A versatile and simplified non-random strategy for nucleotide sequencing

We describe a versatile and simplified non-random strategy for nucleotide sequencing. This procedure avoids exposure of the vector DNA to endo- or exonucleases during construction of the subfragment library. The advantages resulting from this strategy are: (1) minimal manipulation in construction of the subfragment library, (2) no need for compatible restriction sites, (3) no insert size limitation, (4) can be used with both chemical and enzymatic sequencing methods. Hence, the procedure provides simplicity, universality and versatility for non-random nucleotide sequencing. We demonstrate the usefulness of this procedure by obtaining the nucleotide sequence of a goat 3.7-kb EcoRI fragment, which is 5' of the epsilon globin gene.     

A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments

The strategy of shotgun cloning with M13 is based on obtaining random fragments used for the rapid accumulation of sequence data. A strategy, however, is sometimes needed for obtaining subcloned sequences preferentially out of a mixture of fragments. Shotgun sequencing experiments have shown that not all DNA fragments are obtained with the same frequency and that the redundant information increases during the last third of a sequencing project. In addition, experiments have shown that particular fragments are obtained more frequently in one orientation, allowing the use of only one of the two DNA strands as a template for M13 shotgun sequencing. Two new M13 vectors, M13mp8 and M13mp9, have been constructed that permit the cloning of the same restriction fragment in both possible orientations. Consequently, each of the two strands becomes a (+) strand in a pair of vectors. The fragments to be cloned are cleaved with two restriction enzymes to produce a fragment with two different ends. The insertion of such a fragment into the vector can occur only in one orientation. Since M13mp8 and M13mp9 have their array of cloning sites in an antiparallel order, either orientation for inserting a double-digest fragment can be selected by the choice of the vector.     

Genes 55, alpha gt, 47 and 46 of bacteriophage T4: the genomic organization as deduced by sequence analysis

The nucleotide sequence of T4 genes 55, alpha gt, 47 and 46 was determined by a combination of 'classical' procedures and a shotgun approach. Small DNA fragments generated by frequent cleavage with restriction enzymes or by sonication of restriction fragments were cloned in phage M13 vectors and sequenced by the dideoxy method. The positions of the genes were determined by marker rescue between the corresponding T4 amber mutants and the cloned T4 DNA fragments used in the sequencing experiments. The sequence gives an insight into the organization of this 7.1-kb early region of the T4 genome and shows that genetically 'silent' portions within this region are not void of genetic information.     

Bidirectional sequencing of supercoiled plasmid DNA

In this paper we show that restriction DNA fragments can prime DNA synthesis of a homologous supercoiled plasmid DNA. Using the dideoxyribonucleotide chain terminator method, newly synthesized truncated chains can be detached from the primers by restriction enzyme digestion. Therefore, by choosing DNA fragments flanked by two different restriction enzymes sites, nucleotide sequence information can be simultaneously obtained on both regions of the DNA surrounding the restriction fragment. The advantage of this sequencing approach over current methods is that no prior knowledge of the primary sequence is needed to find the nucleotide sequence of a given DNA fragment. Thus, synthetic primers are not required and internal sequences of a given clone can be easily accessed without the need of fragmenting the original construct. The method has been used with rapid plasmid preparations, thus considerable time and effort can be saved in the gathering of nucleotide sequence information.     

Extending the diversity of cytochrome P450 enzymes by DNA family shuffling

The cytochrome P450 enzymes involved in xenobiotic metabolism are an excellent starting point for the directed evolution of novel biocatalysts due to their wide substrate specificity. A shuffled library of three highly homologous mammalian genes (for P450 2C9, P450 2C11 and P450 2C19) was constructed by applying a modified DNA family shuffling procedure. The modifications made to the traditional DNA shuffling protocols involved non-random digestion via the use of different combinations of restriction enzymes (REs) followed by isolation of fragments under 300 bp by size-selective filtration. Shuffled cytochrome P450 mutants were co-expressed in Escherichia coli with their redox partner, NADPH-cytochrome P450 reductase (NPR). We report here how non-random fragmentation may help in chimeragenesis within the areas of low sequence similarity such as substrate recognition sites (SRSs) that are generally underrepresented in recombination using the random fragmentation process. Size-selective filtration was used to limit recovery of incompletely digested fragments and consequently minimize the chances for contamination of the shuffled library with parental forms. No parental forms could be detected in the shuffled library using restriction fragment length polymorphism (RFLP) analysis, suggesting the library was free of parental contamination. Sequencing of randomly selected mutants demonstrated a high level of chimeragenesis with on average of 8.0+/-2.2 crossovers and a low level of mutagenesis with 5.2+/-2.8 spontaneous mutations per approximately 1.5 kbp of the full-length P450 sequence. The proportion of properly folded protein as indicated by the observation of characteristic Fe(II).CO vs. Fe(II) difference spectra was 15% (4/27) of analysed mutants. Screening of the shuffled library for indole oxidation revealed four clones with similar or higher levels of indigo pigment production to those of the parental P450s and two clones with elevated P450 expression. In this paper we present a method for the effective family shuffling of cytochrome P450 enzymes, applicable to the creation of mutant libraries with expanded metabolic diversity and with a significant proportion of functional clones.     

A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing

A set of plasmid vectors was developed which allows fast sequencing by the chemical degradation method. These high-copy-number vectors are derivatives of the plasmid pUC8 containing different multiple-purpose cloning sites flanked by unique recognition sequences for the restriction enzymes BstEII, Tth111I and Eco81I as sites for end-labelling DNA. Due to their partially asymmetric recognition sequences, each of these three restriction sites can be singly end-labelled by a filling-in reaction with selected nucleotides. This allows easy single end-labelling of any cloned DNA fragment for sequencing by the chemical degradation method without any isolation and purification step after the labelling reaction. In addition, the nucleotide sequence of the complementary strand from the same end can be determined by the dideoxy chain termination procedure using the universal M13 primers. In most of the new vectors, the reading frame of the lacZ' gene is retained, allowing identification of cloned fragments.     

A new methodology combining PCR, cloning, and sequencing of clones discriminated by RFLP for the study of microbial populations: application to an UASB reactor sample

This work describes a methodology combining DNA extraction, polymerase chain reaction amplification with primers targeting 16S ribosomal RNA genes, cloning, and sequencing of clones previously analyzed by restriction fragment length polymorphism (RFLP), which can be applied to study the microbial diversity in a given habitat. The methodology allows the minimization of the sequencing effort, which is particularly relevant when analyzing large numbers of clones. The methodology does not require particularly skilled personnel and can easily be adaptable to the molecular characterization of virtually any particular microbial population, provided that both adequate primers and suitable restriction enzymes for RFLP analysis of the clone library have been chosen. An example of application is presented, in which a sample taken from a continuously operating upflow anaerobic sludge blanket reactor was analyzed. RFLP analysis of the initial 162 clones with HaeIII allowed the identification of only 28 distinct profiles. As expected, identical RFLP profiles corresponded to identical nucleotide sequences.     

A systemic DNA sequencing strategy

A systematic DNA sequencing strategy is presented. Instead of the sequencing of randomly selected DNA fragments (the “shotgun” method), the nucleotide order is progressively determined along a DNA chain using the dideoxynucleotide termination sequencing system and the single-stranded bacteriophage vector M13 derivatives. The length of DNA along which the sequence data can be progressively read appears to be limited only by the insertion capacity of the vector. As an example of this strategy a recombinant replicative form with a 2327 nucleotide long HindIII fragment from the restriction enzyme digest of bacteriophage λ DNA was prepared. The replicative form of the recombinant was partially digested with DNAase I in the presence of Mn²⁺. As the replication origin of the phage vector was located near one end of the inserted DNA and the priming site of the vector at the other, the breaks outside the inserted DNA either destroyed the phage or removed the priming site. With the use of a unique restriction site close to the priming site, the breaks within the inserted DNA gave rise to a recombinant mixture with the inserted DNA fixed at one end and sequentially shortened at the other. Using the ddT reaction screening procedure, 11 recombinants were identified in which the inserted DNA varied in length by about 200 nucleotides. Sequencing these recombinants by the dideoxynucleotide sequencing system covered the whole 2327 nucleotides of the HindIII DNA fragment. The average number of nucleotides read from a gel was 210, which fell into the most readable region of a sequencing gel: the overlapping regions between two gels were of 33 to 48 nucleotides.     

Sequence heterogeneity of murine complementary DNA clones related to the C4 and C4-Slp isoforms of the fourth complement component

Two classes of mRNA encoding the murine C4 protein were identified by sequence analysis of clones isolated from a liver complementary DNA library. The divergence found within a 357 base pair sequence available for comparison is limited to five nucleotide replacements located in the region corresponding to the carboxy-terminal end of the C4d peptide fragment. One of the nucleotide substitutions influences the presence of a site for the Hind III restriction endonuclease. That this restriction site indeed discriminates the two non-allelic genes encoding the mouse C4 and C4-Slp isoforms has been demonstrated by Southern blot analysis and nucleotide sequencing at the genomic level. Circumstantial evidence supports the identification of the gene lacking the Hind III site in the region corresponding to the carboxy-terminal end of the C4d fragment as the one encoding the C4-Slp isotype.     

甲基营养菌MP681基因组测序文库的构建

目的:建立甲基营养菌MP681基因组文库,用于鸟枪法测序。方法:提取MP681基因组DNA,经超声随机片段化及T4 DNA聚合酶末端修平处理后,与经SmaⅠ酶切、小牛肠碱性磷酸酶(CIP)去磷酸化处理的pUC19载体连接,电击转化大肠杆菌DH5α感受态,并通过末端双向测序对文库质量进行评价。结果:分别构建了2～4 kb和4～6 kb基因组文库,电泳结果显示插入片段长度与预期符合,文库库容均在10万以上。结论:构建了插入片段大小和库容符合要求的甲基营养菌MP681全基因组鸟枪法2～4 kb、4～6 kb测序文库。     

The restriction scaffold problem.

Most shotgun sequencing projects undergo a long and costly phase of finishing, in which a partial assembly forms several contigs whose order, orientation, and relative distance is unknown. We propose here a new technique that supplements the shotgun assembly data by experimentally simple and commonly used complete restriction digests of the target. By computationally combining information from the contig sequences and the fragment sizes measured for several different enzymes, we seek to form a "scaffold" on which the contigs will be placed in their correct orientation, order, and distance. We give a heuristic search algorithm for solving the problem and report on promising preliminary simulation results. The key to the success of the search scheme is the very rapid solution of two time-critical subproblems that are solved to optimality in linear time. Our simulations indicate that with noise levels of some 3% relative error in measuring fragment sizes, using six enzymes, most datasets of 13 contigs spanning 300kb can be correctly ordered, and the remaining ones have most of their pairs of neighboring contigs correct. Hence, the technique has a potential to provide real help to finishing. Even without closing all gaps, the ability to order and orient the contigs correctly makes the partial assembly both more accessible and more useful for biologists.     

An improved strategy for generating a family of unidirectional deletions on large DNA fragments

A modification of the Barnes "kilo-sequencing" method is described. The procedure presented here makes it possible to obtain a series of nested deletions on large DNA fragments in only two days. It applies to double-stranded DNA, and thus can be used with plasmids as well as the M13mp series of bacteriophages. The main improvements are the use of a second restriction enzyme, which makes it possible to begin the deletions at any site on the DNA fragment, and the use of mung bean nuclease for trimming the DNA edges so that any restriction enzyme can be used. This method, using a pUC vector and sequencing on double-stranded DNA, would make it possible to read a DNA nucleotide sequence on both strands starting with only one construction.     

Electron microscopy and restriction enzyme mapping reveal additional intervening sequences in the chicken ovalbumin split gene

The Eco RI fragment “b” of chicken DNA (Breathnach, Mandel and Chambon, 1977), which contains the sequences coding for the 5′ quarter of ovalbumin mRNA (ov mRNA), has been isolated by molecular cloning using a “shotgun” approach. Electron microscopy and restriction enzyme analysis have revealed that the sequences coding for the 5′ quarter (～500 nucleotides) of ov mRNA are split into four regions separated by three intervening sequences. The cloning procedure seems to be reliable, since the restriction enzyme pattern of the cloned Eco RI fragment “b” is similar to that of the corresponding chromosomal DNA fragment. There is no evidence supporting the existence of a 150–200 nucleotide long sequence at the 5′ end of the ov mRNA similar to the “leader” sequences found at the 5′ end of some adenovirus and SV40 mRNAs.     

Mitochondrial DNA analysis of Scottish populations of Arctic charr, Salvelinus alpinus (L.)

Mitochondrial DNA analysis of a total of 82 individuals from seven Scottish populations of Arctic charr ( Salvelinus alpinus L.) using restriction fragment length polymorphism and direct nucleotide sequencing of the cytochrome b gene revealed little variation within or between populations. One of 19 restriction enzymes showed a polymorphism that was present in three of the seven populations. Only one nucleotide substitution was detected in a 288 base pair segment of the cytochrome b gene examined in 26 fish. Approximately 4% of the mitochondrial genome was surveyed with these complementary procedures.     

Ovine mitochondrial DNA: restriction enzyme analysis, mapping and sequencing data

Restriction endonuclease fragment patterns of mitochondrial DNA (mtDNA) in sheep were analysed with 11 enzymes. Four breeds (Merinolandschaf, Rhoenschaf, Schwarzkoepfiges Fleischschaf and Skudde) of domestic sheep and European Mouflon were examined. A restriction map with 28 cleavage sites of seven enzymes was established. KpnI and PstI do not cut ovine mtDNA. Two EcoRI fragments of Merinolandschaf, Rhoenschaf and Mouflon each were cloned and partially sequenced. Intraspecific nucleotide sequence differences within 1.101 kb ranged from 0.09 to 0.27%. Hybridization analysis with a fragment of porcine mtDNA along with sequencing data from cloned fragments was used for orientation of the restriction map along the bovine sequence. Ovine mtDNA sequences encompassing parts of the Cyt.b-, ND5-, CoIII- and ATPase6 genes were compared with the corresponding sequences of the bovine mtDNA.     

鳞翅目基因组IIB型内切酶位点的统计分析

IIB型限制内切酶能够识别并切割特异酶切位点两端特定距离的DNA,形成粘性末端的30 bp左右的等长DNA片段。利用其特性与限制性酶切位点关联测序技术(RAD)相结合发展出2b-RAD简化基因组测序技术,应用于遗传图谱构建、种群遗传结构分析、性状定位以及细菌分型等多种研究领域。构建2b-RAD测序文库之前,需要对基因组中的IIB型限制内切酶位点进行预测与统计分析,制定有效的测序文库构建方案。本文利用Python语言构建分析基因组中IIB型限制内切酶位点的流程,预测并统计6个鳞翅目代表物种基因组含有的8个商业化IIB型限制内切酶的酶切位点,比较了各个基因组与IIB型限制内切酶之间含有的酶切位点总量、重复序列数量以及酶切间隔长度的关系,为在昆虫基因组中进一步试行2b-RAD研究提供了参考。     

Specific single-cell isolation and genomic amplification of uncultured microorganisms

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.     

A phagemid vector library for cloning DNA with four-nucleotide 5' or 3' overhangs

A phagemid vector library for cloning DNA with four nucleotide 5' or 3' overhangs has been constructed. This library is based on the pT7T3 vector (Pharmacia) which is a modification of the phagemid pTZ18U vector. We have chosen pT7T3 as the parent vector because it can be used for Sanger's dideoxy sequencing and for the generation of RNA probes with either the T7 or T3 promoter. Each member of the cloning vector series pBM has recognition sites for both of the restriction enzymes BspM1 and BstX1 in addition to the basic multiple cloning sites. BspM1 recognizes the sequence 5'...ACCTGC NNNN/NNNN...3' whereas BstX1 recognizes the sequence 5'...CCAN NNNN/NTGG...3'. Thus these two sites can be overlapped, so that only 256 vectors (instead of 512 vectors) need be constructed to cover all the theoretical possible combinations of sites which give complementary cohesive ends for cloning DNA with four nucleotide 5' or 3' overhangs. This vector library can be used for amplification cloning of DNA in a tandem array by choosing appropriate vectors which have nonpalindromic sequences. We have obtained approximately 200 members of the 256 possible clones and have organized the vectors using a MacIntosh HyperCard program for easy retrieval.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.