BIOL 2500 - Topic 10 (part 2)

Restriction mapping

The process of mapping the restriction sites on a specific piece of DNA

Retriction enzymes

Specific enzymes that cuts DNA at specific points/sequencies, resulting double stranded breaks

Where do restriction enzymes come from?

They come from bacteria, and are named after the organism from which they come from

Advantages of restriction mapping

1.) Cheaper than DNA sequencing

2.) Can compare the DNA fragments across multiple organisms

3.) Allows you to obtain start and end sequences, which would otherwise need primers in DNA sequencing

Advantages of restriction enzymes

It can be used to insert cloned DNA or PCR products into plasmids

Restriction enzyme cut ends

It can either create blunt ends or sticky ends

Sticky ends

Ends created by restriction enzymes that have overhangs

Advantage of sticky ends

Two molecules that are cut by the same restriction enzymes will have complementary sticky ends, therefore allowing for those two pieces to be joined together via ligation

Restriction mapping provides…

It provides a foundation for further manipulation of the DNA fragments, as it allows you to see where you can make more cuts to turn the DNA into fragments

Main steps of restriction mapping

1.) Digest the DNA with specific restriction enzymes (which results in fragments of various lengths)

2.) Separate the fragments via gel electrophoresis

3.) Visualize the DNA fragments (using a stain or a probe that fluoresces under UV light)

Linear vs. circular DNA molecules (two fragments)

1.) If a linear molecule that is cut has two fragments, that means it has one cut site

2.) If a circular molecule that is cut has two fragments, that means it has two cut sites

Polymerase chain reaction

1.) An in vitro process that amplifies DNA (i.e. makes multiple copies of DNA)

2.) It was developed by kary mullis

Importance of PCR

It is the first step in DNA sequencing and gene cloning, as these types of processes require lots of copies of the DNA molecule in question

PCR takes place in a…

In a tiny tube filled with the reaction mix

PCR ingredients

1.) ds genomic DNA, from the organism of interest (i.e. the template)

2.) Thermostable Taq DNA polymerase

3.) Primers specific to the sequence of interest

4.) Buffer solution with cofactors

5.) dNTPs

Taq DNA polymerase

The thermostable enzyme used for PCRs, isolated from Thermus aquaticus, a thermophilic bacterium isolated from hot springs