Diagnosis of HNF-1alpha mutations on a PNA zip-code microarray by single base extension

In the present study, we exploited the superior features of peptide nucleic acids (PNAs) to develop an efficient PNA zip-code microarray for the detection of hepatocyte nuclear factor-1alpha (HNF-1alpha) mutations that cause type 3 maturity onset diabetes of the young (MODY). A multi-epoxy linker compound was synthesized and used to achieve an efficient covalent linking of amine-modified PNA to an aminated glass surface. PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in a subsequent multiplex single base extension reaction using chimeric primers with 3' complementarity to the specific mutation site and 5' complementarity to the respective PNA zip-code sequence on the microarray. The primers were extended by a single base at each corresponding mutation site in the presence of biotin-labeled ddNTPs, and the products were hybridized to the PNA microarray. Compared to the corresponding DNA, the PNA zip-code sequence showed a much higher duplex specificity for the complementary DNA sequence. The PNA zip-code microarray was finally stained with streptavidin-R-phycoerythrin to generate a fluorescent signal. Using this strategy, we were able to correctly diagnose several mutation sites in exon 2 of HNF-1alpha with a wild-type and mutant samples including a MODY3 patient. This work represents one of the few successful applications of PNA in DNA chip technology.     

Diagnosis of HNF-1α mutations on a PNA zip-code microarray by single base extension

In the present study, we exploited the superior features of peptide nucleic acids (PNAs) to develop an efficient PNA zip-code microarray for the detection of hepatocyte nuclear factor-1α (HNF-1α) mutations that cause type 3 maturity onset diabetes of the young (MODY). A multi-epoxy linker compound was synthesized and used to achieve an efficient covalent linking of amine-modified PNA to an aminated glass surface. PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in a subsequent multiplex single base extension reaction using chimeric primers with 3′ complementarity to the specific mutation site and 5′ complementarity to the respective PNA zip-code sequence on the microarray. The primers were extended by a single base at each corresponding mutation site in the presence of biotin-labeled ddNTPs, and the products were hybridized to the PNA microarray. Compared to the corresponding DNA, the PNA zip-code sequence showed a much higher duplex specificity for the complementary DNA sequence. The PNA zip-code microarray was finally stained with streptavidin-R-phycoerythrin to generate a fluorescent signal. Using this strategy, we were able to correctly diagnose several mutation sites in exon 2 of HNF-1α with a wild-type and mutant samples including a MODY3 patient. This work represents one of the few successful applications of PNA in DNA chip technology.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus?

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Amplification and methylation of an esterase gene associated with insecticide-resistance in greenbugs, Schizaphis graminum (Rondani) (Homoptera: Aphididae)

The greenbug aphid, Schizaphis graminum (Rondani) has developed resistance to organophosphorus insecticides by the over-production of esterases that have been classified as Type I and Type II. The first twenty N-terminal amino acids of the Type I esterase were determined and used to design an oligonucleotide, which in conjunction with an active site primer derived from conserved sequences of other insect esterases and two internal primers specific for esterases from another aphid species resulted in a 0.85 kb genomic DNA fragment from resistant greenbugs. This was extended by 5′ RACE which provided approximately 1.2 kb of the 5′ end of the esterase gene. The 5′ DNA sequence corresponded to 19 of the 20 known amino acids of the Type I esterase, with the last needing only a one base change (probably resulting from a PCR artifact). Furthermore, the sequence showed very close similarity to the amplified E4/FE4 esterase genes of Myzus persicae (Sulzer). A comparison of sequences suggested that the S. graminum gene has introns in the same positions as the first two introns of E4/FE4, with the second intron being considerably larger in S. graminum. Probing of Southern blots with the 0.85 kb esterase fragment showed that the gene encoding the Type I esterase is amplified 4- to 8-fold in resistant S. graminum and that the amplified sequences contain 5-methylcytosine at MspI/HpaII sites, again in agreement with previous findings for M. persicae genes.     

结合单酶切位点的融合PCR技术对SCN1A基因的定点诱变

目的:利用结合单酶切位点的融合PCR技术对癫痫相关基因SCN1A进行定点突变。方法:首先设计两对引物PF1/PR1和PF2/PR2,PF1和PR2均位于突变位点最近的单酶切位点处,而突变位点设计在第一对反向引物(PR1)和第二对正向引物上(PF2)。通过重叠延伸法两次PCR扩增:第一次用PF1/PR1和PF2/PR2分开扩增,以扩增产物作模板,PF1/PR2作引物进行融合PCR,得到的扩增产物即含有所需要的突变位点,最后将扩增片段克隆入pMD18-T载体,经测序筛选阳性克隆。结果:DNA测序表明SCN1A基因所编码的第946位密码子由精氨酸(Arg)突变为组氨酸(His),再通过酶切和连接反应将重组质粒上的突变片段替换SCN1A表达质粒上的对应片段,成功构建了SCN1A突变载体。结论:与现在常用的长距离PCR定点诱导突变相比较,结合单酶切位点的融合PCR定点突变技术具备扩增距离短的优点,大大降低了自发突变的概率,适合于大肠杆菌中易自发突变的较大载体的定点诱变。     

Three-step PCR mutagenesis for 'linker scanning'.

'Linker scanning' has been used as an efficient method for systematically surveying a segment of DNA for functional elements by mutagenesis. A three-step PCR method was developed to simplify this process. In this method, a set of 'mutation primers' was made with 6 to 8 base substitutions in the center of the primers. In the first PCR reaction, these 'mutation primers' are paired with an 3' primer from the opposite end of the analyzed sequences to form a 'ladder' of fragments containing the base pair substitutions. These are used as templates in the second PCR with the 3' primer as the only primer to generate single stranded sequences, which are used as primers in the third PCR paired with an 5' primer to complete the mutagenesis. We have tested the method in a mutation screen of the steroid sulfatase promoter. Its application to general site specific mutagenesis is discussed.     

Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus

AFLR is a Zn₂Cys₆-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5′-TCGN₅CGA-3′. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5′-TCGN₅CGA-3′ binding site within 200 bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5′-TCGCAGCCCGG-3′ at −110 bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7 bp of the 5′-TCGN₅CGA-3′ motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5′-TCGSWNNSCGR-3′.     

SNPs detection by a single-strand specific nuclease on a PNA zip-code microarray

In this report, a reliable peptide nucleic acid (PNA) microarray-based method for accurately detecting single nucleotide polymorphism (SNP) in human genes is described. The technique relies on the mismatched cleavage activity of a single-strand specific (SSS) nuclease. PCR amplification was performed to prepare gene fragments containing the mutation sites. The amplified fragments were then employed as templates for the SSS nuclease reaction using chimeric probes, modified with biotin at the 5' end and extended with a unique anchoring zip-code complement sequence at the 3' end. The SSS nuclease promotes cleavage of heteroduplex DNAs at base mismatched positions to produce crumbled chimeric probes in the presence of imperfectly matching template strands. In contrast, the probes remain intact when they interact with perfectly matched template strands. Only the non-fragmented probes generate fluorescence signals after treatment with streptavidin-Cy3 on the PNA zip-code array. This methodology was used to successfully genotype selected Korean-specific BRCA mutation sites with wild type and mutant samples. The investigation has led to the development of a reliable SSS nuclease-based system for the diagnosis of human genetic mutations or SNPs.     

Deconstructing the Polymerase Chain Reaction: Understanding and Correcting Bias Associated with Primer Degeneracies and Primer-Template Mismatches

The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3–12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled “Polymerase-exonuclease (PEX) PCR”, in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3’ end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.     

Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays

Detection of DNA sequence variation is critical to biomedical applications, including disease genetic identification, diagnosis and treatment, drug discovery and forensic analysis. Here, we describe an arrayed primer extension-based genotyping method (APEX-2) that allows multiplex (640-plex) DNA amplification and detection of single nucleotide polymorphisms (SNPs) and mutations on microarrays via four-color single-base primer extension. The founding principle of APEX-2 multiplex PCR requires two oligonucleotides per SNP/mutation to generate amplicons containing the position of interest. The same oligonucleotides are then subsequently used as immobilized single-base extension primers on a microarray. The method described here is ideal for SNP or mutation detection analysis, molecular diagnostics and forensic analysis. This robust genetic test has minimal requirements: two primers, two spots on the microarray and a low cost four-color detection system for the targeted site; and provides an advantageous alternative to high-density platforms and low-density detection systems.     

Structure of the murine lactotransferrin gene is similar to the structure of other transferrin-encoding genes and shares a putative regulatory region with the murine myeloperoxidase gene

The structure and nucleotide sequence of the murine lactotransferrin-encoding gene (LTF) deduced partly by direct sequencing of genomic clones in the λ phage vector and partly by enzymatic amplification of genomic DNA segments primed with the oligodeoxyribonucleic primers homologous to the cDNA sequence. The λ phage clones contained the 5′ half of the gene corresponding to the first eight exons and an incomplete ninth exon interrupted by eight introns. Genomic clones corresponding to the 3′ half of the LTF gene could not be obtained on repeated attempts from two different mouse genomic libraries, suggesting the possible presence of unclonable sequences in this part of the gene. Hence, PCR was used to clone the rest of the gene. Four out of the presumed eight remaining introns were cloned along with the flanking exons using PCR. Comparison of the structure of the LTF gene with those of the two other known transferrin-encoding genes, human serum transferrin-encoding gene and chicken ovotransferrin-encoding gene reveals that all three genes have a very similar intron-exon distribution pattern. The hypothesis that the present-day transferrin-encoding genes have originated from duplication of a common ancestral gene is confirmed here at the gene level. An interesting finding is the identification of a region of shared nucleotides between the 5′ flanking regions of the murine LTF and myeloperoxidase-encoding genes, the two genes expressed specifically in neutrophilic granulocytes.     

Molecular cloning and characterization of soybean peroxidase gene families

Plant peroxidases play major roles in many physiological processes. A soybean seedbud (21 days after flowering) Uni-ZAP XR cDNA library was screened with a peroxidase-specific probe. The probe was generated by 3′ rapid amplification of cDNA ends with soybean seedbud total RNA and a degenerate primer derived from a plant peroxidase conserved amino acid region (distal heme ligand). Positive clones were recovered by PCR using the degenerate peroxidase-specific primer and the vector primer T₇ flanking the cloning site. Four cDNAs, designated GmEpa1, GmEpa2, GmEpb1, and GmEpb2, contained 1298, 1326, 1171, and 1145 nucleotides, excluding poly(A) tail, and encoded mature proteins of 303, 303, 292, and 292 amino acids, respectively. The four predicted amino acid sequences showed homology to other peroxidases. GmEpa1 and GmEpa2 exhibited 97% amino acid identity, GmEpb1 and GmEpb2 exhibited 93% amino acid identity, and GmEpa1 and GmEpb1 exhibited 47% amino acid identity. GmEPa1 and GmEPb1 were expressed as fusion proteins in Escherichia coli. The recombinant fusion proteins were sequestered in inclusion bodies and active forms of the two denatured proteins were recovered after in vitro folding in a medium containing hemin, urea and Ca²⁺. GmEpa1 and GmEpa2 messages were detected in developing seed and root, while GmEpb1 and GmEpb2 messages were present in root, leaf, stem and seed pod. These cDNAs and cDNA-specific primers will allow investigations into peroxidase’s role in development, stress response and in other physiological processes.     

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis

In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3′ end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.     

Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones.

The 264 bp mini-transposon Tn5supF was constructed to sequence DNAs cloned in phage lambda without extensive shotgun subcloning or primer walking. Unique sequences near each transposon end serve as primer binding sites, and a supF gene is used to select transposition to lambda. We describe here PCR methods that facilitate Tn5supF-based sequencing. In a first pass, insertions are mapped relative to the ends of the cloned fragment using pairs of primers specific for vector DNA next to the cloning site and for a Tn5supF end. Most insertions not mapped in this step are near the center of the cloned fragment or in the vector arms, and are then mapped relative to the two innermost insertions by 'crossover' PCR. This involves amplification from primers on different DNA molecules, and generates hybrid DNA products whose lengths correspond to the distances between the two insertions. We routinely amplified more than 6 kb in direct PCR and 3 kb in crossover PCR; at the limit we amplified up to approximately 10 kb in direct PCR and approximately 6 kb in crossover PCR, but not reproducibly. Crossover PCR products were also obtained with insertions separated by only 200 bp, indicating that no rare sites are needed to switch templates. PCR products were purified by adsorption and then elution from glass slurry, and sequenced directly. Ladders of more than 400 bp were obtained from primer sites on each DNA strand; 2 kb was read from crossover PCR products, and showed that they were amplified with fidelity. In conclusion, direct and crossover PCR methods expedite transposon insertion mapping, and yield templates for accurate sequencing of both DNA strands.     

Structure, organization and expression of the eukaryotic translation initiation factor 5, eIF-5, gene in Zea mays

The maize genomic DNA sequence encoding the eukaryotic translation initiation factor 5 (eIF-5) has been isolated from genomic library of maize seedlings and the exon–intron structure determined (accession number AJ132240). The length of genomic DNA sequenced was about 7 kb and contained two exons with the translation start site in exon 2. The only intron is located in the non-coding 5′ region and it is 1298 bp long with the splice acceptor and donor sites conforming to the AG/GT rules. Repetitive sequence fragments are located in the 5′ and 3′ intergenic region. The accumulation of eIF-5 mRNA was studied by RNA blot and in situ hybridization. The observed distribution of mRNA may correlate with the function of the protein, as it appears to be highly abundant in tissues where the proportion of cells actively dividing is very high, such as meristematic regions.     

Primer design versus PCR bias in methylation independent PCR amplifications

Many protocols in methylation studies utilize one primer set to generate a PCR product from bisulfite modified template regardless of its methylation status (methylation independent amplification MIP). However, proportional amplification of methylated and unmethylated alleles is hard to achieve due to PCR bias favoring amplification of unmethylated relatively GC poor sequence. Two primer design systems have been proposed to overcome PCR bias in methylation independent amplifications. The first advises against including any CpG dinucleoteides into the primer sequence (CpG-free primers) and the second, recently published by us, is based on inclusion of a limited number of CpG sites into the primer sequence. Here we used the Methylation Sensitive High Resolution Melting (MS-HRM) technology to investigate the ability of primers designed according to both of the above mentioned primer design systems to proportionally amplify methylated and unmethylated templates. Ten “CpG-free” primer pairs and twenty primers containing limited number of CpGs were tested. In reconstruction experiments the “CpG-free” primers showed primer specific sensitivity and allowed us to detect methylation levels in the range from 5 to 50%. Whereas while using primers containing limited number of CpG sites we were able to consistently detect 1–0.1% methylation levels and effectively control PCR amplification bias. In conclusion, the primers with limited number of CpG sites are able to effectively reverse PCR bias and therefore detect methylated templates with significantly higher sensitivity than CpG free primers.