A quantum mechanical model of adaptive mutation

The principle that mutations occur randomly with respect to the direction of evolutionary change has been challenged by the phenomenon of adaptive mutations. There is currently no entirely satisfactory theory to account for how a cell can selectively mutate certain genes in response to environmental signals. However, spontaneous mutations are initiated by quantum events such as the shift of a single proton (hydrogen atom) from one site to an adjacent one. We consider here the wave function describing the quantum state of the genome as being in a coherent linear superposition of states describing both the shifted and unshifted protons. Quantum coherence will be destroyed by the process of decoherence in which the quantum state of the genome becomes correlated (entangled) with its surroundings. Using a very simple model we estimate the decoherence times for protons within DNA and demonstrate that quantum coherence may be maintained for biological time-scales. Interaction of the coherent genome wave function with environments containing utilisable substrate will induce rapid decoherence and thereby destroy the superposition of mutant and non-mutant states. We show that this accelerated rate of decoherence may significantly increase the rate of production of the mutated state.     

DNA聚合酶与引物/模板的相互作用对PCR效率的影响

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

Influence of DNA structure on DNA polymerase beta active site function: extension of mutagenic DNA intermediates

In the ternary substrate complex of DNA polymerase (pol) beta, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol beta on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k(cat)/K(m), dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C approximately A/T > T/A approximately C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K(d)((dGTP)) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand.     

Interactions of calf thymus DNA polymerase alpha with primer/templates.

The interactions of calf thymus DNA polymerase alpha (pol alpha) with primer/templates were examined. Simply changing the primer from DNA to RNA had little effect on primer/template binding or dNTP polymerization (Km, Vmax and processivity). Surprisingly, however, adding a 5'-triphosphate to the primer greatly changed its interactions with pol alpha (binding, Vmax and Km and processivity). While changing the primer from DNA to RNA greatly altered the abilit of pol alpha to discriminate against nucleotide analogs, it did not compromise the ability of pol alpha to discriminate against non-cognate dNTPs. Thus the nature of the primer appears to affect 'sugar fidelity', without altering 'base fidelity'. DNase protection assays showed that pol alpha strongly protected 9 nt of the primer strand, 13 nt of the duplex template strand and 14 nt of the single-stranded template from hydrolysis by DNase I and weakly protected several bases outside this core region. This large DNA binding domain may account for the ability of a 5'-triphosphate on RNA primers to alter the catalytic properties of pol alpha.     

Norovirus RNA‐dependent RNA polymerase: A computational study of metal‐binding preferences

Norovirus (NV) RNA‐dependent RNA polymerase (RdRP) is essential for replicating the genome of the virus, which makes this enzyme a key target for the development of antiviral agents against NV gastroenteritis. In this work, a complex of NV RdRP bound to manganese ions and an RNA primer‐template duplex was investigated using X‐ray crystallography and hybrid quantum chemical/molecular mechanical simulations. Experimentally, the complex crystallized in a tetragonal crystal form. The nature of the primer/template duplex binding in the resulting structure indicates that the complex is a closed back‐tracked state of the enzyme, in which the ‐end of the primer occupies the position expected for the post‐incorporated nucleotide before translocation. Computationally, it is found that the complex can accept a range of divalent metal cations without marked distortions in the active site structure. The highest binding energy is for copper, followed closely by manganese and iron, and then by zinc, nickel, and cobalt. Proteins 2017; 85:1435–1445. © 2017 Wiley Periodicals, Inc.     

Primer-mediated inhibition of the hydrolysis of template DNA by T5-induced DNA polymerase.

Bacteriophage T5-induced DNA polymerase has an associated 3′→5′ exonuclease activity for which both single-stranded and duplex DNA serve as substrate (1). In this report, we demonstrate that hydrolysis of single-stranded DNA homopolymers (template) is inhibited in the presence of complementary (Watson-Crick sense) oligonucleotides (primer). Almost complete inhibition is observed at a primer/template ratio of ? 0.1. Formation of “H-bonded” primer-template complex seems to be necessary for the inhibition of template hydrolysis because (a) similar amounts of noncomplementary oligonucleotides have no detectable effect on the rate of template hydrolysis, and (b) complementary oligonucleotides lose their inhibitory potential at temperatures where the H-bonded primer-template complex is expected to be unstable. From our data, it appears that the inhibition of template hydrolysis in the presence of primer molecules is due to the preferential binding of the enzyme at the 3′-OH terminus of the primer in the primer-template complex.     

Indispensable role of quantum theory in the brain dynamics

Recently, Tegmark pointed out that the superposition of ion states involved in the superposition of firing and resting states of a neuron quickly decohere. It undoubtedly indicates that neural networks cannot work as quantum computers, or computers taking advantage of coherent states. Does it also mean that the brain can be modeled as a neural network obeying classical physics? Here we show that it does not mean that the brain can be modeled as a neural network obeying classical physics. A brand new perspective in research of neural networks from quantum theoretical aspect is presented.     

Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides

The RNA world hypothesis states that the early evolution of life went through a stage where RNA served as genome and as catalyst. The replication of RNA world organisms would have been facilitated by ribozymes that catalyze RNA polymerization. To recapitulate an RNA world in the laboratory, a series of RNA polymerase ribozymes was developed previously. However, these ribozymes have a polymerization efficiency that is too low for self-replication, and the most efficient ribozymes prefer one specific template sequence. The limiting factor for polymerization efficiency is the weak sequence-independent binding to its primer/template substrate. Most of the known polymerase ribozymes bind an RNA heptanucleotide to form the P2 duplex on the ribozyme. By modifying this heptanucleotide, we were able to significantly increase polymerization efficiency. Truncations at the 3'-terminus of this heptanucleotide increased full-length primer extension by 10-fold, on a specific template sequence. In contrast, polymerization on several different template sequences was improved dramatically by replacing the RNA heptanucleotide with DNA oligomers containing randomized sequences of 15 nt. The presence of G and T in the random sequences was sufficient for this effect, with an optimal composition of 60% G and 40% T. Our results indicate that these DNA sequences function by establishing many weak and nonspecific base-pairing interactions to the single-stranded portion of the template. Such low-specificity interactions could have had important functions in an RNA world.     

Annealing control primer system for improving specificity of PCR amplification

A novel primer designed to improve the specificity of PCR amplification, called the annealing control primer (ACP), comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3' end target core sequence and the 5' end nontarget universal sequence. We show that this ACP linker prevents annealing of the 5' end nontarget sequence to the template and facilitates primer hybridization at the 3' end to the target sequence at specific temperatures, resulting in a dramatic improvement of annealing specificity. The effect of this linker is demonstrated by the incorporation of ACP sequences as primers during the amplification of target nucleotide sequence and as hybridization probes in the genotyping of single nucleotide polymorphisms. This is the first report to show that a poly(dI) linker between two different sequences of ACP forms a bubble-like structure and disrupts or destabilizes DNA duplex formation at certain annealing temperatures.     

On genetic information uncertainty and the mutator phenotype in cancer

Recent evidence supports the existence of a mutator phenotype in cancer cells, although the mechanistic basis remains unknown. In this paper, it is shown that this enhanced genetic instability is generated by an amplified measurement uncertainty on genetic information during DNA replication. At baseline, an inherent measurement uncertainty implies an imprecision of the recognition, replication and transfer genetic information, and forms the basis for an intrinsic genetic instability in all biological cells. Genetic information is contained in the sequence of DNA bases, each existing due to proton tunnelling, as a coherent superposition of quantum states composed of both the canonical and rare tautomeric forms until decoherence by interaction with DNA polymerase. The result of such a quantum measurement process may be interpreted classically as akin to a Bernoulli trial, whose outcome X is random and can be either of two possibilities, depending on whether the proton is tunnelled (X=1) or not (X=0). This inherent quantum uncertainty is represented by a binary entropy function and quantified in terms of Shannon information entropy H(X)=-P(X=1)log(2)P(X=1)-P(X=0)log(2)P(X=0). Enhanced genetic instability may either be directly derived from amplified uncertainty induced by increases in quantum and thermodynamic fluctuation, or indirectly arise from the loss of natural uncertainty reduction mechanisms.     

Asymmetric exponential amplification reaction on a toehold/biotin featured template: an ultrasensitive and specific strategy for isothermal microRNAs analysis

The sensitive and specific analysis of microRNAs (miRNAs) without using a thermal cycler instrument is significant and would greatly facilitate biological research and disease diagnostics. Although exponential amplification reaction (EXPAR) is the most attractive strategy for the isothermal analysis of miRNAs, its intrinsic limitations of detection efficiency and inevitable non-specific amplification critically restrict its use in analytical sensitivity and specificity. Here, we present a novel asymmetric EXPAR based on a new biotin/toehold featured template. A biotin tag was used to reduce the melting temperature of the primer/template duplex at the 5′ terminus of the template, and a toehold exchange structure acted as a filter to suppress the non-specific trigger of EXPAR. The asymmetric EXPAR exhibited great improvements in amplification efficiency and specificity as well as a dramatic extension of dynamic range. The limit of detection for the let-7a analysis was decreased to 6.02 copies (0.01 zmol), and the dynamic range was extended to 10 orders of magnitude. The strategy enabled the sensitive and accurate analysis of let-7a miRNA in human cancer tissues with clearly better precision than both standard EXPAR and RT-qPCR. Asymmetric EXPAR is expected to have an important impact on the development of simple and rapid molecular diagnostic applications for short oligonucleotides.     

A polymerase chain reaction mediated by a single primer: cloning of genomic sequences adjacent to a serotonin receptor protein coding region.

Under appropriate conditions, specific double-stranded DNA product was generated after amplification of genomic DNA sequences in a polymerase chain-like reaction that contained only a single primer. This type of amplification reaction was performed with a variety of primers and substrate DNAs. In addition to nonspecific heterogeneous products, 5 of 11 primers reproducibly directed synthesis of double-stranded DNA that corresponded to the region of the template that contained the authentic primer annealing site. Three of these amplified products were cloned and their ends were sequenced. All three contained a copy of the primer at both 5' ends, and the position of one of the primers represented the authentic primer binding site. In each case, the location of the second copy of the primer indicated that it had initially hybridized to a partially homologous sequence in the template DNA. This single primer reaction makes it possible to amplify and clone a DNA region of unknown sequence that is adjacent to a known DNA sequence. One of the single primer reaction products described here included sequence to the 5' side of the coding region of a serotonin receptor gene that contained a functional promoter.     

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.     

Impact of template overhang-binding region of HIV-1 RT on the binding and orientation of the duplex region of the template-primer

Fingers domain of HIV-1 RT is one of the constituents of the dNTP-binding pocket that is involved in binding of both dNTP and the template-primer. In the ternary complex of HIV-1 RT, two residues Trp-24 and Phe-61 located on the β1 and β3, respectively, are seen interacting with N + 1 to N + 3 nucleotides in the template overhang. We generated nonconservative and conservative mutant derivatives of these residues and examined their impact on the template-primer binding and polymerase function of the enzyme. We noted that W24A, F61A, and F61Y and the double mutant (W24A/F61A) were significantly affected in their ability to bind template-primer and also to catalyze the polymerase reaction while W24F remained unaffected. Using a specially designed template-primer with photoactivatable bromo-dU base in the duplex region at the penultimate position to the primer terminus, we demonstrated that F61A, W24A, F61Y as well as the double mutant were also affected in their cross-linking ability with the duplex region of the template-primer. We also isolated the E–TP covalent complexes of these mutants and examined their ability to catalyze single dNTP incorporation onto the immobilized primer terminus. The E–TP covalent complexes from W24F mutant displayed wild-type activity while those from W24A, F61A, F61Y, and the double mutant (W24A/F61A) were significantly impaired in their ability to catalyze dNTP incorporation onto the immobilized primer terminus. This unusual observation indicated that amino acid residues involved in the positioning of the template overhang may also influence the binding and orientation of the duplex region of the template-primer. Molecular modeling studies based on our biochemical results suggested that conformation of both W24 and F61 are interdependent on their interactions with each other, which together are required for proper positioning of the +1 template nucleotide in the binary and ternary complexes.     

MethyScan：一种甲基化特异性PCR引物设计及评估工具

DNA甲基化是重要的表观遗传现象,对基因表达发挥重要调控功能.大量研究表明,基因DNA甲基化是重要的临床诊断生物标志物.在临床上,实施快速、准确的DNA甲基化状态检测是诊断应用的前提和关键.甲基化特异性PCR(methylation specific PCR,MSP)通过将两种引物与甲基化、非甲基化模板各自特异性结合和扩增,实现基因甲基化状态的区分,是切实可行、简单便捷的临床诊断实验技术.但是,不同于常规PCR,MSP主要存在如何强化引物-甲基化/非甲基化模板特异性结合、降低引物序列Tm值差异、去除假阳性扩增及提高敏感性等四大难点.尽管大多数MSP引物设计软件对上述难题都提出了各自解决办法,但在引物设计影响因素考虑、设计与评估并行处理及特异性扩增预测等方面仍然存在较大缺陷.为此,本研究通过对MethPrimer、MSPPrimer、MethBlast、BiSearch等现有MSP引物设计软件原理的深入探究,以及对Bowtie、SAMtools和BEDTools等工具的有效综合整合,基于图形库Matplotlib和第三方Python功能库BioPython与Primer3-py实现了具有系列优点的甲基化特异性PCR引物设计与评估可视化工具MethyScan.它具有引物设计、基因组索引、引物评估等三大完整功能模块,不仅可快速进行MSP引物设计,实现巢式(Nested)引物适配,还可基于4种基因组碱基转换模板分析引物结合信息,图形化展示非特异性扩增与目的片段差异,从而综合评估引物特异性-非特异性扩增.同时,对食管癌、结直肠癌等多种恶性肿瘤中6个潜在生物标志物TFPI-2、NDRG4、CDKN2A、CD44、CASP8和SDHD的甲基化引物设计对比结果表明,MethyScan不仅可获得更多CpG位点的检测引物,而且所获得MSP引物位置与其他软件结果相同或相近,且引物间Tm值差值更小.总之,作为首个图形化展示特异性-非特异性扩增差异MSP引物设计工具,MethyScan可有效提高甲基化引物设计准确性,为临床DNA甲基化检测项目开展、检测试验实施及诊断试剂盒研发提供有力支撑.MethyScan工具下载地址:https://github.com/bioinfo-ibms-pumc/MethyScan.     

扩增条件对茶类植物RAPD带的影响

采用梯度分析的方法试验了模板ＤＮＡ、引物、镁离子、ｄＮＴＰ和Ｔａｑ酶的浓度对茶类植物进行ＲＡＰＤ分析中ＤＮＡ扩增结果的影响。实验表明这些条件的变化对扩增出来的ＲＡＰＤ带的数目和强弱会产生影响。经过比较分析,筛选出对于茶类植物进行ＲＡＰＤ分析较理想的扩增条件：２．０ｍｍｏｌ／ＬＭｇＣｌ２,２００ｕｍｏｌ／ＬｄＮＴＰ,１５ｎｇ引物／２０ｕｌ反应体积,４ｎｇ模板ＤＮＡ／ｕｌ反应体积,１ＵＴａｑ酶／２０ｕｌ反应体积。     

黄皮SRAP反应体系优化正交实验研究

以黄皮(Clausena lansium)‘甜黄皮’品种为试材,利用正交设计L₁₆(4⁵)对黄皮SRAP-PCR反应体系中的5因素(Taq聚合酶、Mg²⁺、模板DNA、dNTPs、引物)在4个水平上进行优化试验。结果表明,不同因素对黄皮SRAP反应体系影响从大到小的顺序为:Mg²⁺和Taq聚合酶> 模板DNA> 引物> dNTPs;初步确立了适合黄皮的SRAP-PCR扩增体系为:在25 μl反应体系中,包括10×PCR buffer 2.5 μl、Taq DNA聚合酶0.75U、Mg²⁺ 2.0 mmol/L、模板DNA 60 ng、dNTPs 0.2 mmol/L、引物0.2 μmol/L。     

Polymerase chain reaction amplification of single-stranded DNA containing a base analog, 2-chloradenine.

By utilization of polymerase chain reaction techniques, single-stranded DNA of defined length and sequence containing a purine analog, 2-chloroadenine, in place of adenine was synthesized. This was accomplished by a combination of standard polymerase chain amplification reactions with Thermus aquaticus DNA polymerase in the presence of four normal deoxynucleoside triphosphates, M13 duplex DNA as template, and two primers to generate double-stranded DNA 118 bases in length. An asymmetric polymerase chain reaction, which produced an excess of single-stranded 98-base DNA, was then conducted with 2-chloro-2'-deoxy-adenosine 5'-triphosphate in place of dATP and with only one primer that annealed internal to the original two primers. Standard polymerase chain reaction techniques alone conducted in the presence of the analog as the fourth nucleotide did not produce duplex DNA that was modified within both strands. This asymmetric technique allows the incorporation of an altered nucleotide at specific sites into large quantities of single-stranded DNA without using chemical phosphoramidite synthesis procedures and circumvents the apparent inability of DNA polymerase to synthesize fully substituted double-stranded DNA during standard amplification reactions. The described method will permit the study of the effects of modified bases in template DNA on a variety of protein-DNA interactions and enzymes.     

Development of a multiple internal control for clinical diagnostic real-time amplification assays

A major pitfall in most published genomic amplification methods for the detection and identification of human pathogens is that they do not include an internal amplification control in order to achieve an acceptable level of confidence for the absence of false-negative results. By applying composite primer technology, a single multiple internal amplification control DNA molecule was constructed to detect and quantify the hepatitis B virus, human polyomavirus, Epstein-Barr virus, Toxoplasma gondii and cytomegalovirus using real-time PCR. The multiple internal amplification control contains all forward and reverse primer binding regions targeted in the five distinct duplex PCRs, but with a unique probe hybridization site. Multiple internal amplification control detection sensitivity, assessed by Probit analysis, was 58 copies per PCR, associated with an extremely wide dynamic range (8 log(10) units). Moreover, in testing 614 patient samples, PCR inhibition occurred at a frequency of 0-8.8%. Similar multiple internal amplification controls for quantitative PCR-based assays could be designed to accommodate any infectious profiles in a particular institution as they are easy to make and inexpensive.