genetica super bueno
TRANSCRIPT
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GENOTECAS
Curso 2011-12
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CONCEPTO DE GENOTECA
Una genoteca es una colección de clones
conteniendo fragmentos de DNA
que comprenden la totalidad (o casi)
de las secuencias de ese genoma.
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DIGESTION PARCIAL
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Animación > construcción de genoteca
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La baja eficiencia de transformación (10-4)limita el número de clones que se puedenobtener con vectores plasmídicos
La cantidad de DNA que se pude insertar es pequeña, máximo 15-20 Kb
Para obtener genotecas se emplean vectores virales
3.2 Gb 3.2x106 Kb ~ 2x105 clones
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10-4
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Vectores virales
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Fago λ
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El DNA de λ tiene extremos cohesivos
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El DNA de λ tiene extremos cohesivos
formación de concatémeros
procesamiento de concatémeros
empaquetamiento
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Sistema de empaquetamiento in vitro
Extracto dador de precabezas y colas
(mutante λA-)
+
Proteína A(terminasa)
+Concatémero
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Figure 20-6 step 1
Cloning in phage λ
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Figure 20-6 step 2
Cloning in phage λ
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Figure 20-6 step 3
Cloning in phage λ
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Figure 20-6 step 4
Cloning in phage λ
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Figure 20-6 step 5
Cloning in phage λ
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Figure 20-6 step 6
Cloning in phage λ
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Figure 20-6 step 7
Cloning in phage λ
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ln (1- P)ln (1- f)
Depende de la fracción media de genoma en cada clon (f)
Tamaño medio inserto en pbTamaño del genoma en pb
TAMAÑO DE UNA GENOTECA
Número de clones necesarios, N: Depende de P = probabilidad de que una secuencia determinada esté en un clon
f =
N =
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En λ no se pueden clonar mas de ~20 Kb
Para tamaños mas grandes se emplean cósmidos
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(Polylinker)
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10-4
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En un cósmido no se pueden clonar mas de 45 Kb
Para tamaños mas grandes se emplean BAC
Bacterial Artificial Chromosome
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~300 Kb
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En BAC no se pueden clonar mas de 300 Kb
Para tamaños mas grandes se emplean YAC
Yeast Artificial Chromosome
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Animación > Clonación
Animación > Manipulation >Techniques >Transferring & storing > Storing DNA
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TIPOS DE GENOTECAS
•Genotecas de DNA genómico
•Genotecas de cDNA (copiado de mRNA por transcriptasa inversa)
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Obtención de unagenoteca de cDNA
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Rastreo de genotecas
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Hibridación sondas radiactivas
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Paseo cromosómico
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Código genético
2
4
2
4
2
4
1
1
22
2
2
22
2
3
2
2
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Oligos diseñados en regiones de menor degeneración
2304 posibles secuencias