A new diagnostic system for ultra-sensitive and specific detection and quantification of Candidatus Liberibacter asiaticus, the bacterium associated with citrus Huanglongbing

An ultra-sensitive and quantitative diagnostic system by combining nested PCR and TaqMan® PCR in a single tube was developed for detection of “Candidatus Liberibacter asiaticus”. The procedure involves two PCR steps using the species-specific outer and inner primer pairs. Different annealing temperatures allow both the first and the second rounds of PCR to be performed sequentially in the same closed tube. The first PCR with outer primers was performed at a higher annealing temperature and with limited amount of primers to prevent interference with the inner primers during the second round of PCR. The specificity of the dual primer TaqMan® is high because the fluorescent signal can only be generated from the TaqMan® probes that are homologous to the product amplified by the outer and inner primers. This new detection system can reliably detect as few as single copies of target DNA. The sensitivity of the dual primer system is comparable to the conventional two-tube nested PCR, but it eliminates the potential risk of cross contamination commonly associated with conventional nested PCR. This one-tube dual primer TaqMan® PCR method is gel-free with reduced handling time and is cost effective. At the same time, this system provides significantly increased sensitivity, improved reliability and high through-put capability suitable for routine, large scale diagnoses of clinical plant tissue and insect samples. The technique described here is generic and can be applied to the detection of other plant pathogenic bacteria.     

New Strategy for Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.     

Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs

Background

Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA.

Results

We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge.

Conclusion

We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.     

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.     

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.     

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。     

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5 end of the cDNA was amplified by a 5-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.     

Partially Overlapping Primer-Based PCR for Genome Walking

Current genome walking methods are cumbersome to perform and can result in non-specific products. Here, we demonstrate the use of partially overlapping primer-based PCR (POP-PCR), a direct genome walking technique for the isolation of unknown flanking regions. This method exploits the partially overlapping characteristic at the 3’ ends of a set of POP primers (walking primers), which guarantees that the POP primer only anneals to the POP site of the preceding PCR product at relatively low temperatures. POP primer adaptation priming at the genomic DNA/POP site occurs only once due to one low-/reduced-stringency cycle in each nested PCR, resulting in the synthesis of a pool of single-stranded DNA molecules. Of this pool, the target single-stranded DNA is replicated to the double-stranded form bound by the specific primer and the POP primer in the subsequent high-stringency cycle due to the presence of the specific primer-binding site. The non-target single stranded DNA does not become double stranded due to the absence of a binding site for any of the primers. Therefore, the POP-PCR enriches target DNA while suppressing non-target products. We successfully used POP-PCR to retrieve flanking regions bordering the gadA locus in Lactobacillus brevis NCL912, malQ in Pichia pastoris GS115, the human aldolase A gene, and hyg in rice.     

CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design

We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).     

Efficient procedure for site-directed mutagenesis mediated by PCR insertion of a novel restriction site

Better understanding of proteins'' structure/function relationship and dissecting their functional domains are still challenges yet to be mastered. Site-directed mutagenesis approaches that can alter bases at precise positions on the gene sequence can help to reach this goal. This article describes an efficient strategy that can be applied not only for both deletion and substitution of target amino acids, but also for insertion of point mutations in promoter regions to study cis-regulating elements. This method takes advantage of the plasticity of the genetic code and the use of compatible restriction sites.Key words: site-directed mutagenesis, restriction site, cloning, PCRUnderstanding the proteins structure/function relationship and dissecting their functional domains is one of the biggest challenges to current proteomic studies.¹ This is mainly achieved by site-directed mutagenesis experiments that can alter bases at precise positions on the gene sequence.² Modifying DNA sequences has become feasible with PCR amplification.³ During the last decade, several strategies have been developed to simplify this approach and increase its efficiency.⁴ The introduction of a site-directed mutation can be realized by one or more PCR reactions. Most of the strategies used in site-directed mutagenesis are based on a substitution of a single base, which leads to a change in one amino acid. This article describes an efficient strategy that can be applied for either deletion or substitution of target amino acids. This strategy is based on performing PCR reactions to create a new restriction site in the sequence of origin, corresponding to the desired mutation. The choice of the restriction site to be created depends on the nature of the amino acid that one desires to introduce in the protein sequence. Since such restriction sites may extend beyond the mutated codon. The preservation of the other codon is done by taking advantage of the plasticity of the genetic code where one amino acid can be encoded by multiple codons.This method was performed in two steps (Fig. 1). In the first step, the DNA sequence of interest, cloned in a plasmid, served as a template for two PCR reactions. Two PCR products are generated. The first one consists of the beginning of the sequence, from the start codon to the mutagenized amino acid codon, where the forward primer bears the start codon region and the reverse primer bears the newly introduced restriction site at the same location of the mutagenized codon. The second PCR product consists of the end of the coding sequence, from the mutagenized amino acid codon to the stop codon. This fragment is generated using a forward primer bearing the same new restriction site as the first PCR product''s reverse primer, and a reverse primer bearing the stop codon region. The two PCR products were cloned separately into a vector in the appropriate orientation. In the second step, the cloning vector bearing the first PCR product was digested with a restriction enzyme site in the vector, and by the restriction enzyme corresponding to the restriction site created by the reverse primer used in the PCR reaction. The resulting fragment was cloned into the vector containing the second PCR fragment, predigested with same two restriction enzymes. The whole mutagenized coding sequence is reassembled by in-frame subcloning of the 3′ end of the coding sequence downstream the 5′ end. All the PCR products were generated using the high fidelity Pfu DNA Polymerase (Promega, Madison, WI USA). For any site-directed mutagenesis experiment, this two-step cloning procedure requires the use of appropriate PCR primers that harbor the desired mutation of the target amino acid. These primers are partially overlapping and contain a common or complementary restriction site enabling the in-frame assembly of the whole coding sequence.Open in a separate windowFigure 1Mutagenesis strategy by restriction enzyme site insertion. (A) In the first step, two PCR products were generated using the full length coding sequence as template. The mutation is carried by the two primers b and c, which are flanked by the same or compatible restriction enzyme''s site (white segment). Both PCR products are separately cloned in the cloning vector in the appropriate orientation. In the second step, the whole mutagenized coding sequence is reassembled by in-frame sub cloning of the 3′ end of the coding sequence downstream the 5′ end. (B) Substitution of threonine by arginine as a result of the insertion of a BglII restriction site. DNA sequencing is carried out to make sure that only the desired change is introduced in the coding sequence. (B-1) The sequence of the native cDNA. (B-2) the sequence of the mutagenized cDNA included BglII restriction site sequence.This approach has been used in a recent study to address the structure/function relationship of the STAS domain of the Arabidopsis thaliana Sultr1;2 sulfate transporter.⁵ A good example of this approach is the replacement of the threonine-serine couple at position 587–588 with an arginine-serine couple. The codon for threonine is: TGT, and that for arginine is: TCT. Serine can be encoded by both TCA and AGA codons. The chosen restriction site used for the reassembly of the whole coding sequence is that of the BglII enzyme: TCT AGA. The insertion of this restriction site enables the substitution of the Thr in position 587 with an Arg while preserving the serine residue in position 588. The BglII restriction site is introduced in the reverse primer and the forward primer used to generate the first and second PCR products respectively. The DNA sequence of the reassembled mutagenized cDNA was checked by sequencing. Than it was expressed, under pGAL1O promoter bearing by pYES2 vector, in yeast mutant deficient in sulfate transporter and the mutagenic protein was detected by imunodetection.Bioinformatic study reveals that this method can be applied to checked a large number of substitutions, insertions or deletions and that finding the right restriction site is not a limiting factor (data no shown).In conclusion, this article describes an efficient two-step procedure for site-directed mutagenesis using primers bearing a restriction site, which is absent from the sequence of origin. The primers flanked by sequences introducing the same or compatible restriction sites mediate the incorporation of the mutation at the selection site. The choice of the restriction site depends on the nature of the desired mutation: insertion, substitution or deletion of an amino acid in a particular position. This strategy can be also used to insert point mutations in promoter regions to study cis-regulating elements.     

全雌系苦瓜ACC合成酶基因cDNA克隆及序列分析

以全雌系苦瓜‘X-Hei-d-d’花蕾为材料,根据已报道ACC合成酶(1-aminocyclopropane-1-carboxylic acid synthase,ACS)保守氨基酸序列设计简并引物,采用RT-PCR技术及序列拼接,获得了全雌系苦瓜ACS基因cDNA序列,命名为Mc-ACS4(GenBank登录号:FJ459814)。该序列包含一个1 455 bp的完整开放阅读框,编码484个氨基酸,具有7个保守区;系统进化上与普通苦瓜ACS基因首先聚类,同源性达99%,二者仅有2个氨基酸差异,推测可能与全雌系苦瓜性别分化有关。     

Transposon mutagenesis: Cloning of chromosomal DNA from the site of Tn916 insertion using polymerase chain reaction

A protocol that allows fast recovery and further analyses of chromosomal DNA adjacent to the Tn916 site of insertion is described. The procedure is based on single specific primer PCR amplification using restricted chromosomal DNA ligated into a suitable vector. Two primers, one Tn916-specific and the second vector-specific, allow amplification of the chromosomal DNA flanking the site of insertion.     

Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.     

Class-specific restrictions define primase interactions with DNA template and replicative helicase

Bacterial primase is stimulated by replicative helicase to produce RNA primers that are essential for DNA replication. To identify mechanisms regulating primase activity, we characterized primase initiation specificity and interactions with the replicative helicase for gram-positive Firmicutes (Staphylococcus, Bacillus and Geobacillus) and gram-negative Proteobacteria (Escherichia, Yersinia and Pseudomonas). Contributions of the primase zinc-binding domain, RNA polymerase domain and helicase-binding domain on de novo primer synthesis were determined using mutated, truncated, chimeric and wild-type primases. Key residues in the β4 strand of the primase zinc-binding domain defined class-associated trinucleotide recognition and substitution of these amino acids transferred specificity across classes. A change in template recognition provided functional evidence for interaction in trans between the zinc-binding domain and RNA polymerase domain of two separate primases. Helicase binding to the primase C-terminal helicase-binding domain modulated RNA primer length in a species-specific manner and productive interactions paralleled genetic relatedness. Results demonstrated that primase template specificity is conserved within a bacterial class, whereas the primase–helicase interaction has co-evolved within each species.     

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.     

Generation of polymerase chain reaction-specific probes for library screening using single degenerate primers

Degenerate oligonucleotide primers were made to peptide sequences from hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea. The primers were used singly in PCR reactions to amplify portions of the gene for HAO from genomic DNA. Southern hybridizations using fragments amplified with each primer showed that they labeled the same genomic DNA fragments. The PCR-amplified fragments were successfully used to screen a gene library for clones containing the HAO gene. The method of isolating genes by PCR with single primers has general utility.     

Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Priming Glycosyltransferase Genes of the Exopolysaccharide Locus in Strains of the Lactobacillus casei Group

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3′ degenerate core based on four highly conserved amino acids and a longer 5′ consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS⁻) and EPS-producing (EPS⁺) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS⁺ bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS⁺ strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.     

Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27

The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4 pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases.     

Universal PCR primers for detection of phytopathogenic Agrobacterium strains.

Two PCR primer pairs, based on the virD2 and ipt genes, detected a wide variety of pathogenic Agrobacterium strains. The endonuclease domain of VirD2 protein, which cleaves transferred DNA (T-DNA) border sequences, is highly conserved; primer oligonucleotides specific for the endonuclease portion of virD2 detected all pathogenic strains of Agrobacterium tested. PCR primers corresponding to conserved sequences in ipt, the T-DNA-borne cytokinin synthesis gene, detected only Agrobacterium tumefaciens and distinguished it from Agrobacterium rhizogenes. The virD2 and ipt primer pairs did not interfere with each other when included in the same PCR amplification, and this permitted simultaneous detection of both genes in a single reaction. One nonpathogenic Agrobacterium radiobacter strain contained virD2 but not ipt; we speculate that this strain arose from a pathogenic progenitor through a deletion in the T-DNA. The virD2 primer pair appears to be universal for all pathogenic Agrobacterium species; used together, the primer sets reported here should allow unambiguous identification of Ti plasmid DNA in bacteria isolated from soil and plants.