Princípio do método: O site dirigido mutagênese de plasmídeos toda explicada neste vídeo é um método de mutagênese que permite que você alterar um gene alvo clonado pela exclusão, substituição ou inserção de algumas bases diretamente em um plasmídeo. Ele funciona, amplificando o plasmídeo inteiro, em uma reação termociclagem não baseados em PCR. Durante a reação primers mutagênico, carregando a mutação desejada na forma de incompatibilidades para o plasmídeo original, estão integrados no plasmídeo recém-sintetizada. Após a remoção do plasmídeo original a partir da reação do plasmídeo mutante se transforma em E. coli. Os passos seguintes são para fins de triagem somente, porque a eficiência de mutação deste método não é 100%. O processo todo leva três dias, mas a parte principal pode ser feito dentro de um dia. 1,0 O primeiro dia: 1,1 termociclagem reação: Original Plasmid: Este site dirigido mutagênese protocolo funciona melhor com plasmídeos de até 10kb. Plasmids maiores são um pouco difícil de sofrer mutações com este método e pode levar um pouco de paciência e ajuste das condições de termociclagem e / ou células competentes. Além disso, o plasmídeo que você trabalha com deve ser isolado de uma represa + cepa de bactérias. Para a reação termociclagem você vai precisar 10-60 ngs do plasmídeo você quer se transformar. Primers: Antes de você pode configurar a sua reação termociclagem você tem que ter o seu primers na mão. Você precisa de cerca de 150 ng de cada cartilha mutagênico. É ok se você tirar 1,5 mL de uma ação diluída 1:10 pm 100. Existem algumas orientações simples que você deve considerar ao projetar seu primers mutagénicos: <ul><li> Os primers devem ser complementares um ao outro </li><li> Os primers devem ter entre 25 e 45 nucleotídeos de comprimento </li><li> As mutações na forma de incompatibilidades para o plasmídeo original deve ser contido em ambos os primers </li><li> As incompatibilidades devem ser centrados na cartilha e ladeado por pelo menos 8 nucleotídeos em cada lado </li><li> Os primers devem ter um GC Conteúdo de pelo menos 40% </li><li> Os primers deve terminar 5 principais e 3 privilegiada, com um ou mais Gs ou Cs </li><li> Os primers não precisa ser fosforilada, nem têm de ser purificados FPLC ou PAGE, apenas devem ser dessalinizada. </li><li> Para o cálculo da Tm você não precisa de nenhuma fórmula, mas Tm no certificado de transporte deve ser superior a 60 ° C. </li><li> Para fins de triagem é prático para inserir ou excluir um sítio de restrição com o seu mutação. Por causa da degeneração do código genético há muitas possibilidades para inserir um sítio de restrição com a sua mutação desejada. O site do New England Biolabs fornece uma ferramenta para encontrar tal um sítio de restrição apropriada. Basta digitar a seqüência de cartilha de codificação para a seqüência de aminoácidos que você deseja, utilizando o código ambigüidade de DNA. A ferramenta de localização da enzima irá dizer-lhe que sítios de restrição pode ser introduzido em paralelo à sua mutação desejada. Se você não consegue encontrar nenhum sítio de restrição possível ou prático desta forma, você pode inserir qualquer sítio de restrição novo sem se preocupar o código genético no local da mutação. A partir daí você pode usar este plasmídeo como modelo para uma série de mutações em que este sítio de restrição será eliminado através da inserção do novo primers mutagênico. Esta abordagem pode ser muito conveniente se você estiver planejando fazer uma série de mutações no mesmo local do plasmídeo. </li></ul> DNA polimerase: Para este método você precisa de uma termoestável DNA polimerase, que exibe 3&#39;-5 &#39;atividade de exonuclease e cria termina sem corte. Usamos sempre recombinante Pfu Polimerase a partir Fermentas. Se você tem problemas com as etapas de amplificação, você pode tentar uma polimerase de maior qualidade, mas para a maioria dos propósitos do Pfu padrão será suficiente. Completar a reação com dNTPs tampão da polimerase e água. 1.2 Condições de termociclagem O termociclador deve ser criado da seguinte maneira. A fase de desnaturação inicial e recorrente é definida como 30 seg a 95 ° C. A temperatura de anelamento dos primers não devem ser calculados com fórmulas complicadas. Costumamos definir a temperatura de anelamento a 55 ° C eo tempo de recozimento para um minuto. Para primers de 25 a 30 nucleotídeos que você estará usando em sua maioria, isso funciona para nós 95% do tempo. De qualquer forma se isso não deve funcionar, simplesmente tente variar a temperatura de tratamento térmico entre 50 - e 60 ° C. A temperatura alongamento depende da polimerase que você usa. Nesta demonstração usamos o Pfu Polimerase a partir Fermentas, que apela a uma temperatura de alongamento de 72 ° C. O tempo de alongamento varia de acordo com o tamanho do plasmídeo. Nós sempre calcular 1 min por kb, e adicionar um minuto extra para quetempo. Por exemplo, para um plasmídeo 9kb nós escolheríamos 10min como tempo de alongamento. 18 ciclos são suficientes para criar o suficiente mutante plasmídeo para o uso posterior. Manter o número de ciclos de baixa, também poupa tempo. <img src="/files/ftp_upload/1135/figure1.png" alt="Figura 1" width="600" height="287" /> 1,3 Gelcheck após reação termociclagem A amplificação bem-sucedido deve ser verificado através da realização de uma electroforese. Diretamente após o ciclo terminar, coloque 5 mL da reação em um gel de agarose 1% TAE. Se a amplificação foi bem-sucedido, você deve ver uma banda distinta. No entanto, se o DNA recém sintetizado não é claramente visível no gel, você pode tentar precipitar a reação de todo e usá-lo para a transformação na próxima etapa. Para nós, isso raramente funcionou, mas vale a pena dar uma tentativa. A melhor coisa a fazer se a reação não funciona é para ajustar a temperatura de recozimento. 1,4 digestão DpnI: Antes da transformação do plasmídeo original, que serviu como um modelo deve ser removido da reação para impedir de fundo forte. Isto é feito por digestão com restrição DpnI. Este endonuclease de restrição corta somente metilado plasmídeo. Seu reconhecimento e sítio de restrição é o GATC seqüência ao passo que A tem de ser metilado. Quando digerir com DpnI apenas o plasmídeo originais não mutada que foi isolado de uma represa + tensão é cortada, o plasmídeo recém-sintetizado mutante que não é metilado não é afetado por DpnI. Basta adicionar 1-2 mL de DpnI à reação e incubar pelo menos uma hora a 37 °. Ao usar o Fast digerir DpnI o tempo de incubação pode ser reduzida para cerca de 15 minutos. A qualidade de sua DpnI eo tempo de incubação desta digestão restrição muito forte determina como o seu fundo com plasmídeo não mutada será. 1,5 Transformação: Após a digestão do plasmídeo DpnI está pronto para transformação em E. competente células coli. Basta adicionar 5 mL da reação de digestão DpnI nas células competentes e realizar a transformação, como recomendado no seu laboratório. No nosso caso, usamos o procedimento de choque térmico, incubando a mistura de bactérias plasmídeo em gelo por 30 min e depois de choque térmico é a 42 ° C por 90 seg. Após a adição de 200 mL SOC-solução, as bactérias são incubadas, vigorosa agitação, a 37 ° C por 1 h. Após uma hora as bactérias são semeadas em agar a seleção da mídia. 2,0 segundo dia 2,1 Triagem de clones parte 1: Seleção de clones Porque a eficiência de mutação não é 100% você precisa de tela para o seu mutantes. Fazemos isso por digestão de restrição em que nós verificamos a presença ou ausência do sítio de restrição que nós adicionados ou excluídos por meio da integração primer. Para isso, geralmente pegar oito colônias e cultivá-las durante a noite para a preparação de plasmídeo, no dia seguinte. 3,0 Dia três 3,1 Triagem de clones parte 2: Restrição de digestão com a enzima marcador No terceiro dia você executar um Mini Prep e digestão subsequente restrição da enzima com seu marcador. No gel, em seguida, você pode ver que um de seus clones carregam a mutação desejada e que não aquelas. O método de rastreamento usando a digestão restrição funciona muito bem. No entanto, você deve confirmar a sua mutação bem sucedida por seqüenciamento.

<ol>
	<li>Papworth, C., Bauer, J. C., Braman, J., Wright, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QuikChange+site-directed+mutagenesis.">QuikChange site-directed mutagenesis.</a> Strategies. 9, 3-4 (1996).</li></ol>

The authors have nothing to disclose.

Mutagênese dirigida local é um método de mutagênese, que fornece uma maneira rápida de mutação de um gene transportado por um plasmídeo. A reação geral pode ser feito em apenas um dia. Com este método, ele vai precisar de apenas um par de primers complementares levando a mutações desejadas e uma revisão da polimerase como Pfu Polimerase. O plasmídeo recém-sintetizado pode ser separado do plasmídeo parental por digerir a reação com o DpnI enzima de restrição. Esta enzima digere apenas o DNA metilado....

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		<div>
		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Pfu polymerase (recombinant or native)</td>
<td>&nbsp;</td>
<td>fermentas</td>
<td>EP0571 or EP0501</td>
<td>&nbsp;</td>
<tr>
<td>dNTP mix 10 mM </td>
<td>&nbsp;</td>
<td>fermentas</td>
<td>R0191</td>
<td>&nbsp;</td>
<tr>
<td>DpnI or DpnI Fast digest</td>
<td>&nbsp;</td>
<td>fermentas</td>
<td>ER1701</td>
<td>&nbsp;</td>
<tr>
<td>primers</td>
<td>&nbsp;</td>
<td>invitrogen</td>
<td>&nbsp;</td>
<td>desalted</td>
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homemade site directed mutagenesis of whole plasmids

Mutagênese dirigida local de plasmídeos todo é uma maneira simples de criar variações ligeiramente diferentes de um plasmídeo original. Com este método, o gene-alvo clonados podem ser alterados pela exclusão, substituição ou inserção de algumas bases diretamente em um plasmídeo. Ela funciona por simplesmente ampliar o plasmídeo inteiro, em uma reação termociclagem não baseados em PCR. Durante a reação primers mutagênico, carregando a mutação desejada, são integrados no plasmídeo recém-sintetizada. Neste vídeo tutorial vamos demonstrar uma maneira fácil e de custo eficaz para introduzir substituições de base em um plasmídeo. O protocolo trabalha com reagentes de referência e é independente de kits comerciais, que muitas vezes são muito caros. Aplicando este protocolo pode reduzir o custo total de uma reação a um oitavo do que custa o uso de alguns dos kits comerciais. Neste vídeo também comentar sobre as etapas críticas durante o processo e dar instruções detalhadas sobre como desenhar os iniciadores mutagênicos.

Watch this Scientific Journal Video about Homemade Site Directed Mutagenesis of Whole Plasmids at JoVE.com

Homemade Site Directed Mutagenesis of Whole Plasmids

Mutagênese dirigida local de plasmídeos todo é uma maneira simples de criar variações ligeiramente diferentes de um plasmídeo original. Aqui nós demonstramos uma forma fácil e de custo eficaz para introduzir substituições de base em um plasmídeo reagentes padrão usando.

Mutagênese do site caseiros Direção de Plasmids Whole

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

homemade-site-directed-mutagenesis-of-whole-plasmids

Research

JoVE Journal

Biology

33.0K visualizações.  Johannes Gutenberg-University Mainz, Germany. Mutagênese dirigida local de plasmídeos todo é uma maneira simples de criar variações ligeiramente diferentes de um plasmídeo original. Aqui nós demonstramos uma forma fácil e de custo eficaz para introduzir substituições de base em um plasmídeo reagentes padrão usando.

Vídeo: Homemade Site Directed Mutagenesis of Whole Plasmids

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Princípios do site-specific Recombinase (SSR) Tecnologia

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Biologia

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

Uma Introdução ao Lab Worm: de Worms Cultivar a mutagênese

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Mutagênese e Funcional Protocolos de Seleção para a Evolução Dirigida de proteínas em E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Examinando o Dynamics Conformacional de Proteínas de Membrana In situ Com Labeling-Site dirigido Fluorescência

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Analisando a função de GTPases Pequenas pela microinjeção de Plasmids em Polarized Células Epiteliais

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Mutagênese Bissulfito Único de ovócitos

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

De alta capacidade de Triagem funcional usando um caseiro Dual-brilho ensaio de luciferase

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Geração de<em&gt; Enterobacter</em&gt; Sp. YSU auxotrofos Usando Transposon Mutagênese

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Um Protocolo de Avaliação Funcional da Whole-Protein saturação mutagênese Bibliotecas Utilizando-High Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...