Seminario biologia molecular

A comparison of the efficiency of five

different commercial DNA

extractionkits for extraction of DNA from faecal

samplesby

Shantelle Claassen, Elloise du Toit ,,Mamadou Kaba, Clinton Moodley ,Heather J. Zar and

Mark P. Nicol .

A comparison of the efficiency of five

different commercial DNA

extractionkits for extraction of DNA from faecal

samplesby

Shantelle Claassen, Elloise du Toit ,,Mamadou Kaba, Clinton Moodley ,Heather J. Zar and

Mark P. Nicol .

BY

MARIA CAROLINA RAVE AGUIRRE

MARIA ISABEL PALACIO

MOLECULAR BIOLOGY

MEDICINE STUDENTS 3nd SEMESTER

TEACHER LINA MARIA MARTíNEZ

SÁNCHEZ

AUGUST 23, 2013

MEDELLÍN - COLOMBIA

BY

MARIA CAROLINA RAVE AGUIRRE

MARIA ISABEL PALACIO

MOLECULAR BIOLOGY

MEDICINE STUDENTS 3nd SEMESTER

TEACHER LINA MARIA MARTíNEZ

SÁNCHEZ

AUGUST 23, 2013

MEDELLÍN - COLOMBIA

ARTICULO ORIGINAL

INTRODUCTION

Variations in the composition of the gut microbiots between individuals are recognized for the development of independent analytic techniques.

These techniques have contributed to many studies for investigating the human gut microbiots and its role in health and disease of the people.

For this objetive is necesary obtain enough amounts of high quality genomic DNA.

Extraction of Deoxyribonucleic acid (DNA) from faeces is very complicated because many substances may be co-extracted, resulting in inhibitory effects of past applications.

Also in extracting DNA from faeces is possible has incomplete lysis cells and shearing of DNA which may also affect the process.

Extracted genomic DNA is used for represent all microbial communities present in the sample of faeces.

The human gastrointestinal tract (GIT) is colonized with germs which have diferent cell walls structures and compositions, making some species more difficult to lyse than others, like Bifidobacteria and Lactobacillus.

GENETIC MATERIAL

DNA EXTRACTION

FENOL CLOROFORMO •Peptides and proteins are extracted from the organic phase with phenol and then used the proteinase K•very toxic the agents used•you can lose many shows whether degrades too CHELEX•DNA has single strand •reagents are not toxic•You don’t lost a lot of sample

MICROBIOTS

Known like: The normal microbiots, normal microbial flora or human microbiome

Meaning: is the set of microorganisms that are found frequently in different parts of the human body in healthy people.It is in a symbiotic relationship with the host, because are advantages for the host and the microorganism.

It is the community of living microorganisms resident in a particular ecological niche

Classification of Human microbiots

Indigenous microbiots: Microorganisms that colonize the host for a long time, may participate in the physiological functions and have evolved along with the species

Allochthonous microbiots: Microorganisms that can be found in any habitat and system, usually do not contribute to the physiology of the host and are present transiently or latently

Also it´s classified by the time in the host:

Latent Microbiots: Microorganisms that have the host for most of my life, have no major fluctuations in population and often have symbiotic with the host activity.

Transient Microbiots: It is continuous fluctuations in the population and not essential for the survival of the host. can colonize in changes as habitat change, age, season, use of antibiotics, etc.

The term “flora" is a mistake

The term “flora" is a mistake

in this case refers to the host like a

carrier

in this case refers to the host like a

carrier

This microorganisms are called opportunistic

pathogens when they cause diseases.

This microorganisms are called opportunistic

pathogens when they cause diseases.

The most common are: Staphylococcus aureus, Escherichia

coli and Candida albicans

The most common are: Staphylococcus aureus, Escherichia

coli and Candida albicans

STOOL

MICROORGANISMS PRESENT IN THE STOOL

Bacteroides speciesBifidobacteriesEubacteriesColiformesEnterococo feacalisCandidaE. coli

DNA extraction

DNA extraction

MICROBIOTSMICROBIOTS

STOOLSTOOL

OBJECTIVES

Compare the relative efficacy of extracting bacterial genomic DNA from human faecal samples using five commercial DNA extraction kits.

Evaluate the DNA extraction kits based on their ability to efficiently lyse bacterial cells, cause minimal DNA shearing, produce reproducible results and ensure broad-range representation of bacterial diversity.

MATERIALES Y MÉTODOS

SUJETOS Y RECOLECCIÓN DE MUESTRAS

cuatro niños menores de dos años y cuatro adultos, después de obtener el consentimiento de los individuos sanos o de sus tutores. Después de la recolección, la muestras se transportaron en hielo y se almacenaron a 70 °C antes de su procesamiento.

EXTRACCIÓN DE ADN A PARTIR DE MUESTRAS FECALES

Extracción del ADN utilizando inicialmente

dos cantidades de materia fecal: 100 mg y

200 mg

Extracción del ADN utilizando inicialmente

dos cantidades de materia fecal: 100 mg y

200 mg

La lisis celular mecánica se realizó en BEAD-BEATING por todos los kits a 50 Hz

durante 5 minutos usando la TissueLyser LT™

La lisis celular mecánica se realizó en BEAD-BEATING por todos los kits a 50 Hz

durante 5 minutos usando la TissueLyser LT™

TABLA 1.

Extracción automatica utilizandoQIAsymphony® Virus/BacteriaMidi Kit (kit QS)Fuera del tablero de lisis se incorporo : 750 μl de buffer de ZR Fecal DNA MiniPrep™ kit (kit Z) combinada con la lisis celular mecánica.

EXCEPTO QIAamp® DNA Stool

Mini Kit (kit QA), por

recomendaciones del fabricante

EXCEPTO QIAamp® DNA Stool

Mini Kit (kit QA), por

recomendaciones del fabricante

El lisado se centrifugó a 10000 rpm durante 1 minEl lisado se centrifugó a 10000 rpm durante 1 min

300 μl del sobrenadante resultante fueron usados para la extraccion del DNA con

ayuda del QIAsymphony® SP instrument (Qiagen, Hombrechtikon, Switzerland)

300 μl del sobrenadante resultante fueron usados para la extraccion del DNA con

ayuda del QIAsymphony® SP instrument (Qiagen, Hombrechtikon, Switzerland)

Para el kit de Z, se agrego un paso adicional centrifugación a 14000 rpm durante 1 minPara el kit de Z, se agrego un paso adicional centrifugación a 14000 rpm durante 1 min

Muestras fecales, en Buffer ASL, para el kit de control de calidad mediante el TissueLyser LT ™ (Qiagen, Fritsch GmbH, Idar-Oberstein, Alemania) a 50 Hz durante 1

min

Muestras fecales, en Buffer ASL, para el kit de control de calidad mediante el TissueLyser LT ™ (Qiagen, Fritsch GmbH, Idar-Oberstein, Alemania) a 50 Hz durante 1

min

Calefacción opcional del lisado fecales a 95 °C en lugar de 75 °CCalefacción opcional del lisado fecales a 95 °C en lugar de 75 °C

El ADN extraído se trató con 3,0 g de RNasa A (Sigma-Aldrich, Carlsbad, Estados Unidos de

América)

El ADN extraído se trató con 3,0 g de RNasa A (Sigma-Aldrich, Carlsbad, Estados Unidos de

América)

Todos los DNA extraidos fueron eluidos en 50 μl de agua destilada, excepto el kit QS, donde el volumen de elución minimo que sugiere

el proveedor es 60 μl usando Buffer AVE.

Todos los DNA extraidos fueron eluidos en 50 μl de agua destilada, excepto el kit QS, donde el volumen de elución minimo que sugiere

el proveedor es 60 μl usando Buffer AVE.

LA CALIDAD Y CANTIDAD DE ADN EXTRAÍDO DE LA MUESTRA

FECALEl rendimiento de ADN (ng) y pureza (relación de absorbancia a 260/280) de ADN genómico extraído se determinó espectrofotométricamente utilizando el Nano gota ® ND-1000 (Nanodrop Technologies Inc., Wilmington, Estados Unidos de América)

.

Integridad del ADN genómico se determinó por visualización de 200 ng de ADN en gel de agarosa al 1% (w / v) que contiene 0,25 g / l de bromuro de etidio (EtBr), se ejecutan en 1 x buffer Tris-EDTA a 100 Voltios

Integridad del ADN genómico se determinó por visualización de 200 ng de ADN en gel de agarosa al 1% (w / v) que contiene 0,25 g / l de bromuro de etidio (EtBr), se ejecutan en 1 x buffer Tris-EDTA a 100 Voltios

EVALUACIÓN DE LOS MÉTODOS DE EXTRACCIÓN DE ADN POR

PCR EN TIEMPO REALGENERACIÓN DE UNA CURVA ESTÁNDAR CON EL ADN

GENÓMICO

Cepas: Bacteroides fragilis 638R y Bifidobacterium longum spp. longum JCM 1217

se cultivaron en agar Brucella en un dióxido de carbono (CO2) en condiciones atmosféricas (85% de nitrógeno, 10% de CO2 y 5%

de hidrógeno) a 37 ° C. Escherichia coli ATCC (American Type Culture Collection) 25922

se cultivó aeróbicamente en agar Mueller-Hinton a 37 ° C.

EVALUACIÓN DE LA EFICIENCIA DE EXTRACCIÓN DE ADN MEDIANTE

TÉCNICAS DE TOMA DE HUELLAS DACTILARES

ANÁLISIS DGGE DE 16S RDNA AMPLIFICADOS

Análisis DGGE de 16S rDNA amplificadosDos rondas de PCR se realizaron enfocandose en el gen 16S de rDNA bacteriano

T-RFLP DEL 16S RDNA AMPLIFICADOS

RESULTADOS

•QA comparado con U y P, > mediana de rendimiento de DNA.•QS muchísimo mayor que U•QA < puro que P y Z (100mg), tambn < Z y U

•qPCR diana de las tres bacterias que se encuentran comúnmente en las heces no mostraron diferencias consistentes en la ampliación de los DNA de estas bacterias• Como se ha demostrado mediante pruebas de qPCR utilizando DNA extraído por los kits

DISCUSSION

REFERENCE WHAT THEY SAID AGREE OR DESAGREE

De Vos and De Vos; Fujimura et al.

“Over the past decade, the influence of the human gut microbiota on health and desease has been the focus of numerous studies”

Agree

Maukonen et al.; Penders et al.

“with faecal samples being widely used”

Agree

Hayashi et al. “Even though faeces may only partly represent the complexity of the GIT microbiota”

Agree

Dave et al. “it is sampled non-invasively and easy to acquire at minimal cost”

Agree

CONCLUSIONS

The gut microbiot is different in each person, even in the same person, so it is important to identify various strains to knowledge deseases.

It is important use RNAse to purify the samples. The gastrointestinal tract have a variety of

microorganism living in symbiosis. We have many analysis techniques for DNA and all use

different principles, but all of them get to the same result.

Mapas conceptuales

Maria Isabel Palacio

Maria Carolina Rave Aguirre

BIBLIOGRAPHY

MARTINEZ SÁNCHEZ, Lina María. Biología molecular. 7 ed. Medellín: UPB. Fac. de Medicina, 2013. 275-290 p.

Shantelle Claassen, Elloise du Toit ,,Mamadou Kaba, Clinton Moodley ,Heather J. Zar and Mark P. Nicol “A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples”. 14 Mayo de 2013.

Bibliografía