seminario biologia molecular

Denaturing Gradient Gel Electrophoresis (DGGE) as a Powerful Novel Alternative for Differentiation of

Epizootic ISA Virus Variants

Marisela Carmona, Dagoberto Sepúlveda, Constanza Cárdenas, Luis Nilo, Sergio H. Marshall

Valentina CartagenaDariela Espinosa Navarro

Tercer SemestreFacultad de Medicina

Universidad Pontificia BolivarianaMedellín, Antioquia

2012

INTRODUCTION

A molecular fingerprinting technique that has been used to examine microbial diversity in complex communities.

Polymerase chain reaction (PCR)-generated DNA fragments of the same length but with different base-pair sequences can be fully separated in a fine-tuned gradient gel.

DGGE constitutes a robust procedure by which a single point mutation can be detected.

Denaturing Gradient

Gel Electrophoresis (DGGE)

INTRODUCTION

• The etiological agent, named ISA virus or ISAv Orthomyxoviridae family and constitutes the only member of the genus Isavirus .

• Virus whose genome consists of eight negative-sense, single-stranded RNA segments segments 5 and 6 are considered key elements in defining pathogenicity

VIRUS

• Encoding a fusion (F) protein that appears to be involved in the fusion of viral and cellular membrane.

Segment 5

• Encodes a haemagglutinin-esterase (HE) protein that mediates both receptor-binding and receptor-destroying activities, as well as putatively participates in the fusion process.

Segment 6

INTRODUCTION

Is a viral disease that causes severe losses in the Atlantic salmon (Salmo salar) farming industry.

The disease first spread rapidly along the Norwegian coast, then to Canada and Scotland, later in the Faroe Islands and eventually to the USA . In Chile, ISA was first detected in marine farmed Coho salmon (Oncorhynchus kisutch) in June 1999 and the first outbreak in Salmo salar occurred in 2007.

The virus has also been reported to be present in apparently healthy wild animals

Infectious salmon anemia (ISA)

Determinate the Denaturing Gradient Gel Electrophoresis (DGGE) as a powerful Novel alternative for differentiation of Epizootic ISA Virus Variants.

GENERAL OBJECTIVE

MATERIALES Y MÉTODOS

MUESTRAS

Órganos combinados de salmones infectados:

• Riñón• Corazón• Agallas

Se confirmaron como POSITIVAS o NEGATIVAS al virus a través de PCR.

VARIANTES DEL VIRUS

• GIM-1 33 nucleótidos del segmento 5

• HPR7b GENOTIPO segmento 6

• GIM Y GIM -2-3 carecen de inserción en segmento 5

• HPR2 Y HPRo segmento 6 • HPR5 SOLO segmento 6

Se utilizaron variantes para demostrar la versatilidad

EXTRACCIÓN DEL RNA

• La posibilidad de aislar RNA lo más intacto posible es esencial tanto para el clonado de genes como para el análisis.

• Homogeneización de la muestra

• Separación y Purificación

• Precipitación

MODELO Y DISEÑO DEL PRIMER

• El primer fue diseñado usando primer3 software

• Se seleccionaron en base a:Secuencia de conservaciónEspecificidad estricta Rendimiento de producción Ausencia de estructura secundaria

Herramienta para diseñar y analizar primers para PCR

PCR

• Técnica que nos permite amplificar selectivamente un segmento específico de DNA, hasta obtener una cantidad suficiente para su posterior manipulación.

• Ciclos de desnaturalización• Apareamiento con cebadores

• Extensión por una ADN polimerasa

PCR DGGE

• Herramienta de diagnóstico rápida, barata y sencilla. De alta sensibilidad y especificidad.

• Implica un cambio de un sólo nucleótido responsable de modificar un sólo aminoácido

• Se trabaja combinando las técnicas de la PCR y la DGGE.

DGGE

• Técnica de identificación molecular utilizada en investigaciones para examinar diversidad microbiana, detectando un punto único de mutación.

• Formamida desnaturalizante, porcentaje de acrilamida, puesta a punto del cebador.

Se utilizó en regiones codificantes del virus importantes para la patogenicidad.

RESULTADOS

Figura 1

Segmento 5 (A) NORUEGA• IN1 8 aminoácidos segmento 3• IN2 11 aminoácidos segmento 5 • IN3 10 aminoácidos segmento 5CHILE• IN4 11 aminoácidos segmento 2Segmento 6 (B)• 35 aminoácidos, eliminando la codificación de 7 a 23 residuos,

que representan una región altamente polimórfica (HPR), que ha resultado ser crucial en la determinación de la virulencia.

Posición de índice de acidez de nucleótidos y aminoácidos:• EU130923 (EU851044) segmento 5 • EU118820 segmento 6.

RESULTADOS

Figura 2

DGGE

Análisis de cuatro variantes HPR en la separación DGGE no estandarizado.

Carriles 1-2 dos aislamientos HPR2. independientes

Carriles 3-4 dos aislamientos HPR7b independientes.

Figura 3

• Carril M: ADN 1 - marcador Kb

• Carriles 1-8 corresponden a las letras bandas (A-H) en la Figura 2.

Figura 4

DGGE

Análisis para las diferentes concentraciones de primer de una variante HPR7b clave.

Duplicar reacciones: Carril 1: 25 nMCarril 2: 20 nM Carril 3: 18,6 nMCarril 4: 12,5 nM

Figura 5

Análisis DGGE perpendicular de todas las variantes seleccionadas para ambos segmentos.

• Panel A: Resolución de variantes en el segmento 5 con y sin inserción. • Panel B: Resolución de las cinco variantes (HPR HPR0, HPR2, HPR5, HPR7b

y HPR8, respectivamente).

DISCUSSION

NAME WHAT HE SAID YES NO

35 Top B The rationale behind it is that fine separation is based on the melting behavior of doublestranted DNA and that melting behavior in turn depends on the base-pairs composition of the target DNA.

X

36 Hayes VM We subsequently made selections based on the denaturant reagent (urea/formamide) ratios. Using established DGGE protocols as reference.

X

41 Nagamine CM Or the formation of heteroduplexes during the PCR reaction in which two or more homologous genes or alleles that differ for a point mutation or insertion/deletion are amplifies using the same primers

X

44 Thornhill DJ Or anomalous melting behavior due to either the dragging or fragments through the gel matrix or secondary structure formation of DNA single strands

X

CONCLUSIONS

• This research is interesting because by two well-known and functional techniques science can generate a test that can be more cheap and which can be used in many and the fields of biology.

• It is interesting to see how a mutation can be hidden in the depths of the central dogma and is more interesting how has been developing tests that science use to decipher all these changes so small that can cause diseases that reach not only do so much damage in people but also in animals

• It is also important the learning we acquire when we study this type of procedure on segments of RNA or DNA and then we can know more about their actions.

• Research studies are essential to reaffirm our knowledge with new ideas to help us to develop of their own ideas in the future, or other research more interesting for everyone.

Valentina Cartagena

Dariela Espinosa

GRACIAS