AcePrimer: automation of PCR primer design based on gene structure

AcePrimer is an internet-accessed application based on CGI/Perl programming that designs PCR primers to search for deletion alleles in Caenorhabditis elegans gene knockout experiments and uses electronic PCR to search the entire genomic DNA sequence for potential false priming or multiple PCR amplification targets. Features such as the ability to target specific exons with the 'poison primer' approach and evaluation of primers with electronic PCR provide a flexible, web-based approach to design effective primers whilst minimizing the need for empirical optimization of PCR experiments.     

Universal primers for fluorescent labelling of PCR fragments--an efficient and cost-effective approach to genotyping by fluorescence

Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.     

Immunoglobulin complementarity-determining region grafting by recombinant polymerase chain reaction to generate humanised monoclonal antibodies

We describe an approach to rapidly generate humanised monoclonal antibodies by grafting rodent complementarity-determining regions onto human immunoglobulin frameworks using recombinant polymerase chain reaction (PCR) methodology. The approach was applied to grafting a rat complementarily-determining region onto a human framework and amplifying the entire humanised heavy chain. The terminal oligodeoxyribonucleotide primers incorporated restriction sites to allow forced cloning into plasmid vectors for sequencing and expression. No nucleotide errors were introduced into the 1463-bp sequence even after sequential applications of PCR.     

An Alternative Approach in Gateway® Cloning when the Bacterial Antibiotic Selection Cassettes of the Entry Clone and Destination Vector are the Same

The Gateway^® recombination technology has revolutionized the method of gene cloning for functional analyses and high-throughput ORFeome projects. In general, Gateway cloning is highly efficient because after LR recombination and bacterial transformation, only cells containing the recombinant destination clone are selected on an antibiotic selection plate. However, when the antibiotic resistance gene for bacterial selection is the same in the entry and destination vectors, the direct selection of recombinant destination clones on an antibiotic plate is difficult. Here, we demonstrate an efficient and comprehensive approach to obtain positive destination clones directly on an antibiotic selection plate in this situation. The strategy involves polymerase chain reaction (PCR)-mediated amplification of the entry clone using entry vector-specific primers that bind outside the attL sequences and the subsequent use of this purified PCR product for LR recombination with the destination vector. Our results suggest that cloning of linear DNA fragments into circular destination vectors through LR recombination is an efficient method for inserts up to 7 kb in size. Using this approach, the yield of colony PCR positive destination clones was 100 % for genes of various sizes tested in our experiments.     

Indexed PCR Primers Induce Template-Specific Bias in Large-Scale DNA Sequencing Studies

Massively parallel sequencing is rapidly emerging as an efficient way to quantify biodiversity at all levels, from genetic variation and expression to ecological community assemblage. However, the number of reads produced per sequencing run far exceeds the number required per sample for many applications, compelling researchers to sequence multiple samples per run in order to maximize efficiency. For studies that include a PCR step, this can be accomplished using primers that include an index sequence allowing sample origin to be determined after sequencing. The use of indexed primers assumes they behave no differently than standard primers; however, we found that indexed primers cause substantial template sequence-specific bias, resulting in radically different profiles of the same environmental sample. Likely the outcome of differential amplification efficiency due to primer-template mismatch, two indexed primer sets spuriously change the inferred sequence abundance from the same DNA extraction by up to 77.1%. We demonstrate that a double PCR approach alleviates these effects in applications where indexed primers are necessary.     

PCR method for detecting recombinant baculoviruses with the insertion of a large gene

A PCR strategy was developed using primers specific to an infectious bursal disease virus (IBDV) gene as well as primers flanking the polyhedrin region of baculovirus to verify the presence of IBDV gene in the recombinant baculovirus and confirm the absence of wild-type baculovirus contamination. This method can be applied to detect the presence of large genes in the recombinant baculovirus with greater sensitivity and avoid the need of modifying the typical PCR procedure provided by the manufacturer. © Rapid Science Ltd. 1998     

A convenient and robust method for construction of combinatorial and random mutant libraries

Here we describe a convenient and robust ligase-independent method for construction of combinatorial and random mutant libraries. The homologous genes flanked by plasmid-derived DNA sequences are fragmented, and the random fragments are reassembled in a self-priming polymerase reaction to obtain chimeric genes. The product is then mixed with linearized vector and two pairs of flanking primers, followed by assembly of the chimeric genes and linearized vector by PCR to introduce recombinant plasmids of a combinatorial library. Commonly, it is difficult to find proper restriction sites during the construction of recombinant plasmids after DNA shuffling with multiple homologous genes. However, this disadvantage can be overcome by using the ligase-independent method because the steps of DNA digestion and ligation can be avoided during library construction. Similarly, DNA sequences with random mutations introduced by error-prone PCR can be used to construct recombinant plasmids of a random mutant library with this method. Additionally, this method can meet the needs of large and comprehensive DNA library construction.     

RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3′-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.     

A PCR-after-ligation method for cloning of multiple DNA inserts

Here we present a novel and simple PCR-after-ligation method for efficient assembly of multiple DNA inserts. After initial ligation of multiple inserts and vector, the ligation mixture is used as template for a PCR using a pair of primers flanking the cloning sites on the vector. The fragment with correct size is gel purified and inserted into the vector by conventional two-way ligation. With this method, a recombinant plasmid containing four DNA inserts was correctly constructed. As a control, all of the constructs obtained directly from DNA ligation were found to be self-ligation of the vector.     

Ligation-independent cloning of PCR products (LIC-PCR).

A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.     

食源性致病菌多重PCR快速检测方法建立与应用

利用PCR技术,建立多组多重食源性致病菌PCR快速检测方法。设计受试菌特异性引物,反应体系中加入多对引物和多种DNA模板,采用正交试验优化PCR反应条件,进行特异性引物的PCR扩增。建立了多组多重食源性致病菌PCR快速检测方法,方法中所检测受试菌株和模拟样品均出现特异性扩增条带,结果与实际相符。所建立多组多重PCR快速检测体系符合设计要求,可以应用于食源性突发公共卫生事件的应急检测和日常样品检测工作。     

Human MECP2 gene at Q28 arm of X chromosome as a suitable target for monitoring PCR inhibition in a nested, multiplexed HIV-1 DNA detection protocol

Human MECP2 gene located at q28 arm of X chromosome was identified as target for thermal co-amplification with HIV-1 proviral DNA of infected individuals. The selected MECP2 gene-specific primers functioned at a wide range of annealing temperature, extension time and exhibited no significant interaction with pathogen specific primers. A 466 bp PCR amplicon originating from human MECP2 gene was found to be diagnostic for inhibition-free PCR reaction when co-amplified with the HIV-1 target gene in a multiplexed, nested PCR reaction. The 5' end of the MECP2 primers were engineered to position an EcoRI restriction endonuclease site to facilitate rapid cloning in various DNA vector molecules at the corresponding EcoRI sites. Cell mass of Escherichia coli (XL1Blue) harboring the recombinant plasmid when added to pleural fluid of HIV-1 infected individuals co-infected with Mycobacterium tuberculosis, generated the diagnostic 466 bp MECP2 PCR amplicon as well as the 194 bp PCR amplicon of target gene from M. tuberculosis. The experiment underlined potential of the region spanning nucleotide position 4118099 to 4118552 of human MECP2 gene (NCBI accession number NT_011726.13) as a reliable target for multiplex PCR to accommodate a wide range of thermal cycling and multiplex reaction conditions. In both cases of this study, electrophoresis-based separation of the 466 bp MECP2 fragment and the 232 bp and 194 bp HIV-1 and M. tuberculosis fragments respectively was distinct and unambiguous. The potential of this human MECP2 gene available from human genome or recombinant plasmid as a potent target to monitor PCR inhibition for a range of different PCR reactions is discussed.     

Extending homologous sequence based on the single gene mutants by one-step PCR for efficient multiple gene knockouts

Multiple gene knockouts play an important role in metabolic engineering. The flanked homology length, homologous to the region adjacent to the target gene, of the knockout fragments has a great effect on the efficiency of multiple gene knockouts, whereas the existing gene knockout methods can only supply a very short homology. This article presents a strategy of easily extending homologous sequence based on the available strain library through one-step PCR amplification (the one-step PCR method). In this approach, the library of single gene mutants was used as the templates for PCR to amplify knockout fragments. Thus, the flanked homology can be extended as long as possible by designing primers upstream and downstream far from the target gene. Based on the one-step PCR method, we studied the effect of the homology length and the number of mutations on the efficiency of multiple gene knockouts. Our results indicated that the one-step PCR method permitted rapid and efficient construction of multiple mutants continuously or simultaneously, and a length of 200–300 bp homologous sequence was equal for multiple gene knockouts.     

Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension

Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in separate polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are 'spliced' together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques.     

Development of a 16S rDNA-targeted PCR assay for monitoring of Lactobacillus plantarum and Lact. rhamnosus during co-cultivation for production of inoculants for silages

AIMS: This paper reports a simple, rapid approach for the parallel detection of Lactobacillus plantarum and Lact. rhamnosus in co-culture in order to produce an inoculant mixture for silage purposes. METHODS AND RESULTS: The 16S rDNA-targeted PCR primers were established for parallel detection of Lact. plantarum and Lact. rhamnosus in a single multiplex PCR. A protocol for application of these primers in direct PCR as well as colony-direct (CD) PCR was developed. These primers were also applicable for the estimation of the relative amount of each DNA type in mixed probes (semi-quantitative PCR). CONCLUSIONS: The PCR assay presented in this study is a robust, fast and semi-quantitative approach for detection of Lact. plantarum and Lact. rhamnosus in liquid cultures as well as on agar plates. SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides an effective tool for the establishment of a regime for co-cultivation of Lact. plantarum and Lact. rhamnosus. This would enable faster and thus cost-reduced production of ensiling inoculants.     

定点突变三种方法的比较研究

目的：通过使用优化后的定点突变三种方法分别对一个新基因重组载体进行定点突变研究,比较这几种定点突变方法的优缺点。方法：重叠延伸PCR法使用Stratagene在线定点突变引物设计程序从而使引物设计简化而可靠;MutaBEST定点突变试剂盒采用胶纯化试剂盒代替说明书推荐的酚-氯仿抽提质粒的方法以提高回收率;使用PrimeSTAR高保真DNA聚合酶和超级感受态试剂盒代替Quikchange定点突变试剂盒的相应组分可使产物不受影响同时降低试验费用。结果：三种方法都能够获得单碱基突变重组载体。结论：QuikChange突变策略通过改进是一种高效、简洁、经济和可靠的定点突变方法。     

Multi-fragment site-directed mutagenic overlap extension polymerase chain reaction as a competitive alternative to the enzymatic assembly method

Methods for introducing multiple site-directed mutations are important experimental tools in molecular biology. Research areas that use these methods include the investigation of various protein modifications in cellular processes, modifying proteins for efficient recombinant expression, and the stabilization of mRNAs to allow for increased protein expression. Introducing multiple site-directed mutations is also an important tool in the field of synthetic biology. There are two main methods used in the assembling of fragments generated by mutagenic primers: enzymatic assembly and overlap extension polymerase chain reaction (OE–PCR). In this article, we present an improved OE–PCR method that can be used for the generation of large DNA fragments (up to 7.4 kb) where at least 13 changes can be introduced using a genomic template. The improved method is faster (due to fewer reaction steps) and more accurate (due to fewer PCR cycles), meaning that it can effectively compete with the enzymatic assembly method. Data presented here show that the site-directed mutations can be introduced anywhere between 50 and 1800 bp from each other. The method is highly reliable and predicted to be applicable to most DNA engineering when the introduction of multiple changes in a DNA sequence is required.     

Evaluation of “sequence-tagged-site” PCR products as molecular markers in wheat

The polymerase chain reaction (PCR) is an attractive technique for many genome mapping and characterization projects. One PCR approach which has been evaluated involves the use of randomly amplified polymorphic DNA (RAPD). An alternative to RAPDs is the sequence-tagged-site (STS) approach, whereby PCR primers are designed from mapped low-copy-number sequences. In this study, we sequenced and designed primers from 22 wheat RFLP clones in addition to testing 15 primer sets that had been previously used to amplify DNA sequences in the barley genome. Our results indicated that most of the primers amplified sequences that mapped to the expected chromosomes in wheat. Additionally, 9 of 16 primer sets tested revealed polymorphisms among 20 hexaploid wheat genotypes when PCR products were digested with restriction enzymes. These results suggest that the STS-based PCR analysis will be useful for generation of informative molecular markers in hexaploid wheat.Contribution no. J-2833 of the Montana Agric Exp Stn