Modified DNA bearing 5(methoxycarbonylmethyl)-2'-deoxyuridine: preparation by PCR with thermophilic DNA polymerase and postsynthetic derivatization

A thymidine analogue bearing a methyl ester at the C5 position was accepted as a substrate by the thermophilic family B DNA polymerases, KOD Dash, Pwo, and Vent(exo-), to form the corresponding PCR product, but not by the thermophilic family A DNA polymerases, Taq, Tth, and T7 thermosequenase. Modified DNA containing this analogue was prepared by PCR on a large scale with KOD Dash DNA polymerase and 5(methoxycarbonylmethyl)-2'-deoxyuridine 5'-triphosphate as a substrate. The methyl ester of the modified DNA was further allowed to react with tris(2-aminoethyl)amine or histamine by an ester-amide exchange reaction to form the corresponding derivatized DNA bearing a tris(2-aminoethyl)amine or histamine moiety. Hydrolysis of the methyl ester of the modified DNA gave a functionalized DNA bearing an anionic carboxyl group. The derivatized DNA could act as a template for the PCR with KOD Dash DNA polymerase and the natural 2'-deoxythymidine 5'-triphosphate or the modified thymidine analogue as a substrate. The postsynthetic derivatization of the modified DNA may expand the variety of structurally modified DNA produced by PCR.     

Increased single-nucleotide discrimination of PCR by primer probes bearing hydrophobic 4'C modifications

We report on significantly increased selectivity of real-time PCR through employment of primer probes that bear hydrophobic 4'C modifications at the 3'-terminal nucleotide. The primer probes were designed to bind the target sequences in such a way that the 3'-terminal nucleotide defines whether a matched or a single mismatched basepair is present depending on the respective target sequence. Several commercially available thermostable DNA polymerases belonging to different DNA polymerase families were tested for their efficacy in discriminating between PCR amplification of matched substrates and duplexes that contain a single mismatch. It turned out that, depending on the 4'C modification and the employed DNA polymerase, significantly increased differentiation between single matches and mismatches could be observed with real-time PCR. The degrees of the observed effects varied with the employed 4'C modification and the sequence context studied. The system is robust enough to work faithfully under several buffer conditions. Our approach should be useful for the direct diagnosis of single nucleotide variations within genes, like single nucleotide polymorphisms or mutations, by PCR without the need for further time- and cost-intensive post-PCR analysis.     

Role of disulfide bridges in archaeal family-B DNA polymerases

The family-B DNA polymerases obtained from the order Thermococcales, for example, Pyrococcus furiosus (Pfu-Pol) are commonly used in the polymerase chain reaction (PCR) because of their high thermostability and low error rates. Most of these polymerases contain four cysteines, arranged as two disulfide bridges. With Pfu-Pol C429-C443 forms one of the disulfides (DB1) and C507-C510 (DB2) the other. Although the disulfides are well conserved in the enzymes from the hyperthermophilic Thermococcales, they are less prevalent in euryarchaeal polymerases from other orders, and tend to be only found in other hyperthermophiles. Here, we report on the effects of deleting the disulfide bridges by mutating the relevant cysteines to serines. A variety of techniques, including differential scanning calorimetry and differential scanning fluorimetry, have shown that both disulfides make a contribution to thermostability, with DB1 being more important than DB2. However, even when both disulfides are removed, sufficient thermostability remains for normal (identical to the wild type) performance in PCR and quantitative (real-time) PCR. Therefore, polymerases totally lacking cysteine are fully compatible with most PCR-based applications. This observation opens the way to further engineering of polymerases by introduction of a single cysteine followed by appropriate chemical modification.     

Using a PCR-Based Method To Analyze and Model Large,Heterogeneous Populations of DNA

The study of populations of large size and high diversity is limited by the capability of collecting data. Moreover, for a pool of individuals, each associated with a unique characteristic feature, as the pool size grows, the possible interactions increase exponentially and quickly go beyond the limit of computation and experimental studies. Herein, the design of DNA libraries with various diversity is reported. By using a facile analytical method based on real-time PCR, the diversity of a pool of DNA can be evaluated to allow extraordinarily high heterogenicity (e.g., >1 trillion). It is demonstrated that these DNA libraries can be used to model heterogeneous populations; these libraries exhibit functions such as self-protection, suitability for biased expansion, and the possibility to evolve into amorphous structures. The method has shown the remarkable power of parallel computing with DNA, since it can resemble an analogue computer and be applied in selection-based biotechnology methods, such as DNA-encoded chemical libraries. As a chemical approach to solve problems traditionally for genetic and statistical analysis, the method provides a quick and cost-efficient evaluation of library diversity for intermediate steps through a selection process.     

Directed DNA polymerase evolution: effects of mutations in motif C on the mismatch-extension selectivity of thermus aquaticus DNA polymerase

The selectivity of DNA polymerases for processing the canonical nucleotide and DNA substrate in favor of the noncanonical ones is the key to the integrity of the genome of every living species and to many biotechnological applications. The inborn ability of most DNA polymerases to abort efficient extension of mismatched DNA substrates adds to the overall DNA polymerase selectivity. DNA polymerases have been grouped into families according to their sequence. Within family A DNA polymerases, six motifs that come into contact with the substrates and form the active site have been discovered to be evolutionary highly conserved. Here we present results obtained from amino acid randomization within one motif, motif C, of thermostable Thermus aquaticus DNA polymerase. We have identified several distinct mutation patterns that increase the selectivity of mismatch extension. These results might lead to direct applications such as allele-specific PCR, as demonstrated by real-time PCR experiments and add to our understanding of DNA polymerase selectivity.     

Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells

Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02–0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.     

Nonenzymatic ligation of DNA with a reversible step and a final linkage that can be used in PCR

Nonenzymatic DNA ligation chemistries containing a reversible step allow thermodynamic control of product formation, but they are not necessarily compatible with polymerase enzymes. We report a ligation system that uses commercially available reagents, includes a reversible step, and results in a linkage that can function as a template for PCR amplification with accurate sequence transfer.     

腮腺炎病毒临床口漱液样本的一步法实时荧光定量PCR检测

目的建立腮腺炎病毒(mumps virus,MuV)临床口漱液样本一步法荧光定量PCR检测方法,为腮腺炎的临床诊断及免疫防控提供依据。方法对101份采集自中国南部3个省、自治区(云南、四川、广西)的临床疑似腮腺炎患者口漱液样本,分别采用细胞培养-MuV特异的巢式PCR法及TaqMan探针Real-time PCR法进行检测,比较两种方法检测MuV的灵敏性。结果细胞培养-MuV特异的巢式PCR法共检出31份MuV阳性样本,系统进化分析表明,该31株MuV均属于F基因亚型;TaqMan探针Real-time PCR法共检出41份MuV阳性样本,其中包含了细胞培养-MuV特异的巢式PCR法检出的31份阳性样本,TaqMan探针Real-time RT-PCR法对MuV的检出率(40.59%)高于传统细胞培养-巢式PCR法的检出率(30.69%)。结论 TaqMan探针Real-time PCR法的灵敏性、特异性和简便性均优于传统的细胞培养-MuV特异的巢式PCR法,可应用于腮腺炎的临床诊断及免疫防控。     

Cover Picture: Chemical Restriction: Strand Cleavage by Ammonia Treatment at 8‐Oxoguanine Yields Biologically Active DNA (ChemBioChem 7/2003)

The cover picture shows a chemical restriction method in which DNA double strands are cleaved at the position of the artificial base 8‐oxoguanine (G^oxo). As as result of the mild cleavage conditions, the DNA fragments remain biologically active and can be used in gene cloning experiments. As an example, the lac Z′ gene was amplified by PCR with 8‐oxoguanine‐modified primers, restricted by treatment with ammonia, ligated into a plasmid vector, transformed in Escherichia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure, and allows the design of any 3′ overhang, independent of the sequence of the cloned DNA. Further information can be found in the article by Giese et. al. on pp. 610–614     

Chemical restriction: strand cleavage by ammonia treatment at 8-oxoguanine yields biologically active DNA

Cleavage of DNA single and double strands at an 8-oxoguanine-containing nucleotide occurs in 90 % yield if the modified oligonucleotide is treated with NH(3) and O(2) at 60 degrees C. The mechanism of this oxidative cleavage reaction was studied, and the reaction was applied to the generation of single-stranded overhangs on PCR-amplified DNA that can be ligated. As an example, the lac Z' gene was amplified by PCR with 8-oxoguanine modified primers, restricted by ammonia treatment, ligated into a plasmid vector, transformed in Escherischia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure with restriction enzymes, and it allows the design of any 3'-overhang independent of the sequence of the cloned DNA.     

Protein–Polymer Microcapsules for PCR Technology

Protein–polymer microcapsules have attracted much attention, due to their special features and potential in biological use. How to make the most of this type of bio‐abiotic hybrid material is an intriguing question. Nevertheless, several unsatisfactory technical issues significantly limited the application of these materials. For instance, introducing various biomolecules and crosslinking for the capsules remains challenging and problematic. In this report, recombinant mCherry protein was covalently linked with poly(N‐isopropylacrylamide) (PNIPAAm) to form amphiphilic protein–polymer conjugates, which assembled into microcapsules. These microcapsules are thermoresistant and can be used in the polymerase chain reaction (PCR). In this setting, the reactant molecules can be readily and easily introduced into the microcapsules, and crosslinking and water–oil phase transition are not necessary. This protein–polymer microcapsule PCR system has potential in various biological applications.     

重叠延伸PCR法定点突变微生物产谷氨酰胺转氨酶基因

目的通过重叠延伸PCR法,定点突变微生物产谷氨酰胺转氨酶(Microbial transglutaminase,MTG)基因。方法根据GenBank中登录的Streptomyces sp.H197基因中MTG序列及重叠延伸PCR定点突变技术的原理,利用DNA-MAN5.0软件设计引物,以提取的Streptomyces sp.H197基因组DNA为模板,PCR扩增MTG基因,以扩增的MTG基因片段为模板,采用重叠延伸PCR技术对一个位点进行定点突变,胶回收PCR产物,与克隆载体pMD19-T连接后,转化感受态E.coli DH5α,提取质粒,经PCR及单、双酶切鉴定,并测序。结果重叠延伸PCR产物可见含有酶切位点和突变位点的特异性片段;重组质粒pMD19-T-MTG经PCR及单、双酶切鉴定,证明构建正确;测序结果表明,与野生型序列比对,仅有一个碱基发生改变,即第773位腺嘌呤突变为胞嘧啶,突变位点与预期一致,实现了目标位点的定点突变。结论重叠延伸PCR法对MTG基因序列预定位点突变成功,证明阳性克隆子含突变位点,为下阶段突变型功能表达奠定基础。     

聚合物衬底材料在PCR生物芯片中的应用及相关技术

介绍了聚合物衬底材料在聚合酶链式反应(PCR)生物芯片中的最新应用及相关技术,这些技术包括聚合物衬底材料的表面钝化,PCR生物芯片的微加工(注模、压模及激光刻蚀等)及封接(氧等离子体表面处理及热键合)等。     

Beating Bias in the Directed Evolution of Proteins: Combining High‐Fidelity on‐Chip Solid‐Phase Gene Synthesis with Efficient Gene Assembly for Combinatorial Library Construction

Saturation mutagenesis (SM) constitutes a widely used technique in the directed evolution of selective enzymes as catalysts in organic chemistry and in the manipulation of metabolic paths and genomes, but the quality of the libraries is far from optimal due to the inherent amino acid bias. Herein, it is shown how this fundamental problem can be solved by applying high‐fidelity solid‐phase chemical gene synthesis on silicon chips followed by efficient gene assembly. Limonene epoxide hydrolase was chosen as the catalyst in the model desymmetrization of cyclohexene oxide with the stereoselective formation of (R,R)‐ and (S,S)‐cyclohexane‐1,2‐diol. A traditional combinatorial PCR‐based SM library, produced by simultaneous randomization at several residues by using a reduced amino acid alphabet, and the respective synthetic library were constructed and compared. Statistical analysis at the DNA level with massive sequencing demonstrates that, in the synthetic approach, 97 % of the theoretically possible DNA mutants are formed, whereas the traditional SM library contained only about 50 %. Screening at the protein level also showed the superiority of the synthetic library; many highly (R,R)‐ and (S,S)‐selective variants being discovered are not found in the traditional SM library. With the prices of synthetic genes decreasing, this approach may point the way to future directed evolution.     

Analysis of Modified Nucleotide Aptamer Library Generated by Thermophilic DNA Polymerases

One of the pivotal steps in aptamer selection is the amplification of target-specific oligonucleotides by thermophilic DNA polymerases; it can be a challenging task if nucleic acids possessing modified nucleotides are to be amplified. Hence, the identification of compatible DNA polymerase and modified nucleotide pairs is necessary for effective selection of aptamers with unnatural nucleotides. We present an in-depth study of using 5-indolyl-AA-dUTP (TAdUTP) to generate oligonucleotide libraries for aptamer selection. We found that, among the eight studied DNA polymerases, only Vent(exo-) and KOD XL are capable of adapting TAdUTP, and that replacing dTTP did not have a significant effect on the productivity of KOD XL. We demonstrated that water-in-oil emulsion PCR is suitable for the generation of aptamer libraries of modified nucleotides. Finally, high-throughput sequence analysis showed that neither the error rate nor the PCR bias was significantly affected by using TAdUTP. In summary, we propose that KOD XL and TAdUTP could be effectively used for aptamer selection without distorting the sequence space of random oligonucleotide libraries.