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We demonstrate a novel method for identification of the insertion site of transgenic DNA by construction a circular DNA containing part of the transgenic DNA (tDNA) and its flanking sequence, which would be different from those in the native genomic DNA (gDNA). Using follistatin (FST) as a model of tDNA, we cleaved the sequence of FST gene and the adjacent sequence with a proper combination of restriction endonuclease and nicking endonuclease and isolated the resultant fragments containing the target FST gene from the gDNA background. Two types of previously unknown ligation property of T7 DNA ligase were disclosed in our experiments. One is non-templated single-stranded overhang ligation capability; the other one is the successful ligation of 3’-overhang and 5’-recessed ends in the presence of two mismatched base pairs. By virtue of these efficient ligation reactions, we successfully construct the circular DNA with high cyclization yield. Further coupling with nested inverse PCR, the method enabled identification of transgenic follistatin a (fsta) cDNA in zebrafish embryonic DNA. The established method provides a practical tool to determine the insertion site of tDNA with a sensitivity of 103 copies in 1.0 μg of gDNA and high specificity. It may be further extended for gene doping detection and assessment of the safety of gene therapy and genetically modified organisms.

Hybrid rice breeding offers an important strategy to improve rice production, and therefore is crucial for ensuring worldwide food security. Purity of hybrids is a key factor in keeping the high production of hybrid rice. While another important thing is mechanical weeding of hybrid rice, which could greatly release labor from laborious work and cut the production cost. Both of these two problems could be partly resolved by developing glyphosate-tolerant restorer lines. In this study, a novel glyphosate-tolerant gene I.variabilis-EPSPS* was transferred into elite indica restorer line Minghui 86 (MH86) by Agrobacterium-mediated transformation. By Southern blot and inverse PCR analysis, 3 transgenic restorer lines (MY28, MY50, and MY51) with a single copy of I.variabilis-EPSPS* integrated into the intergenic regions were selected from 72 regenerated transgenic plants. The inheritance of I.variabilis-EPSPS* in the 3 transgenic restorer lines was proved to follow Mendelian inheritance by germinating test. In the following generations, according to Southern blot, Northern blot, 3’RACE, and Western blot, I.variabilis-EPSPS* were proved to stably inherit and express in the candidate transgenic restorer lines. At the same time, glyphosate tolerance assay was performed in the field, in which the candidate transgenic restorer lines kept normal agronomic traits under glyphosate treatments with a dosage as high as 8400 g/ha. Therefore, the candidate transgenic restorer lines were highly tolerant to glyphosate. The 3 candidate transgenic restorer lines were respectively hybridized with the male sterile line II-32A, and their hybrids were respectively named as MY28H, MY50H, and MY51H. According to Southern blot and Western blot analysis, I.variabilis-EPSPS* could stably inherit to F1 hybrids and express in these hybrids, which indicated that the true hybrids could be distinguished by I.variabilis-EPSPS*. In the field test, the transgenic hybrids with I.variabilis-EPSPS* could suffer 3360 g/ha glyphosate without the damage of agronomic traits, which indicated the application potential of these hybrids in direct seeding. Collectively, the transgenic glyphosate-tolerant restore lines developed in this study were highly tolerant to glyphosate, and they had potential to be used in commercial production of hybrid rice.

Intragenic deletion of IKZF1 is a recurrent genomic alteration in acute lymphoblastic leukemia. The deletions are mediated by illegitimate variable(diversity)joining recombination via cryptic recombination signal sequences (RSSs). We developed a fluorescence in situ hybridization (FISH) probe set that can detect any type of IKZF1 deletion, including the commonly deleted exon 4 to 7 region. The probe set consists of a designed probe for the commonly deleted region (Cy3; red) and a bacterial artificial chromosomes clone probe for detecting the 3' flanking region (Spectrum Green). Intact IKZF1 showed a fusion signal, and the deleted allele showed loss of the red signal (0R1G1F). The FISH probes worked correctly for human leukemic cell lines and clinical samples. One case showed an atypical break-apart signal (1R1G1F). Inverse PCR of the case revealed rearrangement of the excised IKZF1 fragment into a legitimate RSS site at Ig κ on chromosome 2, suggesting a pathogenic role of this recombination-activating gene 1/2-mediated event. In this study, we established FISH probe detecting IKZF1 deletion in a quick, quantitative, and cost-effective manner, and the results provided a novel insight into B-cell receptor editing by rearrangement of a cryptic RSS-mediated genomic fragment in acute lymphoblastic leukemia pathology.

An 82-year-old woman presented with generalized lymphadenopathy and skin involvement. Lymph node biopsy revealed diffuse large B-cell lymphoma with a high proliferation index. G-banding and fluorescence in situ hybridization showed a hypertetraploid karyotype with two copies of t(8;22)(q24;q11), generating the fusion of MYC and the immunoglobulin λ chain gene ( IGL ), and two copies of the novel immunoglobulin heavy chain gene ( IGH ) translocation, t(14;15)(q32;q24). A long-distance inverse polymerase chain reaction (PCR) using nested primer combinations designed for each constant gene of IGH showed that Cγ4 was juxtaposed to the downstream sequence of the BCL2A1 (BCL2-related protein A1) gene through the Sγ4 switch region. As a result of t(14;15)(q32;q24), BCL2A1 and IGH Sγ4-Cγ4 were aligned in the same transcriptional orientation at a distance of 64 kb. Reverse transcriptase-mediated PCR showed high BCL2A1 mRNA levels in a lymphoma specimen. Since BCL2A1 , mapped at 15q24.3 or 15q25.1, encodes a protein that is an anti-apoptotic member of the BCL2 protein family, we herein described the novel double-hit, t(8;22)(q24;q11)/ MYC - IGL and t(14;15)(q32;q24)/ IGH - BCL2A1 , in which BCL2A1 is considered to play a role equivalent to that of BCL2 in the most frequent double-hit, MYC / BCL2 .

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

The western corn rootworm (WCR), a major pest of maize, is notorious for rapidly adapting biochemically, behaviourally and developmentally to a variety of control methods. Despite much effort, the genetic basis of WCR adaptation remains a mystery. Since transformation-based applications such as transposon tagging and enhancer trapping have facilitated genetic dissection of model species such as Drosophila melanogaster, we developed a germline-transformation system for WCR in an effort to gain a greater understanding of the basic biology of this economically important insect. Here we report the use of a fluorescent-marked Minos element to create transgenic WCR. We demonstrate that the transgenic strains express both an eye-specific fluorescent marker and piggyBac transposase. We identified insertion-site junction sequences via inverse PCR and assessed insertion copy number using digital droplet PCR (ddPCR). Interestingly, most WCR identified as transgenic via visual screening for DsRed fluorescence proved to carry multiple Minos insertions when tested via ddPCR. A total of eight unique insertion strains were created by outcrossing the initial transgenic strains to nontransgenic WCR mates. Establishing transgenic technologies for this beetle is the first step towards bringing a wide range of transformation-based tools to bear on understanding WCR biology.

Here, we report the genome of a putative densovirus infecting the carrot psyllid Bactericera trigonica, obtained by inverse PCR and named Bactericera trigonica densovirus (BtDNV). The ambisense genome of BtDNV is identical in structure to those of the ambidensoviruses, and its encoded proteins share the highest sequence identity with the Asian citrus psyllid Diaphorina citri densovirus.

Plasmid DNA of Lactobacillus plantarum PC518 was isolated by an improved method which contained a washing step for removing lysozyme. Three plasmid DNA libraries were constructed. A pair of outward primers was designed at both ends of the novel plasmid fragment obtained from plasmid DNA libraries, and the remainder of the circle plasmid was amplified by inverse PCR (iPCR). The whole sequence of plasmid was analyzed by the basic local alignment search tool, Tandem Repeats Finder, DNAMAN V6.0, DNASTAR and MEGA X software. The copy number was measured using quantitative real-time PCR. Plasmid extract showed 7 bands on agarose gel, indicating that L. plantarum PC518 contains multiple plasmids. The complete sequence of plasmid pLP60 was obtained by plasmid DNA libraries and iPCR. pLP60 is 6006 bp in length with a G + C content of 41.19 %, which encodes 8 open reading frames (ORFs). The ori site like theta-type could be located upstream of repB, which contains a short tandem repeats (sTR) and a long tandem repeats (lTR). RepB of pLP60 only had low similarity with Rep protein of known theta-type plasmids, but phylogenetic tree analysis showed that plasmids whose Rep proteins are similar to pLP60 have lTR at ori, and the conservativeness of lTR is consistent with similarity of Rep proteins, suggesting that RepB of pLP60 is a theta-replicating protein. So pLP60 was classified as class A of theta replication. The copy number of pLP60 was measured as 5 copies per cell by qPCR.

Streptomyces is a widely studied bacterial genus, particularly with regard to secondary metabolites and antibiotics production. Streptomyces griseus S4–7 was isolated from a strawberry Fusarium wilt disease suppressive soil, and its biological control ability has been well established. However, the antifungal mechanism of strain S4–7 is not yet fully understood at the molecular and biochemical level. Therefore, in this study we created a random mutant library for strain S4–7 with the Tn5 transposon element to investigate antifungal traits on a genome-wide scale. In total 4646 individual mutant strains were created and 13 mutants were selected based on loss of antifungal activity. The knockout genes were identified as electron transfer oxidoreductase (eto),sigma factor-70(sig70) and nrps by Inverse PCR (I-PCR). eto regulates the geranylgeranyl reductase gene, which is involved in terpenoid-quinone biosynthesis, an important factor in cell fitness. In the △eto strain, expression of wbl, a master regulator of the production of secondary metabolites, was significantly reduced. sig70 is responsible for the cell differentiation sensing mechanism in genus Streptomyces. △nrps showed decreased production of hybrid peptide-polyketide siderophores. These results suggest that S. griseus S4–7 may have various antifungal mechanisms, and each mechanism is essential to maximal antifungal activity.

DNA analysis of biological specimens containing degraded nucleic acids such as mortal remains, archaeological artefacts, forensic samples etc. has gained more attention in recent years. DNA extracted from these samples is often inapplicable for conventional polymerase chain reaction (PCR), so for its amplification the nearby primers are commonly used. Here we report the data that clarify the features of PCR with nearby and abutting primers. We have shown that the proximity of primers leads to significant reduction of the reaction time and ensures the successful performance of DNA amplification even in the presence of PCR inhibitors. The PCR with abutting primers is usually characterized by the absence of nonspecific amplification products that causes extreme sensitivity with limit of detection on single copy level. The feasibility of PCR with abutting primers was demonstrated on species identification of 100 years old rotten wood.

Polymerase chain reaction (PCR) technique is widely used in many experimental conditions, and Taq DNA polymerase is critical in PCR process. In this article, the Taq DNA polymerase expression plasmid is reconstructed and the protein product is obtained by rapid purification, (“Rapid purification of high-activity Taq DNA polymerase” (Pluthero, 1993 [1]), “Single-step purification of a thermostable DNA polymerase expressed in Escherichia coli” (Desai and Pfaffle, 1995 [2])). Here we present the production data from protein expression and provide the analysis results of the production from two different vectors. Meanwhile, the purification data is also provided to show the purity of the protein product.

DNA methylation has been proposed as one of the promising biomarkers for cancer diagnosis. In this study, we developed a DNA methylation detection system utilizing G-quadruplex and i-motif-forming sequences that requires neither sodium bisulfite treatment nor methylated DNA ligands. We hypothesized that G-quadruplex and i-motif structures would be stabilized by DNA methylation and arrest DNA polymerase activity during quantitative polymerase chain reaction (qPCR). The PCR products from VEGF, RET G-quadruplex, and i-motif-forming sequences were used as templates and analyzed by qPCR. Our results indicated that the initial elongation efficiency of PCR decreased with increasing DNA methylation levels in the G-quadruplex and i-motif-forming sequences. Moreover, we demonstrated that the initial elongation efficiency of PCR decreased with increased DNA methylation of the VEGF region on genomic DNA. These results indicated that DNA methylation of the G-quadruplex and i-motif-forming sequences on genomic DNA can be detected by qPCR.

A polymerase chain reaction (PCR) using a thermostable DNA polymerase is the most widely applied method in many areas of research, including life sciences, biotechnology, and medical sciences. However, a conventional PCR incurs an amplification of undesired genes mainly owing to non-specifically annealed primers and the formation of a primer-dimer complex. Herein, we present the development of a Taq DNA polymerase-specific repebody, which is a small-sized protein binder composed of leucine rich repeat (LRR) modules, as a thermolabile inhibitor for a precise and accurate gene amplification by PCR. We selected a repebody that specifically binds to the DNA polymerase through a phage display, and increased its affinity to up to 10 nM through a modular evolution approach. The repebody was shown to effectively inhibit DNA polymerase activity at low temperature and undergo thermal denaturation at high temperature, leading to a rapid and full recovery of the polymerase activity, during the initial denaturation step of the PCR. The performance and utility of the repebody was demonstrated through an accurate and efficient amplification of a target gene without nonspecific gene products in both conventional and real-time PCRs. The repebody is expected to be effectively utilized as a thermolabile inhibitor in a PCR. Biotechnol. Bioeng. 2016;113: 2544-2552. © 2016 Wiley Periodicals, Inc.

Antheraea mylitta arylphorin protein was extracted from the silk gland of fifth instar larvae and purified by ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. The N-terminal sequencing of ten amino acids (NH2-SVVHPPHHEV-COOH) showed similarity with Antheraea pernyi arylphorin. Based on N-terminal and C-terminal A. pernyi arylphorin sequences, primers were designed, and A. mylitta arylphorin cDNA was cloned by RT-PCR from silk gland mRNA. Sequencing of complete cDNA including 25 nucleotides at 5′ UTR (obtained by 5′ RACE) showed that it consisted of an ORF of 2115 nucleotides which could encode a protein of 704 amino acids (predominantly aromatic residues) having molecular weight 83 kDa. Homology modelling was done using A. pernyi arylphorin as a template. Cloned arylphorin cDNA was expressed in E. coli and recombinant His-tagged protein was purified by Ni-NTA affinity chromatography. Analysis of tissue-specific expression of arylphorin by real-time PCR showed maximum expression in the fat body followed by silk gland and integument. 5’ flanking region (759 bp) of arylphorin gene was amplified by inverse PCR and the full length gene (5359 nucleotides) containing five exons and four introns was cloned from the A. mylitta genomic DNA and sequenced. Polyclonal antibody was raised against purified arylphorin and more native arylphorin protein (500 kDa) was purified from the fat body by antibody affinity chromatography. Study of mitogenic effect of native and chymotrypsin hydrolysate of arylphorin on different insect cell lines showed that arylphorin could be used as serum substitute for in vitro cultivation of insect cells.

The standard polymerase chain reaction (PCR) is used to amplify a segment of DNA that lies between two inward-pointing primers. In contrast, inverse PCR (also known as inverted or inside-out PCR) is used to amplify DNA sequences that flank one end of a known DNA sequence and for which no primers are available. Inverse PCR DNA involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region. The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in PCR. The unknown sequence is amplified by two primers that bind specifically to the known sequence and point in opposite directions. The product of the amplification reaction is a linear DNA fragment containing a single site for the restriction enzyme originally used to digest the DNA. This site marks the junction between the previously cloned sequence and the flanking sequences. The size of the amplified fragment depends on the distribution of restriction sites within known and flanking DNA sequences.

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3×10(-5)) was roughly similar to that of Pfu DNA polymerase (4.8×10(-5)), but much lower than those of wild-type Neq DNA polymerase (57.2×10(-5)), Neq A523R DNA polymerase (13.1×10(-5)), and Neq N540R DNA polymerase (37.7×10(-5)). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.

The large fragment of DNA polymerase I fromGeobacillus stearothermophilusGIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified inE. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310by alanine or leucine and residue Asp540by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas.

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (≈375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ≈550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ≈2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

While the QuikChange site-directed mutagenesis method and its later modifications are extremely useful and simple, they suffer from several drawbacks. Here, we propose a new method, named LFEAP mutagenesis (Ligation of Fragment Ends After PCR) for creating various mutations in plasmid by leveraging three existing concepts: inverse PCR, single primer PCR, and sticky-end assembly. The first inverse PCR on the target plasmid yielded linearized DNA fragments with mutagenic ends, and a second single primer PCR resulted in complementary single-stranded DNA fragments with the addition of overhangs at the 5′ end of each strand. The resulting single strands were then annealed to produce double-stranded DNA with free 5′ single-stranded DNA tails. These products with compatible sticky ends were efficiently assembled into a circular, mutagenized plasmid. With this strategy, multiple simultaneous changes (up to 15) and mutations in large plasmids (up to 50 kb) were achieved with high efficiency and fidelity. LFEAP mutagenesis is a versatile method that offers significant advantages for introducing large and multiple changes in plasmid DNA.

DNA analysis of nimplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is characterized by low suitability for research due to the violation of its integrity and chemical structure; thus, the detection of specific nucleic acid fragments can be achieved by PCR with contiguous primers. In this paper, we present the results that clarify the specific characteristics of PCR with abutting primers. The 3'-ends of these primers are annealed at adjacent nucleotides of complementary chains of DNA target. It has been shown that the proximity of primers enables the formation of specific reaction products with a higher sensitivity and less reaction time. Using artificially damaged DNA and DNA from the soil we demonstrated that the abutting primers provide assured detection of specific DNA fragments. The results of this work may be taken into account in PCR with degraded (fragmented) DNA.

The construction of mirror-image biological systems may open the next frontier for biomedical technology development and discovery. Here we have designed and chemically synthesized a mutant version of the thermostable Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) consisting of d-amino acids. With a total peptide length of 358 amino acid residues, it is the largest chemically synthesized d-amino acid protein reported to date. We show that the d-polymerase is able to amplify a 120-bp l-DNA sequence coding for the Escherichia coli 5S ribosomal RNA gene rrfB by mirror-image polymerase chain reaction, and that both the natural and mirror-image systems operate with strict chiral specificity. The development of efficient miPCR systems may lead to many practical applications, such as mirror-image systematic evolution of ligands by exponential enrichment for the selection of therapeutically promising nuclease-resistant l-nucleic acid aptamers.

Many hybridoma researchers have lost an important hybridoma because of instability, mechanical breakdown of a freezer, or contamination. In many cases, however, it is possible to retrieve the heavy- and light-chain sequences from those hybridomas. Although most cloning methods associated with isolation of VH or VL gene sequences require the isolation of intact, nondegraded mRNA, alternative approaches based on genomic DNA have been reported. With genomic DNA, the DNA sample can remain relatively intact in cases in which the mRNA has been degraded. If intact DNA can be obtained from a nonviable hybridoma, the method described here can be used to recover the heavy- and light-chain sequences. The method uses PCR based on a combination of primers in the 5′ untranslated region of VH and VL and in the JH and JL segments. Although a relatively large number of primers are required for this method, the ability to obtain a full-length V gene without having intact mRNA could be of general interest for rescuing lost hybridomas.

Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.

The MeGolS5 promoter fragment (1492 bp) was amplified from the genomic DNA of Manihot esculenta Crantz by inverse polymerase chain reaction. Promoter sequence analysis by PLACE and PlantCARE showed that the cloned fragment contained several putative cis-elements, such as abscisic acid response element (ABRE), heat shock elements (HSE), as well as TATA-Box and CAAT-Box. The expression prfile of MeGolS5 shows that the gene is induced by several abiotic stresses, such as salt, drought, H2O2 and ABA.

Site-directed mutagenesis is one of the most widely used techniques in life sciences. Here we describe an improved and simplified method for introducing mutations at desired sites. It consists of an inverse PCR using a plasmid template and two partially complementary primers. The synthesis step is followed by annealing of the PCR product's sticky ends, which are generated by exonuclease digestion. This method is fast, extremely efficient and cost-effective. It can be used to introduce large insertions and deletions, but also for multiple point mutations in a single step. To show the principle and to prove the efficiency of the method, we present a series of basic mutations (insertions, deletions, point mutations) on pUC19 plasmid DNA.

The polymerase chain reaction (PCR) is an important technology to amplify a single copy or a few copies of DNA segment in genomic DNAs, visualizing the segment as DNA fragment. Thus, PCR is frequently used in various examinations such as detection of bacteria and fungi in the food industry. Here, we report a simple and sensitive method for detection of PCR products using single-strand tag sequence and hybridization of the tag sequence to the complementary tag sequence immobilized on solid material (STH). The detection sensitivity was found to be at least 50 times higher than electrophoresis/ethidium bromide (EtBr) visualization for approximately a 500-bp fragment and higher than the ordinary hybridization, that is, hybridization of denatured PCR product to probe sequence immobilized on solid material.

Aim: The present study was aimed at polymerase chain reaction (PCR) amplification and cloning of NAD-dependent betahydroxybutyryl coenzyme A dehydrogenase (BHBD) gene of Clostridium chauvoei. Materials and Methods: C. chauvoei was cultured and confirmed by 16-23S rDNA spacer region primers. The primers for nad-bhbd gene of C. chauvoei were designed to aid in cloning into pRham-N-His SUMO-Kan vector, and nad-bhbd gene was amplified by PCR. The amplified nad-bhbd gene was purified and cloned into pRham-N-His SUMO-Kan expression vector. The recombinant plasmid was transformed into E. cloni 10 G cells and the clone was confirmed by colony PCR using the pRham-SUMO-NAD-For and pRham-SUMO-NAD-Rev primers and also by sequencing. Results: PCR amplification of nad-bhbd gene yielded a product length of 844 base pairs which was cloned into pRham-NHis SUMO-Kan vector followed by transformation into E. cloni 10G chemically competent cells. The recombinant clones were characterized by colony PCR, sequencing, followed by basic local alignment search tool (BLAST) analysis to confirm the insert. Conclusions: Immunogenic protein NAD- dependent BHBD of C. chauvoei was cloned and the recombinant clones were confirmed by colony PCR and sequencing analysis.

A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.

The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′-mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation-specific PCR approach directly from untreated human genomic DNA.

Quantum dots (QDs) are of great interest due to their unique chemical and physical properties. Recently, a hot start (HS) polymerase chain reaction (PCR) amplification performance based on QDs with a high-fidelity Pfu DNA polymerase has been reported. However, whether QDs can trigger HS effects with other high-fidelity or conventional DNA polymerases is yet to be understood. In the present study, we studied the QD-triggered HS effects with four high-fidelity and three conventional DNA polymerases, and the HS effect comparisons among them were also made. It was found that QDs could trigger a distinct HS PCR amplification performance with all the four tested high-fidelity DNA polymerases, and specific target DNA could be well amplified even if the PCR mixture was pre-incubated for 2 h at 50°C. On the contrary, the HS effects were not prominent with all the three conventional Taq DNA polymerases. Specifically, the fidelity of Pfu is not sacrificed in the presence of QDs, even after a 1 h pre-incubation at 50°C before PCR. Furthermore, the electrophoresis results preliminarily demonstrated that QDs prefer to adsorb high-fidelity polymerases rather than conventional ones, which might result in the QD-triggered HS effects on PCR performance by using high-fidelity DNA polymerases.

Here we describe the identification, purification and characterisation of glycosylated yellow laccase proteins from the basidiomycete fungus Stropharia aeruginosa. Biochemical characterisation of two yellow laccases, Yel1p and Yel3p, show that they are both secreted, monomeric, N-glycosylated proteins of molecular weight around 55kDa with substrate specificities typical of laccases, but lacking the absorption band at 612nm typical of the blue laccase proteins. Low coverage, high throughput 454 transcriptome sequencing in combination with inverse-PCR was used to identify cDNA sequences. One of the cDNA sequences has been assigned to the Yel1p protein on the basis of identity between the translated protein sequence and the peptide data from the purified protein, and the full length gene sequence has been obtained. Biochemical properties, substrate specificities and protein sequence data have been used to discuss the unusual spectroscopic properties of S. aeruginosa proteins in the context of recent theories about the differences between yellow and blue laccases.

With the development of deep sequencing methodologies, it has become important to construct site saturation mutant (SSM) libraries in which every nucleotide/codon in a gene is individually randomized. We describe methodologies for the rapid, efficient, and economical construction of such libraries using inverse polymerase chain reaction (PCR). We show that if the degenerate codon is in the middle of the mutagenic primer, there is an inherent PCR bias due to the thermodynamic mismatch penalty, which decreases the proportion of unique mutants. Introducing a nucleotide bias in the primer can alleviate the problem. Alternatively, if the degenerate codon is placed at the 5' end, there is no PCR bias, which results in a higher proportion of unique mutants. This also facilitates detection of deletion mutants resulting from errors during primer synthesis. This method can be used to rapidly generate SSM libraries for any gene or nucleotide sequence, which can subsequently be screened and analyzed by deep sequencing. (C) 2013 Elsevier Inc. All rights reserved.

Eosinophilia-associated myeloid neoplasms with rearrangement of chromosome bands 5q31-33 are frequently associated with PDGFRB fusion genes, which are exquisitely sensitive to treatment with imatinib. In search for novel fusion partners of PDGFRB, we analyzed three cases with translocation t(5;20)(q33;p11), t(5;14)(q33;q32), and t(5;17;14)(q33;q11;q32) by 5′-rapid amplification of cDNA ends polymerase chain reaction (5′-RACE-PCR) and DNA-based long-distance inverse PCR (LDI-PCR) with primers derived from PDGFRB. LDI-PCR revealed a fusion between CCDC88C exon 25 and PDGFRB exon 11 in the case with t(5;17;14)(q33;q11;q32) while 5′-RACE-PCR identified fusions between CCDC88C exon 10 and PDGFRB exon 12 and between DTD1 exon 4 and PDGFRB exon 12 in the cases with t(5;14)(q33;q32) and t(5;20)(q33;p11), respectively. The PDGFRB tyrosine-kinase domain is predicted to be retained in all three fusion proteins. The partner proteins contained coiled-coil domains or other domains, which putatively lead to constitutive activation of the PDGFRB fusion protein. In vitro functional analyses confirmed transforming activity and imatinib-sensitivity of the fusion proteins. All three patients achieved rapid and durable complete hematologic remissions on imatinib.

Background/Aims: Optimal use of Cre mediated recombination in conditional animal models depends on well characterized Cre driver lines. Unfortunately, some Cre driver lines exhibit unexpected expression patterns hindering their utility in Cre/loxP systems. Thus, systematic assessment of new Cre lines is essential for generating useful Cre driver lines for future studies. Methods: Here, we describe a Cre Transgenic (Tg) mini-pig line in which the expression of Cre is directed by a 3-kb 5' fragment of the kidney-specific aquaporin 2 (AQP2); however, the AQP2-Cre Tg mini-pig line exhibits expression of Cre in alveolar epithelial cells (AECs) instead of collecting duct cells. The specificity of the AQP2-Cre plasmid was validated in vitro, and indicating that the AQP2-Cre was specifically expressed in the transfected LLC-PK1 cells. Absolute quantitative real-time PCR (qRT-PCR) and inverse PCR were performed to determine the copy numbers and integration sites of the AQP2-Cre transgene. Relative qRT-PCR was performed to evaluate variation in Cre expression levels over time. Results: Our data indicated that this AQP2-Cre Tg mini-pig line exhibits stable expression of Cre recombinase over time and in subsequent generations, even though the AQP2-Cre transgene was segregated and reduced in subsequent generations. Conclusion: Combined with our previous studies of the activity of this Cre, we conclude that this Cre Tg mini-pig line will provide a reliable tool for generating lung-specific gene targeting mini-pig models, thereby allowing the investigation of gene functions in lung development and studying the molecular mechanisms of human lung disease.

Strain-specific polymerase chain reaction (PCR) primers for detection of Bacillus mesentericus strain TO-A (BM TO-A) were developed. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. A 991-bp RAPD marker found to be strain-specific was sequenced, and two primer pairs specific to BM TO-A were constructed based on this sequence. In addition, we explored a more specific DNA region using inverse PCR, and designed a strain-specific primer set for use in real-time quantitative PCR (qPCR). These primer pairs were tested against 25 Bacillus subtilis strains and were found to be strain-specific. After examination of the detection limit and linearity of detection of BM TO-A in feces, the qPCR method and strain-specific primers were used to quantify BM TO-A in the feces of healthy volunteers who had ingested 3×10(8) colony forming unit (CFU) of BM TO-A per day in tablets. During the administration period, BM TO-A was detected in the feces of all 24 subjects, and the average number of BM TO-A detected using the culture method and qPCR was about 10(4.8) and 10(5.8) cells per gram of feces, respectively. Using the qPCR method, BM TO-A was detected in the feces of half of the subjects 3 d after withdrawal, and was detected in the feces of only one subject 1 week after withdrawal. These results suggest that the qPCR method using BM TO-A strain-specific primers is useful for the quantitative detection of this strain in feces.

Short interspersed elements (SINEs) are moderately repetitive DNA sequences in eukaryotic genomes. Although eukaryotic genomes contain numerous SINEs copy, it is very difficult and laborious to isolate and identify them by the reported methods. In this study, the inverse PCR was successfully applied to isolate SINEs from Opsariichthys bidens genome in Eastern Asian Cyprinid. A group of SINEs derived from tRNA(Ala) molecular had been identified, which were named Opsar according to Opsariichthys. SINEs characteristics were exhibited in Opsar, which contained a tRNA(Ala)-derived region at the 5' end, a tRNA-unrelated region, and AT-rich region at the 3' end. The tRNA-derived region of Opsar shared 76 % sequence similarity with tRNA(Ala) gene. This result indicated that Opsar could derive from the inactive or pseudogene of tRNA(Ala). The reliability of method was tested by obtaining C-SINE, Ct-SINE, and M-SINEs from Ctenopharyngodon idellus, Megalobrama amblycephala, and Cyprinus carpio genomes. This method is simpler than the previously reported, which successfully omitted many steps, such as preparation of probes, construction of genomic libraries, and hybridization.

We developed a novel electrochemical strategy for ultrasensitive DNA detection using a dual amplification strategy based on the circular strand-displacement polymerase reaction (CSDPR) and the hybridization chain reaction (HCR). In this assay, hybridization of hairpin-shaped capture DNA to target DNA resulted in a conformational change of the capture DNA with a concomitant exposure of its stem. The primer was then hybridized with the exposed stem and triggered a polymerization reaction, allowing a cyclic reaction comprising release of target DNA, hybridization of target with remaining capture DNA, polymerization initiated by the primer. Furthermore, the free part of the primer propagated a chain reaction of hybridization events between two DNA hairpin probes with biotin labels, enabling an electrochemical reading using the streptavidin-alkaline phosphatase. The proposed biosensor showed to have very high sensitivity and selectivity with a dynamic response range through 10 fM to 1 nM, and the detect limit was as low as 8 fM. The proposed strategy could have the potential for molecular diagnostics in complex biological systems.

Genome library of toxic Alexandium minutum were constructed. A 1014bp DNA reconstruction fragment was obtained and PCR method testified that this fragment had strong amplification signal in toxic strains while no signal in non-toxic strains. The external sequence of the DNA fragment was analyzed by inverse PCR method, and a 240bp nucleotide sequence which translated into proteins aminophenol was Methionine Aminopeptidase (MAP). The sequence of aminophenol was BLAST in NCBI, and found that it had 97% similarity with Alexandium fundyense. This sequence was exact uniform with the reported sequence in the coding region, but there were three bases mutated in non-coding region behind stop codon. Probably, in the course of growth metabolism of Alexandium minutum, MAP executed different physiological functions and controlled the production of the toxin. In the meantime, map gene induced encoding products of the related poisonous gene, and enabled it has active expressions to starting toxin synthesis.

Internal tandem duplication (ITD) mutations of the FLT3 gene have been associated with a poor prognosis in acute myeloid leukemia. Detection of ITD-positive minor clones at the initial diagnosis and during the minimal residual disease stage may be essential. We previously designed a delta-PCR strategy to improve the sensitivity to 0.1% ITD-positive leukemia cells and showed that minor mutants with an allele burden of

In this study, Streptococcus gordonii-specific quantitative real-time polymerase chain reaction (qPCR) primers, RTSgo-F2/RTSgo-R2, were developed based on the nucleotide sequences of RNA polymerase β-subunit gene (rpoB). The specificity of the RTSgo-F2/RTSgo-R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity.

Several reports mention the presence of antibiotic resistance genes in natural and polluted environments, but many studies are based on their detection via polymerase chain reaction (PCR amplification of known genes and not on an activity screening. We constructed a metagenomic fosmid bank from DNA isolated from a polluted river in Brussels, Belgium, the Zenne. A total of 120,000 clones were pooled and plated directly on solid media containing different antibiotics. Several clones were isolated which could grow in the presence of ampicillin. The DNA from several clones was extracted and subjected to restriction analysis and, based on their restriction pattern, two different clones were found. One of the clones was selected for further study as it showed a higher level of resistance to different β-lactams antibiotics (ticarcilline and ceftazidime). To find out which gene is responsible for the resistance, an in vitro transposon mutagenesis was performed and clones having lost the resistance phenotype were analyzed via inverse PCR amplification. Several clones had an insert in a gene encoding a new type of β-lactamase. The amplified fosmid DNA was fully sequenced revealing an insert of 41 kb containing 39 open reading frames (ORFs). Transposon insertions inactivating the resistance to β-lactams were also found in the ORF upstream of the blaA gene, encoding an aminotransferase, suggesting a polar effect on the transcription of the gene downstream. In addition, other genes were found such as histidine biosynthesis genes, which were found to be scattered on the insert, a relA/spoT gene, and genes belonging to type II toxin-antitoxin system. This predicted system was experimentally validated in Escherichia coli using an inducible expression system., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

DNA is a typical organic compound with marked differences from other chemicals and biopolymers because DNA can be amplified by the enzyme polymerase. DNA can be, in principle, amplified from a single copy by the polymerase chain reaction (PCR). In this review, we focus our attention on the chemistry of PCR primers. Because PCR is basic technology in biology research fields, we sometimes use chemically labeled primers without any awareness of the chemistry they leave behind. We would like to emphasize that chemically labeled primers contain a lot of potential for different chemistry ideas and much study is still necessary to advance PCR for single-nucleotide polymorphism (SNP) typing, genetic diagnosis, and other fields. Two categories of primers, affinity-capture primers and signaling primers, are discussed from the viewpoints of their chemical concepts and applications. Affinity-capture primers are used for purification, isolation, and manipulation of PCR products by high specificity and affinity to the cognate molecules by moleculemolecule interactions, whereas signaling primers report the hybridization and/or progress of PCR amplification by a signal change, in most cases by a fluorescence change. The content of this review may be useful for a better understanding of the chemistry of PCR primers and, more importantly, for the invention of novel PCR chemistry.

We isolated and functionally characterized a Dendrobium Actin1 (DmACT1) promoter that drives strong gene expression in the orchid genus Dendrobium . A genomic fragment containing the region 3227 bp upstream of the coding region of DmACT1 was obtained by inverse PCR. Detailed comparison of the full-length cDNA and genomic sequences revealed that DmACT1 has a 1374 bp first intron in the 5’ UTR. However, the 5’ flanking sequences upstream of the coding region showed no obvious sequence similarities compared to those of known promoters, including plant actin promoters. Serial deletion constructs of the 5’ flanking region from the translation initiation codon were fused to the coding sequence of a GUS/luciferase fusion reporter to identify the regulatory elements necessary for promoter activity. Transient assays in the flowers of Dendrobium revealed that the 5’ UTR-intron greatly enhanced pro-moter activity. Moreover, the DmACT1 promoter with its 5’ UTR-intron yielded approximately 10-fold higher reporter activity than the rice Act1 promoter-intron. Our data suggest that the

Protecting group chemistry meets molecular biology: Chemically modified dATP carrying a bulky triethylsilylethynyl group was used in a PCR-based synthesis of a gene internally protected against cleavage by restriction endonucleases. The unmodified flanking regions were cleaved for cloning into a plasmid which was replicated by E. coli, and used for protein production.

We previously reported that Tpa -S DNA polymerase (constructed via fusion of the Sso7d DNA binding protein to the C-terminus of Thermococcus pacificus ( Tpa ) DNA polymerase) is more efficient in long and rapid PCR than wild-type Tpa , Taq , or Pfu DNA polymerases. However, Tpa -S DNA polymerase had a low yield of PCR products compared with commercialized Taq or Pfu DNA polymerases. To improve the yield of PCR products, mutant Tpa -S DNA polymerases were created via site-directed mutagenesis. In this study, we have targeted the N213 residue in the Exo II motif and the K501 residue in the Pol III motif. The mutant Tpa -S DNA polymerases showed enhanced PCR yields compared to that of the Tpa -S DNA polymerase. Specifically, the double mutant Tpa -S N213D/K501R DNA polymerase had an approximately three-fold increase in the yield of 8–10 kb PCR products over that of the Tpa -S DNA polymerase, and catalyzed amplification of a 12 kb PCR product using a lambda template with an extension time of 30 s. Even though the mutation is in the Exo II motif, the error rate of the double mutant Tpa -S N213D/K501R (2.79 × 10 −5 ) was nearly the same as that seen in the Pfu DNA polymerase (2.70 × 10 −5 ).

Body fluid DNA sequencing is a powerful noninvasive approach for the diagnosis of genetic defects, infectious agents and diseases. The success relies on the quantity and quality of the DNA samples. However, numerous clinical samples are either at low quantity or of poor quality due to various reasons. To overcome these problems, we have developed T oligo-primed polymerase chain reaction (TOP-PCR) for full-length nonselective amplification of minute quantity of DNA fragments. TOP-PCR adopts homogeneous “half adaptor” (HA), generated by annealing P oligo (carrying a phosphate group at the 5′ end) and T oligo (carrying a T-tail at the 3′ end), for efficient ligation to target DNA and subsequent PCR amplification primed by the T oligo alone. Using DNA samples from body fluids, we demonstrate that TOP-PCR recovers minute DNA fragments and maintains the DNA size profile, while enhancing the major molecular populations. Our results also showed that TOP-PCR is a superior method for detecting apoptosis and outperforms the method adopted by Illumina for DNA amplification.

Polymerase chain reaction (PCR) is an oft-used preparatory technique in amplifying specific DNA regions for downstream analysis. The size of an amplicon was initially limited by errors in nucleotide polymerization and template deterioration during thermal cycling. A variant of PCR, designated long-range PCR, was devised to counter these drawbacks and enable the amplification of large fragments exceeding a few kb. In this chapter we describe a protocol for long-range PCR, which we have adopted to obtain products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples.

The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive "Ming" lethal egg mutants (l-e(m)) lose water and become concaved around 1h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e(m) mutant to 360 kb on chromosome 10 using 2287 F(2) individuals. Using expression analysis and RNA interference, the best l-e(m) candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~1.9 kb region from the 3' untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a100 kb DNA fragment in the l-e(m) mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e(m) mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e(m) mutant. These results indicate that BmEP80 is responsible for the l-e(m) mutation.

Methylation-specific polymerase chain reaction (MS-PCR) is a more rapid way to detect changes in DNA methylation than is bisulfite sequencing. In addition, by incorporating some basic automation, samples can be prepared and analyzed in a 96-well plate format. The method can be used either quantitatively (qRT-PCR-based MethyLight) or qualitatively (using agarose gels) to detect changes in DNA methylation; both are described in this protocol.

Using oligonucleotide primers that hybridize to conserved sequences in the reverse transcriptase (RT) gene, we have amplified by the polymerase chain reaction three sequence variants of HTLV-I from the genomic DNA of five patients with tropical spastic paraparesis (TSP), and a fourth sequence variant from a healthy carrier of HTLV-I. These results unequivocally identify the retrovirus associated with TSP as HTLV-I and suggest that no sequence variant is uniquely responsible for the condition. The same primers served to amplify two novel single-copy endogenous retroviral RT sequences related to the exogenous mammalian leukaemia viruses: and three KpnI (LINE1) family DNA repeats. This strategy, combining the sensitivity of PCR with cross-reactive primers, may be useful in the search for known or novel retroviruses in other diseases of possible retroviral aetiology.