DNA Isolation Lab

To determine an organism’s genotype, we need to purify its DNA

• with purified DNA, we can determine genotype of specific traits, ancestry, test for infectious diseases, food fraud, environmental DNA, and identification for forensics.

How do we isolate DNA after collecting it?

1. DNA Extraction - the process of isolating, purifying, and releasing DNA from within cell nuclei, breaking down membranes, and removing proteins to make the genetic material available for analysis

   • use soap to break down the membrane, as it creates a hypotonic environment that causes the lipid bilayer to burst

   • use salt to break down the proteins, as it denatures them by disrupting the ionic and hydrogen bonds that maintain their structure

2. Polymerase Chain Reaction (PCR) - a technique to make many copies of a specific DNA region in vitro (in a test tube rather than an organism). It quickly amplifies DNA from small amounts in order to be analyzed

   • begin by heating DNA to 94 degrees celsius to break hydrogen bonds and separate template strands

   • next, add in primers, nucleotides, and thermophilic polymerase into the test tube

     • primers bind to the template

     • once we reduce the heat to 60 degrees celsius, hydrogen bonds bind to the primers

     • in 90 seconds, the copies are seen

3. DNA Polymerase binds and makes new strands of DNA

   • here, we reduce the temp down to 78 degrees celsius

How can we visualize the DNA?

Gel electrophoresis - a technique used to separate DNA fragments according to their size and charge

• when heterozygous, two different bands (alleles) and sizes are shown

• when homozygous, one band (allele) is shown