Modeling DNA mutation and recombination for directed evolution experiments

Directed evolution experiments rely on the cyclical application of mutagenesis, screening and amplification in a test tube. They have led to the creation of novel proteins for a wide range of applications. However, directed evolution currently requires an uncertain, typically large, number of labor intensive and expensive experimental cycles before proteins with improved function are identified. This paper introduces predictive models for quantifying the outcome of the experiments aiding in the setup of directed evolution for maximizing the chances of obtaining DNA sequences encoding enzymes with improved activities. Two methods of DNA manipulation are analysed: error-prone PCR and DNA recombination. Error-prone PCR is a DNA replication process that intentionally introduces copying errors by imposing mutagenic reaction conditions. The proposed model calculates the probability of producing a specific nucleotide sequence after a number of PCR cycles. DNA recombination methods rely on the mixing and concatenation of genetic material from a number of parent sequences. This paper focuses on modeling a specific DNA recombination protocol, DNA shuffling. Three aspects of the DNA shuffling procedure are modeled: the fragment size distribution after random fragmentation by DNase I, the assembly of DNA fragments, and the probability of assembling specific sequences or combinations of mutations. Results obtained with the proposed models compare favorably with experimental data.     

Random DNA fragmentation with endonuclease V: application to DNA shuffling

The enzyme endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3′ to uracil sites. I applied the enzyme to random fragmentation of DNA to revise the complex DNA shuffling protocol. The merit of using endonuclease V is that cleavage occurs at random sites and the length of the fragments can easily be adjusted by varying the concentration of dUTP in the polymerase chain reaction. Unlike the conventional method using DNase I, no partial digestion or gel separation of fragments is required. Therefore, labor is dramatically reduced and reproducibility ensured. I applied this method to recombine two truncated green fluorescent protein (GFP) genes and demonstrated successful DNA shuffling by the appearance of the fluorescent full-length GFP genes.     

In vitro rapid evolution of fungal immunomodulatory proteins by DNA family shuffling

Fungal immunomodulatory proteins (FIPs) found in a wide variety of mushrooms hold significant therapeutic potential. Despite much research, the structural determinants for their immunomodulatory functions remain unknown. In this study, a DNA shuffling technique was used to create two shuffled FIP protein libraries: an intrageneric group containing products of shuffling between FIP-glu (FIP gene isolated from Ganoderma lucidum) and FIP-gsi (FIP gene isolated from Ganoderma sinense) genes and an intergeneric group containing the products of shuffling between FIP-glu, FIP-fve (FIP gene isolated from Flammulina velutipes), and FIP-vvo (FIP gene isolated from Volvariella volvacea) genes. The gene shuffling generated 426 and 412 recombinant clones, respectively. Using colony blot analysis, we selected clones that expressed relatively high levels of shuffled gene products recognized by specific polyclonal antibodies. We analyzed the DNA sequences of the selected shuffled genes, and testing of their protein products revealed that they maintained functional abilities to agglutinate blood cells and induce cytokine production by splenocytes from Kunming mice in vitro. Meanwhile, the relationships between protein structure and the hemagglutination activity and between the changed nucleotide sites and expression levels were explored by bioinformatic analysis. These combined analyses identified the nucleotide changes involved in regulating the expression levels and hemagglutination activities of the FIPs. Therefore, we were able to generate recombinant FIPs with improved biological activities and expression levels by using DNA shuffling, a powerful tool for the generation of novel therapeutic proteins and for their structural and functional studies.     

Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase

The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced λ DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 × 10⁻⁶) than Taq DNA polymerase (11.98 × 10⁻⁶). Uniquely, Neq DNA polymerase also amplified λ DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two- to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.     

Solution structure and dynamics of DNA duplexes containing the universal base analogues 5-nitroindole and 5-nitroindole 3-carboxamide

Universal bases hybridize with all other natural DNA or RNA bases, and have applications in PCR and sequencing. We have analysed by nuclear magnetic resonance spectroscopy the structure and dynamics of three DNA oligonucleotides containing the universal base analogues 5-nitroindole and 5-nitroindole-3-carboxamide. In all systems studied, both the 5-nitroindole nucleotide and the opposing nucleotide adopt a standard anti conformation and are fully stacked within the DNA duplex. The 5-nitroindole bases do not base pair with the nucleotide opposite them, but intercalate between this base and an adjacent Watson–Crick pair. In spite of their smooth accommodation within the DNA double-helix, the 5-nitroindole-containing duplexes exist as a dynamic mixture of two different stacking configurations exchanging fast on the chemical shift timescale. These configurations depend on the relative intercalating positions of the universal base and the opposing base, and their exchange implies nucleotide opening motions on the millisecond time range. The structure of these nitroindole-containing duplexes explains the mechanism by which these artificial moieties behave as universal bases.     

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage.The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2 h (fresh) or 5 days at 4 °C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.     

Genomic distribution of repeated and single copy DNA sequences in Penicillium funiculosum

Study of the reassociation kinetics of Penicillium funiculosum DNA (fragment size > 20 kb) revealed an interspersion of repeated and single-copy DNA sequences. The fragment length of interspersed single-copy DNA was estimated to be more than 9 kb from a curve relating the fraction of DNA binding to hydroxyapatite as a function of DNA fragment length. The length of interspersed short and long repeated nucleotide sequences was determined to be 0.55 kb and > 20 kb, respectively, by agarose gel electrophoresis. The genomic organization of P. funiculosum is thus significantly different from that in other fungi.     

The Efficiency of DNA Labeling with Near-Infrared Fluorescent Dyes

The efficiency of DNA labeling was assessed for 2'-deoxyuridine 5'-triphosphate (dUTP) derivatives containing the Cy7 cyanine dye as a fluorophore. Two fluorescent Cy7-labeled dUTP analogs differed in the chemical structure of the linker between the fluorophore and nucleotide moieties. The efficiency of the polymerase chain reaction (PCR) and inhibition with modified nucleotides were estimated by real-time PCR. The efficiency of labeled nucleotide incorporation in PCR products was measured by quantitative electrophoresis. The efficiency of target DNA labeling was evaluated by binding the fluorescently labeled PCR products to a microarray of oligonucleotide probes immobilized in hydrogel drops (a biochip). The near-infrared hybridization signal was detected by digital luminescence microscopy. An increase in linker length was found to provide more efficient incorporation of the labeled nucleotide. Both of the compounds provided high sensitivity and high specificity of DNA testing via allele-specific hybridization on a biochip.

     

Simultaneous assessment of average fragment size and amount in minute samples of degraded DNA

There is currently no method allowing routine characterization of minute amounts of degraded DNA samples such as those encountered in forensic science, archived tissues, ancient DNA, extracellular or stool DNA, and processed food. Here we describe and directly validate such a method based, on the one hand, on a generalized DNA random fragmentation model and, on the other, on two quantitative polymerase chain reaction (PCR) experiments using two different target sizes. The model also makes it possible to determine the minimum sample amount, the minimum mass average fragment size, and the maximum degradation time necessary to obtain a positive PCR.     

DNA repair of 8-oxo-7,8-dihydroguanine lesions in Porphyromonas gingivalis

The persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. DNA damage is a major consequence of oxidative stress. Unlike the case for other organisms, our previous report suggests a role for a non-base excision repair mechanism for the removal of 8-oxo-7,8-dihydroguanine (8-oxo-G) in P. gingivalis. Because the uvrB gene is known to be important in nucleotide excision repair, the role of this gene in the repair of oxidative stress-induced DNA damage was investigated. A 3.1-kb fragment containing the uvrB gene was PCR amplified from the chromosomal DNA of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic cassette and used to create a uvrB-deficient mutant by allelic exchange. When plated on brucella blood agar, the mutant strain, designated P. gingivalis FLL144, was similar in black pigmentation and beta-hemolysis to the parent strain. In addition, P. gingivalis FLL144 demonstrated no significant difference in growth rate, proteolytic activity, or sensitivity to hydrogen peroxide from that of the parent strain. However, in contrast to the wild type, P. gingivalis FLL144 was significantly sensitive to UV irradiation. The enzymatic removal of 8-oxo-G from duplex DNA was unaffected by the inactivation of the uvrB gene. DNA affinity fractionation identified unique proteins that preferentially bound to the oligonucleotide fragment carrying the 8-oxo-G lesion. Collectively, these results suggest that the repair of oxidative stress-induced DNA damage involving 8-oxo-G may occur by a still undescribed mechanism in P. gingivalis.     

Purification and Amplification of DNA from Penaeus monodon-Type Baculovirus (MBV)

The purpose of this paper was to purify and amplify the DNA fragment of Penaeus monodon -type baculovirus (MBV). Using 30-50% caesium chloride gradients, MBV virions and occlusion bodies with density parameters of 1.28-1.29 and 1.32-1.33 g/ml, respectively, were purified. Two oligonucleotide primers have been successfully designed and utilized for the amplification of a DNA fragment of MBV. After 35 amplification cycles of the MBV DNA fragment, a large amount of amplified product with an approximate molecular weight of 600 bp was obtained. This is the first successfully published work on the amplification of MBV using the polymerase chain reaction (PCR). Using the same primers, DNA extracted from MBV noninfected P. monodon, P. japonicus, and P. orientalis had a negative PCR response. However, a positive PCR response was obtained from DNA extracted from MBV-infected postlarval P. monodon. DIG-dot blot hybridization technique using PCR product obtained from the present study as a probe further confirmed that the product is originated from a portion of MBV polyhedrin gene. It is also suggested that PCR product may be beneficial for an accurate and early diagnosis of MBV infection in larval shrimp.     

DNA shuffling method for generating highly recombined genes and evolved enzymes

We introduce a method of in vitro recombination or "DNA shuffling" to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.0 crossovers per gene, a several-fold higher level of recombination than observed for other methods. We also observed an unprecedented four crossovers per gene in regions of 10 or fewer bases of sequence identity. These properties allow generation of chimeras unavailable by other methods. We detected no unshuffled parental clones or duplicated "sibling" chimeras, and relatively few inactive clones. We demonstrated the method by molecular breeding of a monooxygenase for increased rate and extent of biodesulfurization on complex substrates, as well as for 20-fold faster conversion of a nonnatural substrate. This method represents a conceptually distinct and improved alternative to sexual PCR for gene family shuffling.     

Enhancement of a hybridization analysis efficiency by the controlled DNA fragmentation

The influence of a DNA amplicon fragmentation on the efficiency of detecting a specific sequence by heterophase hybridization analysis was investigated. The nucleotide sequence was detected colorimetrically after biotinylation of an oligonucleotide probe immobilized on a solid carrier via limited elongation in complex with the sample DNA with the use of Taq polymerase. Two simple and reproducible techniques of amplicon fragmentation were proposed. The techniques are based on the introduction of apurinic/apyrimidinic sites in DNA and their subsequent thermal degradation. DNA was depurinated by a mild acidic treatment. Apyrimidinic sites were generated by treating a DNA fragment containing dUMP in place of some dTMP in various proportions with uracil-DNA glycosylase (UDG). The DNA sample treated by either method proved to be suitable for hybridization analysis with Taq polymerase without additional purification. The efficiency of hybridization analysis was higher with the fragmented than with the native DNA amplicon. DNA fragmentation makes it possible to use bridged oligonucleotides, having a lower hybridization ability, as highly selective hybridization probes.     

Analysis of the enterohemorrhagic Escherichia coli O157 DNA region containing lambdoid phage gene p and Shiga-like toxin structural genes.

In this study, we determined the nucleotide sequence of the p gene contained within a 5-kb EcoRI restriction fragment cloned from Shiga-like toxin II (SLT-II)-converting phage 933W of Escherichia coli O157:H7 strain EDL933. The p gene was 702 bp long and had 95.3% sequence similarity to the p gene of phage lambda. Multiple hybridization patterns were obtained when genomic DNA fragments were hybridized with both p and slt-I, slt-II, or slt-IIc sequences. All O157 isolates also possessed an analog of lambda gene p which was not linked with either slt-I or slt-II. Restriction fragment length polymorphism comparisons of clinical O157 isolates and derivates undergoing genotype turnover during infection were made, and loss of large DNA fragments that hybridized with slt-II and p sequences was observed. To further analyze the DNA region containing the p and slt genes, we amplified fragments by using a PCR with one primer complementary to p and the other complementary to either the slt-I or the slt-II gene. PCR analysis with enterohemorrhagic E. coli O157 and non-O157 strains yielded PCR products that varied in size between 5.1 and 7.8 kb. These results suggest that even within O157 isolates, the genomes of SLT-converting phages differ. The methods described here may assist in further investigation of SLT-encoding phages and their role in the epidemiology of infection with enterohemorrhagic E. coli.     

应用RACE法克隆鸽恒定链基因的研究

为比较禽类恒定链的结构和功能, 应用RACE (Rapid Amplification of cDNA Ends) 技术首次克隆并鉴定了鸽恒定链基因。首先用一对含高度保守的DNA片段的简并引物, 从鸽脾细胞RNA扩增部分恒定链片段, 接着测序并设计新引物分别从5′和3′RACE扩增延长该片段。最后根据全基因的序列设计上、下游引物,获得大小为1 050 bp的全长cDNA。比较核苷酸序列, 鸽与鸡的Ii链同源性达到82.8％, 而与人等其它动物的同源性则在52.0％以上; 其中633 bp的开放阅读框编码211个氨基酸残基的前体蛋白。推导和分析氨基酸序列表明, 分子结构与鸡恒定链相似, 其中有些氨基酸残基表现出较高的保守性。     

Mapping of peroxyl radical induced damage on genomic DNA

We have examined the DNA damage produced by reaction of peroxyl radicals with human fibroblast DNA. DNA damage consisted of both strand breaks and base modifications. The extent of strand breaks and base modifications induced as a function of peroxyl radical concentration was determined by quantitation of fragment size distributions using denaturing glyoxal-agarose gel electrophoresis. Both strand breaks and base modifications increased in a log linear fashion with respect to peroxyl radical concentration. Oxidative base modifications were observed to occur to a greater extent than strand breaks at every concentration measured. The sequence-specific distribution of peroxyl radical induced base damage was mapped for 803 nucleotide positions using the method of ligation mediated PCR. A total of 87% of all guanine positions in the examined sequences was found to be significantly oxidized. The order of reactivity of DNA bases toward oxidation by peroxyl radicals was found to be G > C > T. Adenine is essentially unreactive. The yield of oxidative base modifications at guanines and cytosines by peroxyl radicals depends on the exact specification of 5' and 3' flanking bases in a polarity dependent manner. Every guanine in the 5'XGC3' motif was found to be oxidized, where X is any 5' neighbor. In contrast, 5' and 3' purine flanks drastically reduced the extent of peroxyl radical G oxidation. The pattern of base modification and the influence of nearest neighbors differs substantially from that previously reported for hydrogen peroxide damage mediated by low valent transition metal ions for the identical DNA sequences.     

分段DNA shuffling: 一种大分子海藻糖合酶有效的定向进化方法

[目的]红色亚栖热菌(Meiothermus ruber)海藻糖合酶(Trehalose synthase,M-TreS)将麦芽糖转化生成海藻糖只需一步反应,且具有很好的热稳定性及pH耐受性,是潜在的工业生产海藻糖的酶源.为了提高该酶的性能,有必要对其进行定向进化.[方法]M-TreS基因(M-treS)大小为2 889bp.该蛋白质分子本身具有很大的进化空间,但是却不宜进行全长基因Shuffling.分段DNA shuffling是为大分子蛋白质(基因≥2 000 bp)的进化而设计的一种方法.该方法分为三步:(1)用两对引物分别扩增目的基因的上游片段和下游片段;(2)上下游片段各自进行Shuffling; (3)利用重叠延伸PCR连接上下游突变群,建立完整基因的突变文库.[结果]结合易错PCR,通过该方法经一轮进化获得一株酶活力是野生型1.6倍、催化效率是野生型2倍的突变株.序列分析表明,该突变株共有6个位点发生了氨基酸的替代,其中一个来自易错突变,2个来自同源重组,3个为随机突变.[结论]分段DNA shuffling是进化大分子蛋白质的有效方法.     

Inclusion of biotinylated analogs of dUTP and dCTP in DNA by DNA-polymerases. Cloning DNA fragments, containing biotinylated deoxyribouridine in E. coli

The synthesis of a biotinylated derivative of dCTP, viz. N4-[(N-biotinyl)-4-amino-butoxyl]-2'-deoxycytidine 5'-triphosphate (I), is described. DNA polymerase I (Klenow fragment) incorporates (I) in DNA chains instead of thymidine, although with a lower efficiency than previously described biotinylated dUTP derivative (II), whereas highly purified DNA polymerase alpha from human placenta uses as substrate derivative (II) but not (I). A DNA fragment bearing biotin residues in one of strands was synthesized with the use of DNA polymerase alpha and dUTP derivative (II); its cloning in the plasmid vector pBR322 revealed that the DNA nucleotide sequence remained intact.     

Nucleotide sequence of BamHI family satellite DNA and its unit length polymorphism in bluegill sunfish Lepomis macrochirus

We have isolated and sequenced a member of tandem repetitive DNA containing BamHI site (BamHI family satellite DNA) from bluegill sunfish Lepomis macrochirus. PCR amplification with specific primers was performed to define the size of unit length repeat of the BamHI family satellite DNA, revealing that there were two distinct size of DNA fragments (0.9 kb and 1.3 kb) in the PCR products. The longer fragment (1.3 kb) consisted of internal sub-duplication of shorter fragment (0.9 kb). We have compared the size of PCR products among four fish populations, and found that both fragments co-existed in one population whereas the longer fragment was dominant in other three populations. The results may reflect ongoing homogenization of satellite DNA type over a short evolutionary time scale.     

Correcting errors in synthetic DNA through consensus shuffling

Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from ~60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of ~1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences.