Molecular Biology

Lecture 10

Chapter 5

Molecular Tools for Studying Genes and

Gene Activity

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- Western blot

- Protein-DNA interactions

- Knockout mice

- Genomic or cDNA library screeningDNA hybridizationAntibody screening

Lecture outline

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Immunoblots

Immunoblots (also called Western blots) use a similar process to Southern blots

– Electrophoresis of proteins– Blot the proteins from the gel to a membrane– Detect the protein using antibody or antiserum

to the target protein– Labeled secondary antibody is used to bind

the first antibody and increase the signal

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Western Blots

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Genomic library construction (very complex library)

Dna extractionDigestion with restriction endonucleaseLigation in vectorSelection

cDNA library construction (simplified library)

Extract mRNAReverse transcription to make cDNALigation in vectorSelection

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DNA hybridization

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Selection of clone of interest

DNA hybridizationMust have a probe to screen the library

Antibody screeningMust have antibodies specific for your protein

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Antibody screening

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Assaying DNA-Protein Interactions

• Study of DNA-protein interactions is of significant interest to molecular biologists

• Types of interactions often studied:– Protein-DNA binding– Which bases of DNA interact with a

protein

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Filter Binding

Filter binding to measure DNA-protein interaction is based on the fact that double-stranded DNA will not bind by itself to a filter, but a protein-DNA complex will

– Double-stranded DNA can be labeled and mixed with protein

– Assay protein-DNA binding by measuring the amount of label retained on the filter

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Nitrocellulose Filter-Binding Assay• dsDNA is labeled and mixed with protein• Pour dsDNA through a nitrocellulose filter• Measure amount of radioactivity that passed

through filter and retained on filter

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Gel Mobility Shift• DNA moves through a gel faster when it is not

bound to protein• Gel shift assays detect interaction between

protein and DNA by reduction of the electrophoretic mobility of a small DNA bound to a protein

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Footprinting

• Footprinting shows where a target lies on DNA and which bases are involved in protein binding

• Three methods are very popular:– DNase footprinting– Dimethylsulfate footprinting– Hydroxyl radical footprinting

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DNase Footprinting

Protein binding to DNA covers the binding site and protects from attack by DNase• End label DNA, 1 strand only• Protein binds DNA• Treat complex with DNase I mild conditions for average of 1 cut per molecule• Remove protein from DNA, separate strands and run on a high-resolution polyacrylamide gel

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Knockouts

• Probing structures and activities of genes does not answer questions about the role of the gene in the life of the organism

• Targeted disruption of genes is now possible in several organisms

• When genes are disrupted in mice the products are called knockout mice

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News

Biologists claim Nobel prize with a knock-out

The architects of a technique that has allowed biologists to identify the function of genes easily have been rewarded for their efforts with this year's Nobel Prize in Physiology or Medicine

Mario Capecchi University of Utah in Salt Lake City Martin EvansCardiff University in WalesOliver SmithiesUniversity of North Carolina at Chapel Hill,

From: http://www.nature.com/news/2007/071009/full/449642a.html

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Making a Knockout Mouse: Stage 1

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Making a Knockout Mousse: Stage 2

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Knockout Results

• Phenotype may not be obvious in the progeny, but still instructive

• Other cases can be lethal with the mice dying before birth

• Intermediate effects are also common and may require monitoring during the life of the mouse