Multi-priming sequencing: a DNA sequencing method involving restriction enzyme-digested DNA fragments as primers.

An improved strategy for fluorescence-labeled dideoxy chain termination sequencing involving restriction enzyme-digested DNA fragments as primers, which are prepared from the DNA to be sequenced, is described. By using modified nucleoside triphosphates for strand protection in chain termination reactions, newly synthesized chains were detached from a primer at the regenerated recognition site by means of suitable restriction enzyme digestion. The digests could be analyzed with commercial automated DNA sequencers. Thus, by using restriction DNA fragments (double-stranded) as primers, sequence information was obtained from both "minus" and "plus" single-stranded DNA templates without subcloning. Nor is the synthesis of oligonucleotide primers needed. This method, named "Multi-Priming Sequencing," was proven to be time-saving, economical, and effective compared to conventional methods.     

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.     

New rapid methods for DNA sequencing based in exonuclease III digestion followed by repair synthesis.

We describe improve enzymatic methods for sequencing method for sequencing DNA. They are based on partial digestion of duplex DNA with exonuclease III to produce DNA molecules with 3' ends shortened to varying lengths, followed by repair synthesis to extend and label the 3' ends. After asymmetrical cleavage of the DNA with a restriction enzyme, the labeled products are separated by gel electrophoresis and the sequence read from the autoradiogram. The entire procedures, beginning with unrestricted DNA and followed through gel electrophoresis, takes only one day for sequencing both strands of the DNA molecule. These methods are especially suitable for sequencing DNA cloned in plasmid vectors, and they greatly extend the usefulness of the dideoxynucleotide chain termination method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74, 5463, 1977). Using these methods we have determined the sequence of a 410 base pair fragment which includes the yeast SUP3 tyrosine tRNA gene.     

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.     

Consecutive DNA terminator sequencing by using enzymatically generated primers

An improved strategy for the dideoxy chain termination sequencing of M13 recombinant DNA using enzymatically generated primers is presented. It involves synchronous extension of the universal primer with the Klenow fragment of DNA polymerase I at an average rate of 200 deoxynucleoside triphosphates per minute to the immediate downstream region of a preselected restriction enzyme site. Subsequent cleavage with the restriction enzyme generates the short primers with homogeneous 5′ ends which can be used for further sequencing. The next restriction sites are selected in the newly sequenced regions of DNA by means of a microcomputer. By repeating this primer extension-cleavage-sequencing strategy sequences altogether about 6 kb long from several recombinant single-stranded M13 DNAs have been determined by using twenty restriction enzymes with different specificities.     

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。     

A method for cloning mixtures of long,synthetic oligodeoxynucleotides

A method is described for cloning synthetic oligodeoxynucleotides, which can theoretically be of any length. The method requires only a single oligodeoxynucleotide strand and a vector with two unique restriction sites, one of which is for an enzyme that generates 3′ protruding ends. A mixture of unpurified oligonucleotides containing a wild-type genetic regulatory sequence of the Escherichia coli gnd gene and two mutations of it was cloned into a plasmid carrying a gnd-lacZ protein fusion. Individual cloned oligonucleotides were readily identified by direct DNA sequencing of plasmid templates. The method is rapid, efficient, and has application to gene synthesis and site-directed mutagenesis.     

Subtracted restriction fingerprinting--a tool for bacterial genome typing

Reproducible, discriminative, high-throughput methods are required for the identification of bacterial strains and isolates in a clinical environment. A new molecular typing method for bacteria was developed and tested on Salmonella and E. coli species. The technique is called subtracted restriction fingerprinting and is based on double restriction enzyme digestion of genomic DNA followed by end labeling. The "detection" enzyme produces TTAA overhangs that are filled in with digoxigenated nucleotides for subsequent detection, while the "subtraction" enzyme produces GCGC overhangs that are filled in with biotinylated nucleotides that permit the removal of this subset of fragments with either streptavidin-coated magnetic particles or AffiniTip streptavidin columns. The two restriction enzymes are selected to produce a fragment size profile suitable for a specific analytical system. In this demonstration of the principle of subtracted restriction fingerprinting, analysis of Salmonella enterica subsp. enterica serovar Dublin and E. coli on a 30-cm 1.2% agarose gel revealed up to 50 sharp evenly spaced bands, which were sufficient for the discrimination between various isolates and substrains. The restriction enzyme combinations suitable for the analysis of Salmonella and E. coli are presented. The method requires fewer enzymatic steps than amplified fragment length polymorphism, does not need the specialized DNA preparation essential for pulsed field gel electrophoresis, and has a higher reproducibility than PCR-based methods.     

Identification and characterisation of PmaCI an endonuclease of novel specificity from Pseudomonas maltophila.

We report the use of MonoQ FPLC (Fast Protein Liquid Chromatography) for the rapid purification of a novel Type II restriction endonuclease PmaCI, from Pseudomonas maltophila, which recognises the sequence 5'-CAC decreases GTG-3'. The resulting enzyme is free of other nucleases to a level suitable for its characterisation by multiple-substrate digestion and DNA sequencing techniques. This method appears to be widely applicable and we have used it for the isolation of restriction endonucleases of comparable purity from a range of other organisms. Also described is a rapid method for screening a library of small inserted regions in recombinant M13 molecules for the presence and subsequent screening of restriction sites of interest.     

Solid-Phase Nested Deletion: A New Subcloning-less Method for Generating Nested Deletions

We have developed a new subcloning-less method for generatingnested deletions which we have termed Solid-Phase Nested Deletion.The basic procedure for this method is as follows. The targetDNA fragment is cloned in the multiple cloning site of a cloningvector, pUC or its derivatives, and amplified by PCR using aset of primers, one of which is 5'-biotinylated. The amplifiedDNA is partially digested by a restriction enzyme with a 4-baserecognition sequence. The digested DNA is ligated with a syntheticadapter DNA. Monodiverse beads coupled with streptavidin (Dynabeads^TMM-280 streptavidin) are added to the mixture and the biotinylatedDNA fragments are separated by applying magnetic field. Theunidirectionally deleted DNA fragments are recovered by PCRfrom the magnetic beads, and size-fractionated by agarose gelelectrophoresis. The DNA fragments are amplified by PCR andused for sequencing. We demonstrate the potential of this methodusinga 4878-bp EcoRI fragment of phage DNA.     

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.     

Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants.

A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells. Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized. By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated. The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites. Escherichia coli cells transformed by these plasmids were subject to large-scale analysis. One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence.     

Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants.

A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells. Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized. By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated. The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites. Escherichia coli cells transformed by these plasmids were subject to large-scale analysis. One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence.     

Chemical DNA sequencing in the opposite direction to the dideoxy chain termination method, using a single preparation of M13 single-stranded DNA

A strategy has been developed allowing the use of a single preparation of single-stranded DNA clones for chemical DNA sequencing in the opposite direction to the classical dideoxy chain termination method. Oligonucleotide complementary to the 5'-end of the multipurpose cloning sequence, with the proper restriction enzyme, is used to cleave specifically the molecules to expose a unique 5'-end, upstream to the inserted DNA, for the kinase labeling reaction. No further treatments are necessary before Maxam-Gilbert chemical sequencing reactions.     

High resolution preparative gel electrophoresis of DNA fragments and plasmid DNA using a continuous elution apparatus

An apparatus designed for preparative gel electrophoretic separation of proteins (M. A. Hediger, (1984) Anal. Biochem. 142, 445-454) has been used successfully for separating DNA restriction fragments. The apparatus displayed yields and resolutions that are higher than those obtainable with commercially available devices. The amounts of DNA applied to the column range from a few micrograms to milligram quantities. Restriction DNA fragments very similar in size were isolated in pure form with the apparatus. After ethanol precipitation, these fragments were successfully used for restriction enzyme cleavages, ligation, or chemical sequencing. Furthermore the apparatus provides a convenient method for the large-scale isolation of plasmid DNA. The method requires only 4 h of electrophoresis and therefore greatly reduces the preparation time compared with the conventional equilibrium centrifugation method which requires centrifugation times of up to 60 h. In contrast to the centrifugation method, contaminants such as RNA, proteins, and chromosomal DNA are efficiently removed by this technique.     

Random Tagging Genotyping by Sequencing (rtGBS), an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome

Genotyping by sequencing (GBS) is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al. method: some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS). By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS) method. The study comprises two types of biological replicates: six different kiwifruit plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, stdGBS vs. rtGBS methods, and two independent library amplifications, each sequenced in separate lanes. A statistically significant unbiased distribution of restriction fragment size by rtGBS showed that this method targeted 49% (39,145) of BamH I sites shared with the reference genome, compared to only 14% (11,513) by stdGBS.     

A new method for constructing NotI linking and boundary libraries using a restriction trapper.

We have developed a novel method for constructing NotI linking and boundary libraries using a modified "solid-supported ligation primer" (restriction trapper). The restriction trapper could be used to purify the DNA fragments with a specific restriction enzyme cutting site(s) at their ends. The method uses a ligation and recutting reaction with double-stranded DNA ends of a hairpin-shaped oligolinker which is connected covalently to the surface of the latex beads. Selectivity is based on the specificity of the restriction enzyme for its recognition site, resulting in efficient purification. We applied this technique to the construction of high-quality NotI linking and NotI boundary libraries, which contain almost all the NotI sites of the genome and, in addition, are free of illegitimately ligated clones.     

水稻OsNCED3基因的RNAi载体构建

水稻OsNCED3基因是水稻抗逆过程中重要的基因之一.以水稻中花10号幼苗为材料,提取基因组DNA.设计引物扩增区段cDNA并引入相应的酶切位点,以基因组DNA作为模板,进行RNAi-OsNCED3顺式和反式目的片段的PCR扩增.将PCR产物连接到pMD19-T载体上,经酶切和PCR检测后进行测序.测序结果表明:RNAi-OsNCED3顺式和反式目的片段均已正确的连接到pMD19-T载体上.然后将RNAi-OsNCED3顺式和反式目的片段通过酶切和连接,连接到含有发夹结构的质粒pFGC5941上.PCR及双酶切结果显示,构建的pFGC5941-OsNCED3即RNAi-OsNCED3载体结构完整.