation and then declines when the DNA becomes highly degraded. We demonstrated that dNTP uptake continues for >10 polymerization cycles and is affected by the quality and quantity of template DNA and by the amount of substrate dNTP. These results suggest that although reconstruction of degraded DNA may allow amplification of large fragments, reconstructive polymerization and amplification polymerization may compete. This was confirmed in PCR where the addition of degraded DNA reduced the resultant product. Because terminal deoxynucleotidyl transferase activity of Taq polymerase may inhibit 3' annealing and restrict the length of template reconstruction, we suggest modified PCR techniques which separate reconstructive and amplification polymerization reactions.     

A novel kinase-based ATP assay using molecular beacon

Quantitation of E6 oncogene sequences of the human papillomavirus type 16 by real-time or quantitative PCR (qPCR) is used to determine the viral load, which correlates with the degree of the cervical neoplastic lesions. In the presence of EvaGreen, a new DNA intercalating fluorochrome, we obtained consistent and reproducible qPCR amplification curves and thermal denaturation profiles identical to those of the authentic E6–HPV16 (human papillomavirus 16) genome from the amplification products derived from a construct carrying the E6–HPV16 oncogene. E6–HPV16 quantitation in the presence of EvaGreen, therefore, is reproducible and specific and may be used to determine HPV16 viral load.     

Chlamydomonas (Chlorophyceae) colony PCR

The ease and effectiveness of colony polymerase chain reaction (PCR) has allowed rapid amplification of DNA fragments and screening of large number of colonies of interest including transformants and mutants with genetic manipulations. Here, we evaluated colony PCR in Chlamydomonas. Individual colonies were treated with 10 mM ethylenediaminetetraacetic acid (EDTA) or Chelex-100 and the resulting clear cell lysate was used for PCR reaction. Either genomic DNA or plasmid DNA incorporated into the genome was equally amplified. We found that the Chelex method is superior to EDTA method in certain cases. This colony PCR technique will bypass the tedious process of isolating genomic DNA for PCR reaction and will make it possible for rapid amplification of genomic DNA fragments as well as rapid large-scale screening of transformants.     

PCR assay based on a microsatellite-containing locus for detection and quantification of Epichloë endophytes in grass tissue.

A PCR assay which allows detection and quantification of Epichloë endophytes in tissues of the grass Bromus erectus is described. PCR with specific primers flanking a microsatellite-containing locus (MS primers) amplified fragments 300 to 400 bp in length from as little as 1.0 pg of fungal genomic DNA in 100 ng of DNA from infected plant material. When annealing temperatures were optimized, all Epichloë and Acremonium strains tested, representing many of the known taxonomic groups, yielded an amplification product, indicating that the MS primers may be useful for in planta detection of a variety of related species, including agronomically important Acremonium coenophialum and Acremonium lolii. No fragments were generated from DNA isolates from uninfected plant material or from unrelated fungi isolated from B. erectus. For diagnostic applications, a B. erectus-specific primer pair was designed for use in multiplex PCR to allow simultaneous amplification of plant and fungal DNA sequences, providing an internal control for PCR failure caused by inhibitory plant compounds present in DNA extracts. For quantitative applications, a heterologous control template in primer binding sites complementary to the MS primers was constructed for use in competitive PCR, allowing direct quantification of Epichloë in plant DNA extracts. The fungal DNA present in infected leaves of B. erectus between 1 and 20 pg per 100 ng of leaf DNA, but the amounts of fungal DNA present in the sheath and blade of a given leaf were correlated, indicating that the degree of infection varied between plant individuals but that leaves were colonized in a uniform way.     

An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3′→5′ exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 10⁷-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.     

Bisulphite Differential Denaturation PCR for Analysis of DNA Methylation

Differential denaturation during PCR can be used to selectively amplify unmethylated DNA from a methylated DNA background. The use of differential denaturation in PCR is particularly suited to amplification of undermethylated sequences following treatment with bisulphite, since bisulphite selectively converts cytosines to uracil while methylated cytosines remain unreactive. Thus amplicons derived from unmethylated DNA retain less cytosines and their lower G + C content allows for their amplification at the lower melting temperatures, while limiting amplification of the corresponding methylated amplicons (Bisulphite Differential Denaturation PCR, BDD-PCR). Selective amplification of unmethylated DNA of four human genomic regions from three genes, GSTP1, BRCA1 and MAGE-A1, is demonstrated with selectivity observed at a ratio of down to one unmethylated molecule in 105 methylated molecules. BDD-PCR has the potential to be used to selectively amplify and detect aberrantly demethylated genes, such as oncogenes, in cancers. Additionally BDD-PCR can be effectively utilised in improving the specificity of methylation specific PCR (MSP) by limiting amplification of DNA that is not fully converted, thus preventing misinterpretation of the methylation versus non-conversion.      

Plastic versus glass capillaries for rapid-cycle PCR

Rapid-cycle PCR uses fast temperature transitions and minimal denaturation and annealing times of "0" s to complete 30 cycles in 10 to 30 min. The most popular platform amplifies samples in glass capillaries arranged around a carousel with circulating air for temperature control. Recently, plastic capillary replacements for glass capillaries became available. We compared the performance of plastic and glass capillaries for rapid-cycle PCR. Heat transfer into plastic capillaries was slowed by thicker walls, lower thermal conductivity, and a lower surface area-to-volume ratio than glass capillaries. Whereas the denaturation and annealing target temperatures were reached by samples in glass capillaries, samples in plastic capillaries fell short of these target temperatures by 6 degrees -7 degrees C. Rapid-cycle PCR was performed on two human genomic targets (APOE and ACVRL1) and one plasmid (pBR322) to amplify fragments of 225-300 bp in length with melting temperatures of 90.3 degrees -93.1 degrees C. Real-time amplification data, end-point melting curves, and end-point gel analysis revealed strong, specific amplification of samples in glass and complete amplification failure in plastic. Only the APOE target was successfully amplified by extending the denaturation and annealing times to 5 or 10 s. A 20 s holding period was necessary to reach target temperatures in plastic capillaries.     

In silico analysis of complete bacterial genomes: PCR, AFLP-PCR and endonuclease restriction

We have developed a website, www.in-silico.com, which runs a software program that performs three basic tasks in completely sequenced bacterial genomes by in silico analysis: PCR amplification, amplified fragment length polymorphism (AFLP-PCR) and endonuclease restriction. For PCR, after selection of the genome and introduction of primers, fragment size, DNA sequence and corresponding open reading frame (ORF) identity of the resulting PCR product is computed. Plasmids of sequenced species may be included in the analysis. Theoretical AFLP-PCR analyzes similar parameters, and includes a suggestion tool providing a list of commercial restriction enzyme pairs yielding up to 50 amplicons in the selected genome. Endonuclease restriction analysis of complete genomes and plasmids calculates the number of restriction sites for endonucleases in a given genome. If the number of fragments is 50 or fewer, pulsed field gel electrophoresis image and restriction maps are illustrated. Other tools that have been included in this site are ORF search by name and DNA to protein translation as well as restriction digestion of user-defined DNA sequences. AVAILABILITY: This is a new molecular biology resource freely available over the Internet at http://www.in-silico.com     

Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA

An innovative combination of various recently described molecular methods was set up to efficiently identify regions flanking a marker DNA in insertional mutants of Chlamydomonas. The technique is named restriction enzyme site-directed amplification PCR (RESDA-PCR) and is based on the random distribution of frequent restriction sites in a genome and on a special design of primers. The primer design is based on the presence of a restriction site included in a low degenerated sequence at the 3' end and of a specific adapter sequence at the 5' end, with the two ends being linked by a polyinosine bridge. Specific primers of the marker DNA combined with the degenerated primers allow amplification of DNA fragments adjacent to the marker insertion by using two rounds of either short or long cycling procedures. Amplified fragments from 0.3 to 2 kb or more are routinely obtained at sufficient purity and quantity for direct sequencing. This method is fast, is reliable (87% success rate), and can be easily extrapolated to any organism and marker DNA by designing the appropriate primers. A procedure involving the PCR over enzyme digest fragments is also proposed for when, exceptionally, positive results are not obtained.     

Genome-scale design of PCR primers and long oligomers for DNA microarrays

During the last years, the demand for custom-made cDNA chips/arrays as well as whole genome chips is increasing rapidly. The efficient selection of gene-specific primers/oligomers is of the utmost importance for the successful production of such chips. We developed GenomePRIDE, a highly flexible and scalable software for designing primers/oligomers for large-scale projects. The program is able to generate either long oligomers (40–70 bases), or PCR primers for the amplification of gene-specific DNA fragments of user-defined length. Additionally, primers can be designed in-frame in order to facilitate large-scale cloning into expression vectors. Furthermore, GenomePRIDE can be adapted to specific applications such as the generation of genomic amplicon arrays or the design of fragments specific for alternative splice isoforms. We tested the performance of GenomePRIDE on the entire genomes of Listeria monocytogenes (1584 gene-specific PCRs, 48 long oligomers) as well as of eukaryotes such as Schizosaccharomyces pombe (5006 gene-specific PCRs), and Drosophila melanogaster (21 306 gene-specific PCRs). With its computing speed of 1000 primer pairs per hour and a PCR amplification success of 99%, GenomePRIDE represents an extremely cost- and time-effective program.     

The new LM-PCR/shifter method for the genotyping of microorganisms based on the use of a class IIS restriction enzyme and ligation mediated PCR

This study details and examines a novel ligation-mediated polymerase chain reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4-base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4- base sequence of the 3' end of the primer(s) determine a subset of the genomic restriction fragments, which are amplified by PCR. The method permits the differentiation of bacterial species strains on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light. The usefulness of the LM-PCR/ Shifter method for genotyping is analyzed by a comparison with the restriction endonuclease analysis of chromosomal DNA by the pulsed-field gel electrophoresis (REA-PFGE) and PCR melting profile (PCR MP) methods for isolates of clinical origin. The clustering of the LM-PCR/Shifter fingerprinting data matched those of the REA-PFGE and PCR MP methods. We found that the LM-PCR/Shifter is rapid, and offers good discriminatory power and excellent reproducibility, making it a method that may be effectively applied in epidemiological studies.     

Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5′ end of the PCR primer and the extended newly synthesized DNA 3′ end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by ‘selection marker swapping’ upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.     

DNA analysis with multiplex microarray-enhanced PCR

We have developed a highly sensitive method for DNA analysis on 3D gel element microarrays, a technique we call multiplex microarray-enhanced PCR (MME-PCR). Two amplification strategies are carried out simultaneously in the reaction chamber: on or within gel elements, and in bulk solution over the gel element array. MME-PCR is initiated by multiple complex primers containing gene-specific, forward and reverse, sequences appended to the 3′ end of a universal amplification primer. The complex primer pair is covalently tethered through its 5′ end to the polyacryl- amide backbone. In the bulk solution above the gel element array, a single pair of unattached universal primers simultaneously directs pseudo-monoplex PCR of all targets according to normal solution-phase PCR. The presence of a single universal PCR primer pair in solution accelerates amplification within gel elements and eliminates the problem of primer interference that is common to conventional multiplex PCR. We show 10⁶-fold amplification of targeted DNA after 50 cycles with average amplification efficiency 1.34 per cycle, and demonstrate specific on-chip amplification of six genes in Bacillus subtilis. All six genes were detected at 4.5 pg of bacterial genomic DNA (equivalent to 10³ genomes) in 60 independent amplification reactions performed simultaneously in single reaction chamber.     

Specific PCR primers to identify arbuscular mycorrhizal fungi within colonized roots

 A set of PCR primers targeted at five major phylogenetic subgroups of arbuscular mycorrhizal fungi (Glomales) was designed to facilitate specific amplification of internal transcribed spacers and 18 S rRNA gene fragments from colonized roots in the absence of spores. The subgroups include the recently discovered deeply divergent lineages of Glomales, which could not be detected by previously reported PCR primers, and the former genus Sclerocystis. Restriction fragment length polymorphism patterns presented allow identification of presently known members of these groups. The resulting PCR products can be used to identify the fungal symbionts at the genus or species level by restriction digests or DNA sequencing. A novel DNA extraction method allows visual control of the analyzed roots by staining procedures after analysis by PCR. Accepted: 2 April 2000     

Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates

Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99°C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or Taq plus Pwo DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.coli exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.     

PCR fragmentation of DNA

A method has been developed to prepare random DNA fragments using PCR. First, two cycles are carried out at 16 degrees C with the Klenow's fragment and oligonucleotides (random primers) with random 3'-sequences and the 5'-constant part containing the site for cloning with the site-specific endonuclease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturation of the DNA and addition of the Klenow's fragment, the random primers can link to newly synthesized DNA strands, and after DNA synthesis single-stranded DNA fragments are produced which have a constant primer sequence at the 5'-end and a complementary to it sequence at the 3'-end. During the third cycle, the constant primer is added and double-stranded fragments with the constant primer sequences at both ends are formed during DNA synthesis. Incubation for 1 h at 37 degrees C degrades the oligonucleotides used at the first stage due to endonuclease activity of the Klenow's fragment. Then routine PCR amplification is carried out using the constant primer. This method is more advantageous than hydrodynamic methods of DNA fragmentation widely used for "shotgun" cloning.