Chapter 5C: Nucleic Acid Hybridization and DNA Sequencing

What does a Northern blot detect? 

RNA sequences

Steps in Northern blotting?

1. Separate RNA

2. Transfer

3. Hybridize with DNA probe

4. Detect

Purpose of Northern blot?

Study when and where a gene is expressed

Why is it called “Northern” blot?

Joke on southern blot inventor’s name

What does a western blot detect? 

Proteins

Another name for Western blot?

Immunoblotting 

Steps in Western Blotting?

1. SDS-PAGE

2. Transfer

3. Primary antibody 

4. Secondary antibody 

5. Detect 

What does a western blot tell us?

Presence and rough quantity of protein

Southern = …

DNA

Northern = …

RNA

Western = …

Protein

Purpose of in situ hybridization? 

Locate specific genes on chromosomes 

Steps for In Situ Hybridization

1. Prepare Chromosomes

2. Label probe

3. Primary antibody 

4. Secondary antibody 

5. Stain 

6. Detect fluorescence 

Solor indicating gene of interest (In Situ Hybridization)

Yellow Fluorescence

Color of the rest of the chromosome (In Situ Hybridization)

Red Fluorescence

Who developed DNA sequencing?

Sanger, Maxam, Gilbert

Nobel Prize year for DNA Sequencing?

1980

Key idea of Sanger sequencing?

Chain termination with ddNTPs

Why ddNTPs stop synthesis? 

lack 3’OH → no phosphodiester bond 

How are fragments detected?

Radioactive or fluorescent labeling

Four reactions in Sanger sequencing (DNA sequencing)

One for each base : A, T, G, C

main improvements over manual Sanger?

Fluorescent ddNTPs → automatic detection

How is the sequence read? 

1. Laser excites dye

2. detector reads colors

3. computer outputs sequence

High-throughput sequencing output?

Millions of nucleotides quickly

Pyrosequencing principle?

1. Nucleotide incorporation

2. light flash 

3. sequence detected 

Purpose of reporter genes

Monitor gene activity

Examples of fluorescent reporters

GFP, DAPI

Examples of luminescent reporters?

Luciferase

Examples of selectable reporters?

lacZ, CAT

Purpose of Site-Directed Mutagenesis?

Introduce specific DNA changes

Types of DNA changes

Insertions, deletions, substitutions

Why make DNA changes?

Study protein function or modify DNA

Nitrocellulose filter-binding assay principle?

DNA-protein complex binds filter; free DNA does not

Gel mobility shift assay principle?

Protein-bound DNA moves slower in gel

Foot printing purpose?

Identify exact DNA binding site of protein

ChIP pupose?

Detect protein-DNA interactions in chromatin

Co-Immunoprecipitation (Co-IP) purpose?

Identify protein-protein interactions

How Co-IP works

Antibody captures target protein → co-precipitated partners detected

knockout mice? 

Gene is deleted or nonfunctional 

Transgenic mice?

Added foreign genes