Principio del método: El sitio de mutagénesis dirigida de los plásmidos todo se explica en este video es un método de mutagénesis que le permite modificar un gen diana clonado por sustitución, supresión o inserción de unas cuantas bases directamente en un plásmido. Funciona mediante la amplificación de todo el plásmido, en una reacción de termociclado no basados ​​en la PCR. Durante la reacción de primers mutagénicos, portadores de la mutación deseada en forma de desajustes en el plásmido original, se integran en el plásmido de nueva síntesis. Después de la eliminación de la plásmido original de la reacción del plásmido mutado se transforma en E. coli. Los siguientes pasos son sólo para propósitos de revisión, porque la eficiencia de la mutación de este método no es 100%. Todo el procedimiento dura tres días, pero la parte principal se puede hacer en un día. 1.0 Primer día: 1.1 reacción termociclado: Plásmido original: Este sitio de mutagénesis dirigida protocolo funciona mejor con plásmidos de hasta 10kb. Los plásmidos son más grandes un poco difícil de mutar con este método y puede tomar un poco de paciencia y ajuste de las condiciones de ciclos térmicos y / o las células competentes. Además, el plásmido que se trabaja con debe estar aislado de una presa + cepa de bacterias. Para la reacción de termociclado tendrá 10 a 60 ngs del plásmido desea mutar. Cebadores: Antes de que pueda configurar su reacción termociclado usted tiene que tener su primers en la mano. Usted necesita cerca de 150 ng de cada cebador mutagénico. Esta bien, si se toma 1,5 l de una 1:10 pm 100 acciones diluidas. Hay algunas pautas sencillas que usted debe considerar al diseñar su primers mutagénicos: <ul><li> Los cebadores deben ser complementarias entre sí </li><li> Los primers debe estar entre 25 y 45 nucleótidos de longitud </li><li> Las mutaciones en la forma de los desajustes que el plásmido original debe estar contenido en los dos primers </li><li> Los desajustes se deben centrar en el primer y flanqueado por al menos 8 nucleótidos en cada lado </li><li> Los primers deben tener un contenido de GC de al menos 40% </li><li> Los primers debe terminar cinco prime y prime 3 con uno o más Gs o C </li><li> Los primers no necesitan ser fosforilados, ni tampoco tienen que ser purificados o FPLC PAGE, simplemente desalinizada debe ser. </li><li> Para el cálculo de Tm no necesita ninguna fórmula, pero Tm en el certificado de envío debe ser superior a 60 ° C. </li><li> Para fines de selección es práctico para insertar o eliminar un sitio de restricción con la mutación. Debido a la degeneración del código genético hay muchas posibilidades para insertar un sitio de restricción con la mutación deseada. El sitio web de la Nueva Inglaterra Biolabs ofrece una herramienta para encontrar ese sitio de restricción apropiadas. Simplemente introduzca su primer secuencia de codificación de la secuencia de aminoácidos que desee, utilizando el código de la ambigüedad de ADN. La herramienta de búsqueda de la enzima le dirá que los sitios de restricción se pueden introducir en paralelo a la mutación deseada. Si usted no puede encontrar ningún sitio de restricción posible o práctico de esta manera, usted puede insertar cualquier nuevo sitio de restricción sin importar el código genético en el sitio de la mutación. A partir de entonces puede utilizar este plásmido como molde para una serie de mutaciones en el que se elimina la restricción de este sitio de la inserción de los primers mutagénicos nuevo. Este enfoque puede ser muy conveniente si usted está planeando hacer una serie de mutaciones en el mismo sitio del plásmido. </li></ul> ADN-polimerasa: Para este método se necesita un ADN-polimerasa termoestable que exhibe 3&#39;-5 &#39;exonucleasa y crea extremos romos. Siempre usamos Pfu recombinante de la polimerasa de Fermentas. Si tienes problemas con los pasos de amplificación puede probar con una polimerasa de alta calidad, pero para la mayoría de los Pfu estándar será suficiente. Completar la reacción de la polimerasa con buffer de dNTPs, y el agua. 1.2 Condiciones de termociclado El termociclador debe establecerse de la siguiente manera. La fase de desnaturalización inicial y recurrente está establecido en 30 segundos a 95 ° C. La temperatura de hibridación de los primers no deben ser calculados con fórmulas complicadas. Por lo general, ajustar la temperatura de recocido a 55 ° C y el tiempo de recocido a un minuto. Para los primers de 25 a 30 nucleótidos que va a utilizar en su mayoría, esto funciona para nosotros el 95% del tiempo. De todos modos, si esto no funciona, simplemente tratar de variar la temperatura de hibridación entre el 50 - y 60 ° C. La temperatura depende de la elongación de la polimerasa que utiliza. En esta demostración se utiliza el PFU-polimerasa de Fermentas, que llama a una temperatura de elongación de 72 ° C. El tiempo de elongación varía según el tamaño del plásmido. Siempre calculamos 1 minuto por cada kb, y añadir un minuto adicional para quetiempo. Por ejemplo, un plásmido 9kb nos volveríamos a elegir 10 minutos como tiempo de elongación. 18 ciclos son suficientes para crear suficiente plásmido mutado para el uso posterior. Mantener el número de ciclos de baja, también le ahorra tiempo. <img src="/files/ftp_upload/1135/figure1.png" alt="Figura 1" width="600" height="287" /> 1.3 Gelcheck después de la reacción termociclado La amplificación de éxito debe ser revisado por la realización de una electroforesis. Inmediatamente después de la bicicleta ha terminado, carga de 5 l de la reacción en un 1% en gel de agarosa TAE. Si la ampliación se ha realizado correctamente, debería ver una banda distinta. Sin embargo, si la nueva síntesis de ADN no es claramente visible en el gel, se puede tratar de precipitar la reacción de todo y lo utilizan para la transformación en el siguiente paso. Para nosotros, esto rara vez ha trabajado, pero vale la pena darle una oportunidad. Lo mejor que puedes hacer si la reacción no funciona es para ajustar la temperatura de recocido. 1.4 DpnI la digestión: Antes de la transformación del plásmido original que sirvió de plantilla debe ser removido de la reacción para evitar que gran experiencia. Esto se realiza mediante digestión de restricción con DpnI. Esta endonucleasa de restricción corta sólo metiladas plásmido. Su reconocimiento y su sitio de restricción es la secuencia GATC, mientras que A tiene que ser desnaturalizado. Durante la digestión con DpnI sólo el plásmido originales sin mutación que fue aislado de una presa + tensión se corta, el plásmido mutado nueva síntesis que no está metilado no se ve afectada por DpnI. Sólo tienes que añadir 2.1 l de DpnI a la reacción e incubar por lo menos una hora a 37 °. Cuando se utiliza el ayuno DpnI digerir el tiempo de incubación puede ser reducida a unos 15 minutos. La calidad de su DpnI y el tiempo de incubación de esta restricción de la digestión en gran medida determina la fortaleza de su fondo con el plásmido sin mutación será. 1.5 Transformación: Después de la digestión del plásmido DpnI está listo para su transformación en las autoridades competentes E. células de E. coli. Sólo tienes que añadir 5 l de la reacción de digestión DpnI en las células competentes y llevar a cabo la transformación como se recomienda en su laboratorio. En nuestro caso usamos el procedimiento de golpe de calor mediante la incubación de la mezcla de bacterias plásmido en hielo durante 30 minutos y después de choque térmico es de 42 ° C durante 90 segundos. Después de la adición de 200 l SOC-solución, las bacterias se incuban, sacudiendo vigorosamente, a 37 ° C durante 1 h. Después de 1 hora la bacteria se colocan en la selección de los medios de agar. 2.0 El segundo día 2.1 Detección de clones parte 1: Selección de los clones Debido a que la eficiencia de la mutación no es 100% que necesita para la detección de su mutantes. Hacemos esto por restricción de la digestión en el que se comprueba la presencia o ausencia del sitio de restricción que se agregan o eliminan mediante la integración de imprimación. Para ello se suelen recoger ocho colonias y hacerlas crecer durante la noche para la preparación de plásmido al día siguiente. 3.0 El tercer día 3.1 Detección de clones parte 2: restricción de la digestión con la enzima marcador En el tercer día se realiza una preparación Mini y restricción de la digestión posterior con el marcador de la enzima. En el gel que se puede ver que uno de sus clones portadores de la mutación deseada y cuáles no. El método de detección sistemática mediante restricción de la digestión funciona muy bien. Sin embargo, usted debe confirmar la mutación del éxito de la secuenciación.

<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.

Mutagénesis dirigida es un método de mutagénesis, que proporciona una forma rápida de mutar un gen transportado por un plásmido. La reacción general se puede hacer en un solo día. Con este método, sólo necesitará un par de cebadores de cortesía llevar a las mutaciones deseadas y una revisión de la polimerasa como PFU-polimerasa. El plásmido de nueva síntesis se puede separar de los padres plásmido por digestión de la reacción con la enzima de restricción DpnI. Esta enzima digiere sólo el ADN metilado....

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<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>
</tr>		</tbody>
		</table>
		</div>

homemade site directed mutagenesis of whole plasmids

Mutagénesis dirigida de los plásmidos es toda una forma sencilla de crear variaciones ligeramente diferentes de un plásmido original. Con este método el objetivo de genes clonados pueden ser alterados por la sustitución, supresión o inserción de unas cuantas bases directamente en un plásmido. Funciona simplemente amplificando el conjunto del plásmido, en una reacción de termociclado no basados ​​en la PCR. Durante la reacción de primers mutagénicos, portadores de la mutación deseada, se integran en el plásmido de nueva síntesis. En este video tutorial se demuestra de una manera fácil y rentable para introducir sustituciones de bases en un plásmido. El protocolo trabaja con reactivos de referencia y es independiente de los kits comerciales, que a menudo son muy caros. La aplicación de este protocolo se puede reducir el costo total de una reacción a una octava parte de lo que cuesta utilizar algunos de los kits comerciales. En este vídeo también comentarios sobre los pasos críticos durante el proceso y dará instrucciones detalladas sobre cómo diseñar los primers mutagénicos.

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

Homemade Site Directed Mutagenesis of Whole Plasmids

Mutagénesis dirigida de los plásmidos es toda una forma sencilla de crear variaciones ligeramente diferentes de un plásmido original. Aquí se demuestra de una manera fácil y rentable para introducir sustituciones de bases en un plásmido usando reactivos estándar.

Mutagénesis dirigida casera de plásmidos enteros

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 vistas.  Johannes Gutenberg-University Mainz, Germany. Mutagénesis dirigida de los plásmidos es toda una forma sencilla de crear variaciones ligeramente diferentes de un plásmido original. Aquí se demuestra de una manera fácil y rentable para introducir sustituciones de bases en un plásmido usando reactivos estándar.

Video: 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.

Principios de la recombinasa específica de sitio (RSS) Tecnología

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

Investigación

Biología

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.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

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.

Introducción al Laboratorio de Worm: El cultivo de gusanos de Mutagénesis

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.

Mutagénesis y funcional protocolos de selección de la evolución dirigida de proteínas en 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.

Examinar la dinámica conformacional de las proteínas de membrana In situ Sitio-dirigida con fluorescencia de etiquetado

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.

El análisis de la función de las pequeñas GTPasas de la microinyección de plásmidos en células epiteliales polarizadas

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.

Único ovocito Mutagénesis bisulfito

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.

Selección de alto rendimiento funcional utilizando un doble resplandor de ensayo de luciferasa Homemade

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.

Generación de<em&gt; Enterobacter</em&gt; Sp. YSU auxótrofos Usando transposón mutagénesis

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.

Un protocolo para la evaluación funcional de la Proteína Total-mutagénesis de saturación Bibliotecas La utilización de secuenciación de alto rendimiento

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