BIOL 2500 - Topic 10 (part 1)

Developing technologies

Molecular biologists use their understanding of cellular processes to develop new technologies

Different types of DNA technologies

1.) PCR

2.) DNA sequencing

3.) Restriction enzymes

4.) Gene editing

DNA technologies

Techniques that obtain, amplify, and manipulate DNA fragments

Applications of DNA technology (example)

1.) Genetic engineering

2.) Genomics

Example of what DNA technologies can allow us to do

It takes a DNA sequence from the human genome and then inserts it into bacteria to be expressed, allowing us to perform gene editing or make bulk amounts of the product

In vitro vs. In vivo

In vitro —> Inside a tube

In vivo —> Inside the cell

What is essential for genetics research

Detecting and quantifying DNA, RNA, and proteins

Why is detecting and quantifying DNA, RNA, and proteins important for genetics research

1.) So that we can determine the size of specific genes

2.) So that we can identify different forms of proteins in humans and/or between organisms

3.) So that we can determine if genes are upregulated or downregulated

In vitro DNA

1.) Southern blots

2.) PCR

In vitro RNA

1.) Northern blot

2.) RT-PCR

In vitro protein

Western blot

In vivo DNA

FISH

In vivo RNA

In situ hybridization

In vivo protein

Immunofluorescence

Regardless of whether it is in vitro or in vivo, detecting and quantifying DNA, RNA, and proteins require…

It requires a probe

Blotting

An in vitro method that detects and quantifies nucleic acids and proteins, which utilizes gel electrophoresis to separate the molecules

Southern Blot

1.) Works with DNA

2.) Named after Edwin Southern (the discoverer)

Northern Blot

Works with RNA

Western Blot

1.) Works with proteins

2.) It uses antibodies as probes to bind to specific proteins, allowing it to be detected via fluorescence or chemiluminescence

Southern blot vs. Northern and Western blot

Northern and Western blots are just modifications of the southern blot, which is what came first

Southern and Northern blot vs. Western blot

1.) Southern and Northern blots use similar probes, usually a nucleic acid fragment

2.) Western blots use antibodies as probes

General blotting steps

1.) Do a gel electrophoresis

2.) Transfer the molecule onto a membrane

3.) Visualize the sample using the radioactive probes and autoradiography

Specific blotting steps (1-4)

1.) Make the gel (usually 1% agarose)

2.) Load the gel onto a buffer

3.) Load the sample into the wells

4.) Run an electrical current to separate the molecules

Specific blotting steps (5-8)

5.) Transfer the molecules onto the special paper (i.e. the membrane)

6.) Add in the probes

7.) After washing away the excess probes, expose it to radiation to activate the fluorescence

8.) Analyze the results