Title: Learn:Testing Your Own DNA: Restriction Digest and Gel Electrophoresis
Course: BTEC-1000-301-302-303-F21 > Section: DNA Analysis

Module 4 Testing Your Own DNA: Restriction Digest and Gel Electrophoresis

The primers you will use in your PCR reaction are specific for a particular region of the TAS2R38 gene. That region contains the SNP that determines whether or not you can taste PTC. After PCR, you’ll have about a billion copies of that TAS2R38 region. But how will you be able to tell whether you have a C (taster) or a G (nontaster) at that particular spot?

To determine whether you have a C or G, you will use a restriction enzyme. Restriction enzymes are like tiny molecular scissors that cut DNA only when they find a particular sequence. There are hundreds of restriction enzymes, and they each have a particular DNA sequence they cut. You will use an enzyme called HaeIII, which cuts wherever it finds the sequence GGCC. If there is not a GGCC, it will not cut the DNA. The SNP we are looking for has either GGCC, in the taster form, or GGGC, in the nontaster allele. Thus, the restriction enzyme HaeIII allows us to distinguish between the two alleles.

The diagram shows how PCR amplification and restriction digestion identifies the G-C polymorphism in the TAS2R38 gene. The “C” allele, on the right, is digested by HaeIII and correlates with PTC tasting.

As you can see from the figure (right side), the 221 bp (base pair) PCR product of the taster allele will be cut into two fragments, 177 bp and 44 bp, by HaeIII. The PCR product from the nontaster allele will not be cut, so it will remain 221 bp. If we can tell which size fragments are in your tube after digesting the PCR product with HaeIII, we can predict whether you have the taster allele, the nontaster allele, or both, and thus how well you will taste the chemical PTC.

So how will you determine the length of your DNA fragments? The technique is called gel electrophoresis. Watch the video below to learn how it works.

Watch

As you saw in the video, gel electrophoresis uses an electric field to separate DNA molecules by size. Opposites attract in electricity, so DNA’s negative charge makes it attracted to the positive end of an electric field. To get to the positive end, the DNA has to pass through the gel. Think of the gel as a dense, vine-filled jungle, the kind humans would use a machete to get through. Who would get through that jungle faster, an elephant or a rabbit?

The rabbit would get through more quickly, because it’s small enough to fit through the spaces between the vines. The elephant’s progress would be very slow, because the vines make it hard for the huge animal to get through. Gel electrophoresis operates in the same way: smaller and more compact molecules travel quickly, while bigger and less compact molecules move more slowly.

When you run your gel, you’ll load samples of your TAS2R38 PCR products, both digested (treated with restriction enzyme HaeIII) and undigested. You will also run a marker or ladder. The ladder is a mix of known sizes of linear DNA. Comparing the migration of your sample with the ladder will allow you to determine the size of your DNA fragments. Below is an image showing the expected results. Your gel will have only three lanes: the ladder, your undigested DNA, and your digested DNA. Your results should look like either the tt nontaster, TT taster, or Tt taster. (A few students will have other patterns, as this test only covers the two most common alleles of TAS2R38; others do exist.)

A gel showing the expected band patterns for tt nontasters, TT tasters, and Tt tasters, along with a ladder (marker) with fragments of known sizes.

Download and read the SNP lab student manual.pdf Note any places you have questions, so you can ask an instructor when you come into the lab.  (An accessible version is here: SNP Student Manual.pdf)