T载体介导的基于attL核心区LR反应的简化快速Gateway克隆系统

Gateway克隆技术已得到广泛的应用。该技术先通过BP反应将目标片段连到带有完整attL特异识别位点的入门载体,然后与终载体通过LR反应得到表达载体。Gateway克隆方法与传统的酶切连接方法相比有快速简单等优点。但是,BP和LR酶都非常昂贵。本研究首先对3个常用Gateway载体的atts特异位点序列比对发现,attL序列核心交换位点“core attL”的21~22 bp长的碱基是保守和必要的。由此,设计含有core-attL序列的引物,通过PCR克隆得到DNA片段并连入pMD18-T载体,然后进行LR反应,可成功得到目标表达载体,并在保守的位点上正确重组。本研究还对其中一个带有绿色荧光蛋白基因的表达载体转化至烟草,能够正常表达该蛋白质。结果表明,通过将含有attL核心位点基因片段连接到pMD18-T载体上,可以省略BP反应而将目标片段连接到终载体上,节约了反应时间和成本。     

Total synthesis of multi-kilobase DNA sequences from oligonucleotides

A method for synthesizing DNA from 40-mer oligonucleotides, which we used to generate a 32-kb DNA fragment, is explained. DNA sequences are synthesized as approximately 500 bp fragments (synthons) in a two-step PCR reaction and cloned using ligation-independent cloning (LIC). Synthons are then assembled into longer full-length sequences in a stepwise manner. By initially synthesizing smaller fragments (synthons), the number of clones sequenced is low compared with synthesizing complete multi-kilobase DNA sequences in a single step. LIC eliminates the need for purification of fragments before cloning, making the process amenable to high-throughput operation and automation. Type IIs restriction enzymes allow seamless assembly of synthons without placing restrictions on the sequence being synthesized. Synthetic fragments are assembled in pairs to generate the final construct using vectors that allow selection of desired clones with two unique antibiotic resistance markers, and this eliminates the need for purification of fragments after digestion with restriction endonucleases.     

Use of the polymerase chain reaction for the differential cross screening of libraries cloned into phage-lambda-based vectors

We describe a simple and rapid method that can be used to identify sequences present in any two DNA libraries (either genomic or cDNA), provided only that the libraries are in different vectors with different cloning sites. This procedure makes use of the polymerase chain reaction (PCR) to amplify the inserts of one library. The product of the PCR reaction is then used to screen a second library to identify sequences which are common to both. We illustrate the use of this method for the systematic isolation of human X-chromosome-linked genomic clones that harbor sequences expressed in human chorioretinal tissue.     

基于PIPE现象的质粒克隆位点序列设计与功能评价

分子克隆是现代生物学研究的核心技术之一,是基因工程、蛋白质工程中的重要手段。为提高分子克隆实验的操作效率,本研究设计并合成基于聚合酶引物不完全延伸（polymerase incomplete primer extension,PIPE）现象的质粒克隆位点序列。并以此为基础统一相关引物的设计方案,避免传统酶切--连接法中需针对不同载体MCS序列设计不同引物的缺点。该方案利用13 bp定长接头序列,在同一体系中使用2对引物、2种线性化模板同时扩增载体和插入片段,通过20个循环,在1次PCR过程中即合成可供转化使用的带缺口质粒产物。在NEB Q5酶系统中,利用此法将3种荧光素酶序列插入pET-15b及pET-21b(+)载体,均获得成功。且利用商品化感受态细胞（转化效率 > 5×108 cfu/μg）转化后所获得转化子数量均在300个以上,其中含插入片段的阳性克隆比例可达85%以上。基于本方案的设计及作用原理,可将其应用于10 kb以内载体和插入片段的快速重组。且具有通用性强、耗时少、阳性克隆得率高和成本低等优点,是传统DNA重组方法的有益补充,可作为各实验室的常规分子克隆手段之一。     

Targeted gene walking polymerase chain reaction.

We describe a modification of a polymerase chain reaction method called 'targeted gene walking' that can be used for the amplification of unknown DNA sequences adjacent to a short stretch of known sequence by using the combination of a single, targeted sequence specific PCR primer with a second, nonspecific 'walking' primer. This technique can replace conventional cloning and screening methods with a single step PCR protocol to greatly expedite the isolation of sequences either upstream or downstream from a known sequence. A number of potential applications are discussed, including its utility as an alternative to cloning and screening for new genes or cDNAs, as a method for searching for polymorphic sites, restriction endonuclease or regulatory regions, and its adaptation to rapidly sequence DNA of lengthy unknown regions that are contiguous to known genes.     

A protocol for the construction of microsatellite enriched genomic library

An improved protocol for constructing microsatellite-enriched libraries was developed. The procedure depends on digesting genomic DNA with a restriction enzyme that generates blunt-ends, and on ligating linkers that, when dimerized, create a restriction site for a different blunt-end producing restriction enzyme. Efficient ligation of linkers to the genomic DNA fragments is achieved by including restriction enzymes in the ligation reaction that eliminate unwanted ligation products. After ligation, the reaction mixture is subjected to subtractive hybridization without purification. DNA fragments containing microsatellites are captured by biotin-labeled oligonucleotide repeats and recovered using streptavidin-coated beads. The recovered fragments are amplified by PCR using the linker sequence as primer, and cloned directly into a plasmid vector. The linker has the sequence GTTT on the 5′ end, which promotes efficient adenylation of the 3′ ends of the PCR products. Consequently, the amplified fragments could be cloned into vectors without purification. This procedure enables efficient enrichment and cloning of microsatellite sequences, resulting in a library with a low level of redundancy.     

cDNA selection with YACs

Identification of expressed sequence tags (ESTs) in large genomic segments is an important step in positional cloning and genomic mapping studies. A simple and efficient polymerase chain reaction (PCR)-based approach is described here to identify coding sequences in large genomic fragments of DNA cloned in vectors such as yeast artificial chromosome (YAC) vectors. The method is based on blocking of sequences such as repetitive and GC rich sequences in the genomic DNA immobilized on nylon paper discs prior to hybridization of the discs to cDNA library, and recovery of the selected cDNAs by the PCR. Single or multiple cDNA libraries can be used in the selection procedure. The procedure has been used successfully also with total yeast DNA containing a YAC.     

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.     

DNA cloning without restriction enzyme and ligase

One common problem in using the traditional DNA cloning procedure is that suitable natural restriction sites are often unavailable for a given task. Creating new restriction sites is often time consuming. Here, I describe a simple technique of producing "customized cohesive ends" by a combination of PCR primer design and lambda exonuclease digestion. These complementary cohesive ends can form hybrids to link two sequences. Because the overhangs created by lambda exonuclease are slightly longer than the complementary sequence, after hybrid formation, a stretch of single-strand gap remains, which then is repaired by Klenow (3'-->5' exo-) enzyme. The repair process also stabilizes the linkage. Because of the independence from natural or artificial restriction sites, this method allows rapid and precise insertion of one DNA fragment into another at virtually any position. It also simplifies the planning of a cloning strategy, increases recombinant frequency and is suitable for automation.     

Seamless gene engineering using RNA- and DNA-overhang cloning

Here we describe two methods for generating DNA fragments with single-stranded overhangs, like those generated by the activity of many restriction enzymes, by simple methods that do not involve DNA digestion. The methods, RNA-overhang cloning (ROC) and DNA-overhang cloning (DOC), generate polymerase chain reaction (PCR) products composed of double-stranded (ds) DNA flanked by single-stranded (ss) RNA or DNA overhangs. The overhangs can be used to recombine DNA fragments at any sequence location, creating "perfect" chimeric genes composed of DNA fragments that have been joined without the insertion, deletion, or alteration of even a single base pair. The ROC method entails using PCR primers that contain regions of RNA sequence that cannot be copied by certain thermostable DNA polymerases. Using such a chimeric primer in PCR would yield a product with a 5' overhang identical to the sequence of the RNA component of the primer, which can be used for directional ligation of the amplified product to other preselected DNA molecules. This method provides complete control over both the length and sequence of the overhangs, and eliminates the need for restriction enzymes as tools for gene engineering.     

Twin PCRs: a simple and efficient method for directional cloning of PCR products

A simple and efficient method was developed for directional cloning of PCR products without any restriction enzyme digestion of the amplified sequence. Two pairs of primers were designed in which parts of two restriction enzyme recognition sequences were integrated, and the primers were used for two parallel PCRs. The PCR products were mixed, heat denatured and re-annealed to generate hybridized DNA fragments bearing sticky ends compatible with restriction enzymes. This method is particularly useful when it is necessary to use a restriction enzyme but there is an additional internal restriction site within the amplified sequence, or when there are problems caused by end sensitivity of restriction enzymes.     

High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli

In this article, we describe a high-throughput cloning method, seamless enzyme-free cloning (SEFC), which allows one-step assembly of DNA fragments in vivo via homologous recombination in Escherichia coli. In the method, the desired open reading frame (ORF) is amplified by use of ORF-specific primers with flanking sequences identical to the two ends of a linearized vector. The polymerase chain reaction (PCR) product and the linearized vector are then cotransformed into E. coli cells, where the ORF is incorporated into the vector in vivo. SEFC is a simple, reliable, and inexpensive method of cloning in which PCR fragments are fused into expression vectors without unwanted amino acids or extra in vitro manipulations apart from the single PCR amplification step. Using this method, we successfully cloned human liver complete ORFs into the yeast AD and DB vectors and generated a clone resource of 4964 AD-ORFs and 4676 DB-ORFs in 3 months. This approach will be useful for daily DNA cloning and for creating proteome-scale clone resources.     

Modified T-RFLP methods for taxonomic interpretation of T-RF

Terminal restriction fragment length polymorphism (TRFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct TRFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs.     

应用反向PCR克隆慢病毒介导的转基因小鼠整合位点序列

目的：为分析慢病毒介导的转基因小鼠中外源基因整合位点的信息,应用反向PCR克隆整合位点序列。方法：小鼠基因组总DNA酶解和自连接后,针对慢病毒载体的特点在LTR附近设计一组特异的PCR引物,优化半巢式PCR的各种参数,提高整合位点序列克隆的效率。结果：克隆了分别携带绿色荧光蛋白（GFP）和转铁蛋白（TF）基因的慢病毒介导的转基因小鼠家系7只小鼠中10个外源基因整合位点序列。结论：本方法可用于慢病毒介导的转基因小鼠整合位点序列的克隆,为分析整合位点与外源基因表达之间的关系等提供了科学依据。     

Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning.

A simple and rapid method for cloning of amplification products directly from the polymerase chain reaction (PCR) has been developed. The method is based on the addition of a 12-base dUMP-containing sequence (CUACUACUACUA) to the 5' end of PCR primers. Incorporation of these primers during PCR results in the selective placement of dUMP residues into the 5' end of amplification products. Selective degradation of the dUMP residues in the PCR products with uracil DNA glycosylase (UDG) disrupts base pairing at the termini and generates 3' overhangs. Annealing of 3' protruding termini to vector DNA containing complementary 3' ends results in chimeric molecules which can be transformed, with high efficiency, without in vitro ligation. Directional cloning of PCR products has also been accomplished by incorporating different dU-containing sequences at the end of each PCR primer. Substitution of all dT residues in PCR primers with dU eliminates cloning of aberrant "primer dimer" products and enriches cloning of genuine PCR products. The method has been applied to cloning of inter-Alu DNA sequences from human placental DNA. Using a single primer, DNA sequences between appropriately oriented Alu sequences were amplified and cloned. Cloning of cDNA for the glyceraldehyde-3'-phosphate dehydrogenase gene from rat brain RNA was also demonstrated. The 3' end region of this gene was amplified by the 3' RACE method and the amplified DNA was cloned after UDG digestion. Characterization of cloned DNAs by sequence analysis showed accurate repair of the cloning junctions. The ligase-free cloning method with UDG should prove to be a widely applicable procedure for rapid cloning of PCR-amplified DNA.     

Rapid generation of nested deletions by differential restriction digestion

A method was devised for generating nested deletions in DNA that exploits the difference in frequency of restriction sites recognized by compatible restriction endonucleases. A cloning vector was constructed that contains no common blunt-end or RsaI restriction sites and two 8-bp blunt-end restriction sites flanking a commodious multiple cloning site. DNA fragments are cloned into the multiple cloning site using blue-white selection, and nested deletions are generated by digesting the resulting plasmid with either SwaI or PmeI and partially digesting the insert DNA with RsaI. The DNAs are ligated and transformed, producing afamily of plasmids with different-sized deletions. The DNA sequence of these inserts can be rapidly determined, and the overlapping sequences can be assembled in silico to produce a large DNA contig. Nested deletions generated in this manner can also be used for the structure-function analysis of proteins.     

A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering

Background  

The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR.     

A universal cloning vector using vaccinia topoisomerase I

Vaccinia DNA topoisomerase I (TOPO) charged vectors with a sticky T are routinely used to clone polymerase chain reaction (PCR) products with an extra A at their 3′ end (TOPO TA Cloning from Invitrogen). TOPO charged blunt vectors are used to clone blunt end PCR products (TOPO Blunt Cloning). Here, we demonstrate that both TOPO TA vectors and TOPO Blunt vectors can be used to clone PCR products with either a blunt end or an extra A at the 3′ end. We further demonstrate that these vectors can be used to clone sticky end DNA generated with restriction enzymes. In summary, these TOPO vectors can be used as universal cloning vectors.