An efficient bipartite PCR technique to introduce specific changes in large plasmids

Amplifying an entire double-stranded plasmid by an inverse polymerase chain reaction (PCR) using a pair of tail-to-tail primers is a particularly efficient approach for introducing changes into DNA sequences. However, the approach generally works best for plasmids less than 5 Kb and it can be difficult to amplify the large multicomponent vectors that are used for protein expression in various eukaryotic cells. We have therefore adopted an alternative approach in which two smaller PCR products are generated and then ligated to produce the complete plasmid. A mutagenic primer is used to introduce the desired change and each reaction includes one of a pair of tail-to-tail primers from within an antibiotic resistance gene contained on the plasmid so that the two PCR products contain complementing parts of the complete gene. Ligating the two products generates various combinations but only the correctly ligated molecules recreate the antibiotic resistance gene and are able to replicate in Escherichia coli. When combined with methods to minimize the carryover of template plasmid, this can be an efficient way of introducing mutations into large plasmids.     

Creating random mutagenesis libraries using megaprimer PCR of whole plasmid

The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it. Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector need optimizing. Even then, the resultant library is often plagued with unwanted plasmids that have no inserts or multiple inserts. These species have to be eradicated to avoid tedious screening, especially when producing a mutant gene library. To overcome these problems, we modified the QuikChange protocol so that each plasmid carries a single insert. Although the QuikChange was originally developed for site-directed mutagenesis using complementary mutagenic oligonucleotide primers in whole plasmid PCR, we found that the protocol also worked for megaprimers consisting of hundreds of nucleotides. Based on this discovery, we used insert fragments, which we wanted to clone, as the primers in the QuikChange reaction. The resultant libraries were virtually free from species with no inserts or multiple inserts. The present method, which we designated MEGAWHOP (megaprimer PCR of whole plasmid), is thus ideal for creating random mutagenesis megalibraries.     

Efficient site-directed saturation mutagenesis using degenerate oligonucleotides.

We describe a reliable protocol for constructing single-site saturation mutagenesis libraries consisting of all 20 naturally occurring amino acids at a specific site within a protein. Such libraries are useful for structure-function studies and directed evolution. This protocol extends the utility of Stratagene's QuikChange Site-Directed Mutagenesis Kit, which is primarily recommended for single amino acid substitutions. Two complementary primers are synthesized, containing a degenerate mixture of the four bases at the three positions of the selected codon. These primers are added to starting plasmid template and thermal cycled to produce mutant DNA molecules, which are subsequently transformed into competent bacteria. The protocol does not require purification of mutagenic oligonucleotides or PCR products. This reduces both the cost and turnaround time in high-throughput directed evolution applications. We have utilized this protocol to generate over 200 site-saturation libraries in a DNA polymerase, with a success rate of greater than 95%.     

荧光标记DNA扩增片段长度多态性方法的改进

采用常规PCR试剂和合成的接头和引物,其中MseⅠ引物为荧光标记物FAM标记,并对扩增片段长度多态性(AFLP)程序进行了改进,优化了PCR反应和电泳条件,建立了一个新的、有效的反应体系,降低了实验费用,经比较实验结果与AFLP荧光标记试剂盒的实验效果一致.     

Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis

To develop a simple, speedy, economical and widely applicable method for multiple-site mutagenesis, we have substantially modified the Quik-Change™ Site-Directed Mutagenesis Kit protocol (Stratagene, La Jolla, CA). Our new protocol consists of (i) a PCR reaction using an in vitro technique, LDA (ligation-during-amplification), (ii) a DpnI treatment to digest parental DNA and to make megaprimers and (iii) a synthesis of double-stranded plasmid DNA for bacterial transformation. While the Quik Change™ Kit protocol introduces mutations at a single site, requiring two complementary mutagenic oligonucleotides, our new protocol requires only one mutagenic oligonucleotide for a mutation site, and can introduce mutations in a plasmid at multiple sites simultaneously. A targeting efficiency >70% was consistently achieved for multiple-site mutagenesis. Furthermore, the new protocol allows random mutagenesis with degenerative primers, because it does not use two complementary primers. Our mutagenesis strategy was successfully used to alter the fluorescence properties of green fluorescent protein (GFP), creating a new-color GFP mutant, cyan-green fluorescent protein (CGFP). An eminent feature of CGFP is its remarkable stability in a wide pH range (pH 4–12). The use of CGFP would allow us to monitor protein localization quantitatively in acidic organelles in secretory pathways.     

一种简便高效的酵母基因组提取方法

提取基因组进行检测是酵母研究过程中的必要步骤之一。以毕赤酵母菌株GS115作为研究对象,主要成分为0.2 mol/L醋酸锂和1% SDS的酵母裂解液能高效的裂解酵母细胞壁。与两种酵母基因组提取试剂盒相比,该方法从相同体积的酵母培养液中获得的基因组的量高5倍以上,并且操作简便、快速,能在2 h内完成一次提取过程,极大地缩短了时间。以GS115中的内源AOX基因为目的基因,对提取的基因组进行PCR检测和Southern杂交检测,进一步验证了基因组的质量。因此,本文建立了一种简便、快速、经济而高效的酵母基因提取方法。     

Sticky PCR: A PCR-based protocol for targeted protein engineering

This article describes a simple but powerful PCR-based protocol for the generation of cohesive ends on linear DNA fragments, permitting the precise engineering of DNA constructs for a variety of applications. These include the introduction of deletion mutations, domain swapping, creating hybrid DNA fusions, or targeted protein engineering. This novel method can also facilitate the cloning of large or complex DNA fragments into a relevant cloning vector independent of the use of internal restriction endonuclease sites. The protocol involves the amplification of the required fragments by polymerase chain reaction through the use of two sets of overlapping desalted oligonucleotide primers. The subsequent mixing, denaturation and re-annealing of these products present correct cohesive terminal ends for ligation. There is no requirement for special vectors, enzymes or bases, suggesting that this protocol provides a unique way of engineering constructs in a rapid and cost-effective way for specific applications, such as precise deletion or swapping of various domains of the epidermal growth factor receptor to determine their role in membrane localization.     

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function.     

A Rapid and Economic In-House DNA Purification Method Using Glass Syringe Filters

Background

Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations.

Methodology/Principal Findings

We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for purification was much less than that required using a commercial kit.

Conclusions/Significance

This method provides DNA yield and quality similar to that obtained with commercial kits, but is more rapid and less costly.     

Simple preparation method of PCR fragments for automated DNA sequencing

In an effort to find a simple and inexpensive purification method of polymerase chain reaction (PCR) reaction before cycle sequencing reaction, we compared a commercial system with a precipitation protocol performed in our laboratory. We found that, particularly with small PCR products, our method works with greater success than the method compared. Our precipitation method may be used on a larger PCR fragment before cycle sequencing reaction as well. Furthermore, it has the advantage of being simple as the well-known dilution method; in contrast to the dilution method, the precipitation method removes excess primers as well as possible primer dimers.     

A polymerization-based method to construct a plasmid containing clustered DNA damage and a mismatch

Exposure of biological materials to ionizing radiation often induces clustered DNA damage. The mutagenicity of clustered DNA damage can be analyzed with plasmids carrying a clustered DNA damage site, in which the strand bias of a replicating plasmid (i.e., the degree to which each of the two strands of the plasmid are used as the template for replication of the plasmid) can help to clarify how clustered DNA damage enhances the mutagenic potential of comprising lesions. Placement of a mismatch near a clustered DNA damage site can help to determine the strand bias, but present plasmid-based methods do not allow insertion of a mismatch at a given site in the plasmid. Here, we describe a polymerization-based method for constructing a plasmid containing clustered DNA lesions and a mismatch. The presence of a DNA lesion and a mismatch in the plasmid was verified by enzymatic treatment and by determining the relative abundance of the progeny plasmids derived from each of the two strands of the plasmid.     

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。     

Whole plasmid mutagenic PCR for directed protein evolution

Protein function can be engineered through iterated cycles of random mutagenesis and screening (directed evolution). Optimization of protein expression is essential for the development of sensitive and precise high throughput assays. Here we optimize the performance of a plasmid-borne Escherichia coli lacZ gene in two rounds of directed evolution. First, its promoter was "randomized" by whole plasmid polymerase chain reaction (PCR) and intra-molecular self-ligation. A genetically stable constitutive expression vector was isolated in an in vivo genetic selection. Second, the entire plasmid was randomly mutated in a slightly mutagenic long polymerase chain reaction. The PCR products were digested with a restriction enzyme, self-ligated by T4 DNA ligase and transformed into E. coli. The resulting library of beta-galactosidase (beta-gal) mutants consisted mostly ( approximately 80%) of hypomorphs, suggesting that the mutation rate was appropriate for directed evolution applications. We isolated and characterized 14 variants with increased activity in reactions with 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal). The purified protein derived from one clone exhibited a 100-fold improvement in k(cat) over its parent in reactions with para-nitrophenyl-beta-d-galactopyranoside (pNP-gal). This latter result clearly demonstrates the utility of whole plasmid mutagenic PCR for directed protein evolution.     

Multi-node graphs: a framework for multiplexed biological assays.

Multiplex polymerase chain reaction (PCR) is an extension of the standard PCR protocol in which primers for multiple DNA loci are pooled together within a single reaction tube, enabling simultaneous sequence amplification, thus reducing costs and saving time. Potential cost saving and throughput improvements directly depend on the level of multiplexing achieved. Designing reliable and highly multiplexed assays is challenging because primers that are pooled together in a single reaction tube may cross-hybridize, though this can be addressed either by modifying the choice of primers for one or more amplicons, or by altering the way in which DNA loci are partitioned into separate reaction tubes. In this paper, we introduce a new graph formalism called a multi-node graph, and describe its application to the analysis of multiplex PCR scalability. We show, using random multi-node graphs that the scalability of multiplex PCR is constrained by a phase transition, suggesting fundamental limits on efforts to improve the cost-effectiveness and throughput of standard multiplex PCR assays. In particular, we show that when the multiplexing level of the reaction tubes is roughly theta(log (sn)) (where s is the number of primer pair candidates per locus and n is the number of loci to be amplified), then with very high probability we can 'cover' all loci with a valid assignment to one of the tubes in the assay. However, when the multiplexing level of the tube exceeds these bounds, there is no possible cover and moreover the size of the cover drops dramatically. Simulations using a simple greedy algorithm on real DNA data also confirm the presence of this phase transition. Our theoretical results suggest, however, that the resulting phase transition is a fundamental characteristic of the problem, implying intrinsic limits on the development of future assay design algorithms.     

双磁珠法质粒DNA提取技术及应用

质粒是基因合成与测序领域中使用最为频繁的基因运载工具,然而传统的质粒DNA提取方法面临提取通量低、生产成本高等问题,无法满足日益增长的需求。本研究基于质粒提取原理,开发了双磁珠法(double-magnetic-bead method, DMBM)质粒提取技术,探究了磁珠投入量、质粒DNA片段大小、菌液投入量等因素对质粒提取的影响,并且对比了本技术与商业化质粒DNA提取试剂盒提取DNA质量、提取通量及提取成本。结果表明,双磁珠法质粒DNA提取技术可满足不同细胞密度、不同片段长度的质粒DNA提取。此外,该技术搭载96通道全自动核酸提取仪,提取的质粒DNA纯度更高、提取时间缩短80%、提取成本缩减57.1%,从而实现了质粒DNA提取的高通量、低成本,有效助力基因合成与测序。     

TAMS technology for simple and efficient in vitro site-directed mutagenesis and mutant screening

Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. We now describe a method that allows mutagenesis on any DNA template (eg. cDNA, genomic DNA and plasmid DNA), and is highly efficient for multiple-site mutagenesis (up to 100%). The technology takes advantage of the requirement that, in order for DNA polymerases to elongate, it is crucial that the 3′ sequences of the primers match the template perfectly. When two outer mutagenic oligos are incorporated together with the desired mutagenic oligos into the newly synthesised mutant strand, they serve as anchors for PCR primers which have 3′ sequences matching the mutated nucleotides, thus amplifying the mutant strand only. The same principle can also be used for mutant screening.     

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis.     

基于PCR的质粒快速定点诱变方法建立与评价

基因突变对生命的进化具有重要意义,针对质粒的DNA定点诱变技术也是基因工程、蛋白质工程研究中的重要手段之一。为了提高质粒定点诱变的效率,本研究利用引物部分重叠的设计方案,使用Tm值相对固定的引物设计模式,将同一引物分为重叠区（Tm=50±2℃）和非重叠区（Tm=60±2℃）,并在严格控制模板用量（2 pg/kb）的基础上,通过20个PCR循环对目标质粒进行定点诱变扩增,随后取0.5 μL产物直接用于转化。在FastPfu Fly酶系中,利用此法构建了6个含碱基替换、缺失和插入的质粒,均获得成功,突变效率可达96%以上,阳性克隆获得数达70个以上。此外,利用4种不同PCR酶系对该法的适用性进行了评价,结果表明突变效率均可达93%以上,阳性克隆获得数均在10个以上。通过适当增加PCR模板用量（10 pg/kb）并使用纯化后的PCR产物进行转化,该法可适用于转化效率大于106（cfu/μg）的任意感受态细胞,对应的突变效率可大于91%,阳性克隆获得数大于20。根据本法的作用原理,该方案适合质粒中10～20 bp（因重叠区GC含量及碱基序列的不同而改变）以内的任意碱基替换和插入,以及任意长度的DNA片段缺失。且具有通用性强、耗时少、诱变成功率高、成本低、对感受态及转化效率无特殊要求等优点,适合各实验室的日常研究使用。     

An extended boiling method for small-scale preparation of plasmid DNA tailored to long-range automated sequencing

Automated fluorescence sequencing depends on high-quality plasmid DNA, which is conveniently prepared by minipreparation procedures. While those procedures are effective for high-copy number plasmids, purity and yields of low-copy number plasmids are often not sufficient to achieve reasonable sequencing results. Here, we describe a reproducible and cheap procedure for the small-scale preparation of plasmid DNA, which is based on the original Holmes and Quigley protocol, comprising a boiling and two selective precipitation steps. Besides various other modifications, this procedure utilizes polyethylene glycol (PEG) precipitation as a key step to further purify plasmid DNA tailored to automated fluorescence sequencing. Independent of the plasmid size and copy number, the modified procedure yields plasmid DNA, which gives average reading lengths of 800 and more bases with a standard fluorescence cycle sequencing protocol. To demonstrate the efficiency and reproducibility of the method, sequencing data of various human interleukin-6 gene variants cloned in different vectors are presented. This procedure offers an economical alternative to commercial miniprep kits, utilizing silica resins or anion-exchanger matrices and, moreover, is more reliable and consistent with respect to reading lengths and accuracy in automated fluorescence sequencing.     

Two different PCR approaches for universal diagnosis of brown rot and identification of Monilinia spp. in stone fruit trees

AIMS: To design a protocol for the universal diagnosis of brown rot by polymerase chain reaction (PCR) in plant material and subsequently Monilinia spp. identification. METHODS AND RESULTS: Primers for discrimination of Monilinia spp. from other fungal genera by PCR were designed following a ribosomal DNA analysis. Discrimination among species of Monilinia was subsequently achieved by developing primers using SCAR (Sequence Characterised Amplified Region) markers obtained after a random amplified polymorphic DNA study. In addition, an internal control (IC) based on the utilization of a mimic plasmid was designed to be used in the diagnostic protocol of brown rot to recognize false negatives due to the inhibition of PCR. CONCLUSIONS: The four sets of primers designed allowed detection and discrimination of all Monilinia spp. causing brown rot in fruit trees. Addition of an IC in each PCR reaction performed increased the reliability of the diagnostic protocol. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection protocol presented here, that combined a set of universal primers and the inclusion of the plasmid pGMON as an IC for diagnosis of all Monilinia spp., and three sets of primers to discriminate the most important species of Monilinia, could be an useful and valuable tool for epidemiological studies. The method developed could be used in programmes to avoid the spread and introduction of this serious disease in new areas.