Conserved PCR primer set designing for closely-related species to complete mitochondrial genome sequencing using a sliding window-based PSO algorithm

Background

Complete mitochondrial (mt) genome sequencing is becoming increasingly common for phylogenetic reconstruction and as a model for genome evolution. For long template sequencing, i.e., like the entire mtDNA, it is essential to design primers for Polymerase Chain Reaction (PCR) amplicons which are partly overlapping each other. The presented chromosome walking strategy provides the overlapping design to solve the problem for unreliable sequencing data at the 5′ end and provides the effective sequencing. However, current algorithms and tools are mostly focused on the primer design for a local region in the genomic sequence. Accordingly, it is still challenging to provide the primer sets for the entire mtDNA.

Methodology/Principal Findings

The purpose of this study is to develop an integrated primer design algorithm for entire mt genome in general, and for the common primer sets for closely-related species in particular. We introduce ClustalW to generate the multiple sequence alignment needed to find the conserved sequences in closely-related species. These conserved sequences are suitable for designing the common primers for the entire mtDNA. Using a heuristic algorithm particle swarm optimization (PSO), all the designed primers were computationally validated to fit the common primer design constraints, such as the melting temperature, primer length and GC content, PCR product length, secondary structure, specificity, and terminal limitation. The overlap requirement for PCR amplicons in the entire mtDNA is satisfied by defining the overlapping region with the sliding window technology. Finally, primer sets were designed within the overlapping region. The primer sets for the entire mtDNA sequences were successfully demonstrated in the example of two closely-related fish species. The pseudo code for the primer design algorithm is provided.

Conclusions/Significance

In conclusion, it can be said that our proposed sliding window-based PSO algorithm provides the necessary primer sets for the entire mt genome amplification and sequencing.     

An integrated pipeline for the development of novel panels of mapped microsatellite markers for Leishmania donovani complex, Leishmania braziliensis and Leishmania major

A panel of microsatellites mapped to the Leishmania genome might make it possible to find associations between specific loci and phenotypic traits. To identify such loci, a Perl programme was written that scans the sequence of a genome and writes all loci containing microsatellites to a MySQL database. The programme was applied to the sequences of the L. braziliensis, L. infantum and L. major genomes. The database is publicly available over the internet: http://www.genomics.liv.ac.uk/tryps/resources.html 'Microsatellite Locus Extractor', and allows the selection of mapped microsatellites that meet user-defined criteria from a specified region of the selected genome. The website also incorporates a primer design pipeline that will design primers to amplify the selected loci. Using this pipeline 12 out of 17 primer sets designed against the L. infantum genome generated polymorphic PCR products. A tailed primer protocol was used to label all microsatellite primers with a single set of labelled primers. To avoid the culture of parasites prior to genotyping, sets of nested PCR primers were developed to amplify parasite DNA eluted from microscope slides. The limit of detection was approximately 1.6 parasite equivalents. However, only 6/56 DNA from slides stored at ambient temperature for over 6 months gave positive PCR results.     

GONOME: measuring correlations between GO terms and genomic positions

Background

The design of oligonucleotides and PCR primers for studying large genomes is complicated by the redundancy of sequences. The eukaryotic genomes are particularly difficult to study due to abundant repeats. The speed of most existing primer evaluation programs is not sufficient for large-scale experiments.

Results

In order to improve the efficiency and success rate of automatic primer/oligo design, we created a novel method which allows rapid masking of repeats in large sequence files, for example in eukaryotic genomes. It also allows the detection of all alternative binding sites of PCR primers and the prediction of PCR products. The new method was implemented in a collection of efficient programs, the GENOMEMASKER package. The performance of the programs was compared to other similar programs. We also modified the PRIMER3 program, to be able to design primers from lowercase-masked sequences.

Conclusion

The GENOMEMASKER package is able to mask the entire human genome for non-unique primers within 6 hours and find locations of all binding sites for 10 000 designed primer pairs within 10 minutes. Additionally, it predicts all alternative PCR products from large genomes for given primer pairs.     

SNPbox: a modular software package for large-scale primer design

SUMMARY: We developed a modular software package SNPbox that automates and standardizes the generation of PCR primers and is used in the strategy for constructing single nucleotide polymorphisms (SNPs) maps. In this strategy, the focus of primer design can be either on the validation of annotated public SNPs or on the SNP discovery in exon regions or extended genomic regions, both by resequencing. SNPbox relies on Primer3 for the primer design and combines this program with other publicly available software tools such as BLAST, Spidey and RepeatMasker, and newly developed algorithms. Primer conditions were chosen such that PCR amplifications are uniform for each PCR amplicon facilitating the use of high-throughput genetic platforms. SNPbox can also be used for the design of primer sets for mutation analysis, STR marker genotyping and microarray oligo design. Of the 2500 primer sets designed by SNPbox, 95% successfully amplified genomic DNA under uniform PCR conditions. AVAILABILITY: The software is available from the authors upon request. SUPPLEMENTARY INFORMATION: SNPbox_supplement.     

Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes.

We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.     

用于聚合酶链式反应(PCR)引物设计的计算机程序

 本文报道了两个用于PCR引物设计的计算机程序PCRDESN和PCRDESNA。PCRDESN程序主要从以下4个方面评价用户自己设计的一对引物的质量:(1)引物内的碱基反向重复或发夹结构,(2)两个引物之间的碱基互补配对,(3)两个引物之间的同源性,(4)引物的碱基组成及特点和T_m值计算。通过用多例文献发表的及本院有关实验室提供的引物对序列的验证,确定了程序的运算参数,证明该程序能较好地检验引物对的质量和解释某些PCR实验失败的原因。PCRDESNA程序采用逐级优化的方法和比PCRDESN所选用的更严紧的引物选择参数对用户提供的核酸序列进行快速检索,以确定所有可能的和合适的引物对。     

MethPrimer: designing primers for methylation PCRs

MOTIVATION: DNA methylation is an epigenetic mechanism of gene regulation. Bisulfite- conversion-based PCR methods, such as bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP), remain the most commonly used techniques for methylation mapping. Existing primer design programs developed for standard PCR cannot handle primer design for bisulfite-conversion-based PCRs due to changes in DNA sequence context caused by bisulfite treatment and many special constraints both on the primers and the region to be amplified for such experiments. Therefore, the present study was designed to develop a program for such applications. RESULTS: MethPrimer, based on Primer 3, is a program for designing PCR primers for methylation mapping. It first takes a DNA sequence as its input and searches the sequence for potential CpG islands. Primers are then picked around the predicted CpG islands or around regions specified by users. MethPrimer can design primers for BSP and MSP. Results of primer selection are delivered through a web browser in text and in graphic view.     

Nuclear DNA-based markers for plant evolutionary biology

While DNA-based markers can provide a wealth of information for the study of plant evolutionary biology, progress is limited by the lack of primers available for PCR. To overcome this limitation, we outline a protocol for developing oligonucleotide primers targeting regions of low copy-number nuclear genes. This protocol is intended to lead to universally useful primer sets. To test our approach, we designed eight primer sets and tested their abilities to amplify targets from representatives of each dicot and one monocot subclass. Five of the eight primer sets amplified targets from at least five of the seven taxa and thus exhibited broad taxonomic usefulness; the remaining primers were rather specific, however, and amplified targets from at most three taxa. In only one primer-taxon combination was a complex multiple-banded amplification produced. Overall, the protocol outlined proved quite useful at identifying broadly applicable primers targeted to low copy-number nuclear genes. Wider application of this approach should be effective at greatly increasing the amount of genetic information available for a diversity of plant nuclear genomes.     

Systematic Design of 18S rRNA Gene Primers for Determining Eukaryotic Diversity in Microbial Consortia

High-throughput sequencing of ribosomal RNA gene (rDNA) amplicons has opened up the door to large-scale comparative studies of microbial community structures. The short reads currently produced by massively parallel sequencing technologies make the choice of sequencing region crucial for accurate phylogenetic assignments. While for 16S rDNA, relevant regions have been well described, no truly systematic design of 18S rDNA primers aimed at resolving eukaryotic diversity has yet been reported. Here we used 31,862 18S rDNA sequences to design a set of broad-taxonomic range degenerate PCR primers. We simulated the phylogenetic information that each candidate primer pair would retrieve using paired- or single-end reads of various lengths, representing different sequencing technologies. Primer pairs targeting the V4 region performed best, allowing discrimination with paired-end reads as short as 150 bp (with 75% accuracy at genus level). The conditions for PCR amplification were optimised for one of these primer pairs and this was used to amplify 18S rDNA sequences from isolates as well as from a range of environmental samples which were then Illumina sequenced and analysed, revealing good concordance between expected and observed results. In summary, the reported primer sets will allow minimally biased assessment of eukaryotic diversity in different microbial ecosystems.     

The degenerate primer design problem

A PCR primer sequence is called degenerate if some of its positions have several possible bases. The degeneracy of the primer is the number of unique sequence combinations it contains. We study the problem of designing a pair of primers with prescribed degeneracy that match a maximum number of given input sequences. Such problems occur when studying a family of genes that is known only in part, or is known in a related species. We prove that various simplified versions of the problem are hard, show the polynomiality of some restricted cases, and develop approximation algorithms for one variant. Based on these algorithms, we implemented a program called HYDEN for designing highly-degenerate primers for a set of genomic sequences. We report on the success of the program in an experimental scheme for identifying all human olfactory receptor (OR) genes. In that project, HYDEN was used to design primers with degeneracies up to 10(10) that amplified with high specificity many novel genes of that family, tripling the number of OR genes known at the time.     

The degenerate primer design problem: theory and applications.

A PCR primer sequence is called degenerate if some of its positions have several possible bases. The degeneracy of the primer is the number of unique sequence combinations it contains. We study the problem of designing a pair of primers with prescribed degeneracy that match a maximum number of given input sequences. Such problems occur when studying a family of genes that is known only in part, or is known in a related species. We prove that various simplified versions of the problem are hard, show the polynomiality of some restricted cases, and develop approximation algorithms for one variant. Based on these algorithms, we implemented a program called HYDEN for designing highly degenerate primers for a set of genomic sequences. We report on the success of the program in several applications, one of which is an experimental scheme for identifying all human olfactory receptor (OR) genes. In that project, HYDEN was used to design primers with degeneracies up to 10(10) that amplified with high specificity many novel genes of that family, tripling the number of OR genes known at the time.     

Species-specific detection of hydrocarbon-utilizing bacteria

Rapid detection and quantitative assessment of specific microbial species in environmental samples is desirable for monitoring changes in ecosystems and for tracking natural or introduced microbial species during bioremediation of contaminated sites. In the interests of developing rapid tests for hydrocarbon-degrading bacteria, species-specific PCR primer sets have been developed for Pseudomonas aeruginosa, Stentrophomonas (Xanthomonas) maltophilia, and Serratia marsescens. Highly variable regions of the 16S rRNA gene were used to design these primer sets. The amplification products of these primer sets have been verified and validated with hemi-nested PCR and with ligase chain reaction (LCR) techniques, and have been applied to the analyses of environmental water samples. These species-specific primer sets were also chosen to amplify in conjunction with a universal set of PCR primers chosen from highly conserved neighboring sequences in the same gene. These multiplex or competitive PCR procedures enable testing with an internal marker and/or the quantitative estimation of the relative proportion of the microbial community that any one of these species occupies. In addition, this universal PCR primer set amplified the same size amplicon from a wide spectrum of procaryotic and eucaryotic organisms and may have potential in earth biota analyses.     

Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis

The polymerase chain reaction is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. We have developed and tested efficient tools for PCR primer and probe design, which also predict oligonucleotide properties based on experimental studies of PCR efficiency. The tools provide comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, unique, group-specific, bisulphite modification assays, Overlap-Extension PCR Multi-Fragment Assembly, as well as a programme to design oligonucleotide sets for long sequence assembly by ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator.     

Characterization of CR1 repeat random PCR markers for mapping the chicken genome

Polymerase chain reaction (PCR) primers complementary to portions of the chicken repetitive element CR1 have been used previously to generate useful markers on the chicken genome linkage map. To understand better the genetic basis for this technique and to convert CR1–PCR loci to markers useful in physical genome mapping, five polymorphic CR1–PCR-generated DNAs were cloned and partially sequenced. Inverse PCR was then employed to clone the corresponding region of the genomes of both the Jungle Fowl (JF) and White Leghorn (WL) parental DNA templates. Our results demonstrate that some of the CR1–PCR-generated DNAs arise from priming at an endogenous CR1 element, whereas others are due to chance complementarity between the CR1–PCR primer in use and random annealing sites in the genome, unrelated to a demonstrable CR1 element. In all five instances, it was possible to identify the sequence difference between the JF and WL parental DNAs that gave rise to the initial polymorphism and design allele-specific PCR primer sets that uniquely detect that polymorphism. In four of the five instances, the polymorphism was a one or two basepair sequence difference within the primer annealing site, but in the fifth case the responsible difference was outside, but very close to, the annealing site. In all instances the allele-specific PCR for the sequence polymorphism mapped identically with the corresponding CR1–PCR amplification polymorphism. We conclude that CR1–PCR provides an efficient and reliable mechanism for genome mapping in avians that can correlate linkage and physical mapping approaches.     

DOGSET: pre-designed primer sets for fine-scale mapping and DNA sequence interrogation in the dog

DOGSET is an online resource that provides access to primer sequences that have been computationally mined from the reference genome using heuristic algorithms. The electronic repository includes PCR primers corresponding to 32 135 markers for genetic mapping and 334 657 sequence-tagged gene elements for targeted re-sequencing and mutation discovery. A customized report that tailors primer design to wet bench protocols can be exported for a region of interest by specifying genome coordinates in a graphical user interface.     

MRPrimer: a MapReduce-based method for the thorough design of valid and ranked primers for PCR

Primer design is a fundamental technique that is widely used for polymerase chain reaction (PCR). Although many methods have been proposed for primer design, they require a great deal of manual effort to generate feasible and valid primers, including homology tests on off-target sequences using BLAST-like tools. That approach is inconvenient for many target sequences of quantitative PCR (qPCR) due to considering the same stringent and allele-invariant constraints. To address this issue, we propose an entirely new method called MRPrimer that can design all feasible and valid primer pairs existing in a DNA database at once, while simultaneously checking a multitude of filtering constraints and validating primer specificity. Furthermore, MRPrimer suggests the best primer pair for each target sequence, based on a ranking method. Through qPCR analysis using 343 primer pairs and the corresponding sequencing and comparative analyses, we showed that the primer pairs designed by MRPrimer are very stable and effective for qPCR. In addition, MRPrimer is computationally efficient and scalable and therefore useful for quickly constructing an entire collection of feasible and valid primers for frequently updated databases like RefSeq. Furthermore, we suggest that MRPrimer can be utilized conveniently for experiments requiring primer design, especially real-time qPCR.     

Genome-specific primer sets for starch biosynthesis genes in wheat

Common wheat (Triticum aestivum L., 2n=6x=42) is an allohexaploid composed of three closely related genomes, designated A, B, and D. Genetic analysis in wheat is complicated, as most genes are present in triplicated sets located in the same chromosomal regions of homoeologous chromosomes. The goal of this report was to use genomic information gathered from wheat–rice sequence comparison to develop genome-specific primer sets for five genes involved in starch biosynthesis. Intron locations in wheat were inferred through the alignment of wheat cDNA sequences with rice genomic sequence. Exon-anchored primers, which amplify across introns, allowed the sequencing of introns from the three genomes for each gene. Sequence variation within introns among the three wheat genomes provided the basis for genome-specific primer design. For three genes, ADP-glucose pyrophosphorylase (Agp-L), sucrose transporter (SUT), and waxy (Wx), genome-specific primer sets were developed for all three genomes. Genome-specific primers were developed for two of the three genomes for Agp-S and starch synthase I (SsI). Thus, 13 of 15 possible genome-specific primer sets were developed using this strategy. Seven genome-specific primer combinations were used to amplify alleles in hexaploid wheat lines for sequence comparison. Three single nucleotide polymorphisms (SNPs) were identified in a comparison of 5,093 bp among a minimum of ten wheat accessions. Two of these SNPs could be converted into cleaved amplified polymorphism sequence (CAPS) markers. Our results indicated that the design of genome-specific primer sets using intron-based sequence differences has a high probability of success, while the identification of polymorphism among alleles within a genome may be a challenge.     

DNA聚合酶与引物/模板的相互作用对PCR效率的影响

PCR是体外酶促合成特异DNA片段的一种方法,引物的优劣直接关系到PCR的特异性与成功与否。传统的PCR引物设计软件基本上忽略了DNA聚合酶与引物/模板的亲和性对PCR效率的影响。为揭示DNA聚合酶与引物/模板的相互作用是否对PCR的效率有影响,通过构建Taq DNA 聚合酶与不同序列引物/模板DNA相互作用的三维结构模型,采用MM/GBSA方法计算复合物的结合自由能,以结合自由能为参数,为人血清白蛋白基因(Human Serum Albumin gene,HSA gene)和结核杆菌pyrF基因(Mycobacterium tuberculosis pyrF gene)设计了PCR引物。PCR实验结果表明,引物的PCR效率与结合自由能相关：引物与聚合酶的结合自由能越低,PCR实验的效率相对越高。这说明DNA聚合酶与引物/模板的相互作用对PCR效率有重要影响。因此,引物/模板DNA与聚合酶的结合自由能可以作为PCR引物设计的新参数。     

Identification and phylogenetic sorting of bacterial lineages with universally conserved genes and proteins

Molecular characterizations of bacteria often employ ribosomal DNA (rDNA) to establish the identity and relationships among organisms, but the use of rRNA sequences can be problematic as the result of alignment ambiguities caused by indels, the lack of informative characters, and varying functional constraints over the molecule. Although protein-coding regions have been used as an alternative to rRNA, there is neither consensus among the genes examined nor ways to rapidly obtain sequence information for such genes from uncharacterized bacterial species. To standardize the set of protein-coding loci assayed in bacterial genomes, we examined over 100 widely distributed genes to identify sets of universal primers for use in the PCR amplification of protein coding regions that are common to virtually all bacteria. From this set, we developed primer sets that each target of 10 genes spanning an array of genomic locations and functional categories. Although many of the primers contain sequence degeneracies that aid in targeting genes across diverse taxa, most are adequate for direct sequencing of amplification products, thereby eliminating intermediate cloning before sequence determination. We foresee the analysis of these protein-coding regions as being complementary to ribosomal DNA for answering questions pertaining to bacterial identification, classification, phylogenetics and evolution.     

Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library

Non-immune phage scFv library is one of the most attractive resources for therapeutics, diagnostics and basic research. As a matter of fact, quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers. PCR using this type of primers is difficult to optimize conditions for efficient amplification, and therefore causes loss of antibody diversities. To overcome this problem, we described a novel two-step amplification of V_κ and V_H genes with newly designed primer sets. Initially, we amplified V_κ and V_H genes from their signal sequences to the joining region to keep antibody diversity as large as possible. Thereafter, highly degenerated primers were used to amplify the V_κ and V_H genes from the framework region 1 to the joining region. The V_κ and V_H genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library. Fifteen clones from the library were randomly picked and sequenced, and the diversity of full-length scFvs was confirmed. Expression capability of clones in the library was 80% after confirmation using colony hybridization. The results demonstrated the efficiency of this strategy and the primer sets for construction of the scFv library.