​ Figure 9. Comparison of two-row (A, DH39) and six-row (B, DH46) phenotypes in Oregon Wolfe barley Informative and Spectacular Subset (OWB-ISS). These phenotypes are controlled by alternate alleles of the Vrs1 gene (Komatsuda et al., 2007). The six-rowed phenotype originated multiple times and in different regions through independent mutations of Vrs1. The gain in yield established barley as a founder crop for Near Eastern Neolithic civilization. ​

​​ Figure 10. Restriction digest of Vrs1 amplicons. A, Restriction enzymes cut DNA at specific sequences. If you change the sequence, the cut site is not recognized, so the DNA remains intact. B, Restriction enzyme cut sites for two-row (top) and six-row (bottom) allele of the Vrs1 locus in Oregon Wolfe barley (OWB).​

Learning Objectives

• Upon completion of this exercise, students will be able to
• Explain the limitations of PCR and gel electrophoresis to detect differences in alleles.
• Outline the process of restriction digestion and explain how it can detect DNA sequence changes.
• Interpret gel electrophoresis data to identify allelic differences in the Vrs1 gene.
• Discuss and evaluate evidence to support claims concerning GMOs and domestication of plants by artificial selection.

Introduction: Restriction Digests

Since the two alleles are the same length in terms of DNA base pairs, we cannot simply use PCR and gel electrophoresis to differentiate between the two-row and six-row plants. To determine the genotype at the Vrs1 locus, we will amplify the plant DNA using PCR and then digest it with a restriction enzyme (Fig. 10). Restriction enzymes cut DNA at specific sequences. The enzyme used in this activity, Ncil, cleaves the dominant two-row allele in three places, resulting in four DNA bands during electrophoresis. The change in DNA sequence in the vrs1 (six-row) allele happens to be at one of the Ncil cut sites, eliminating this cut site, so Ncil will cut the recessive six-row allele in two places, resulting in only three fragments.

PCR of the Vrs1 Gene

Materials

• Thermal Cycler
• Centrifuge Tubes (1.5 ml)
• Cup, Ice
• Micropipettes
• Pipette Tips
  
• Vrs1 PCR Primers
• Molecular-Grade Water
• DNA Template(s)
• Vortex, Markers
• Regular PCR Tubes Protocol: 0.2-ml PCR Tubes, Taq DNA Polymerase, and Master Mix
• PCR Tubes with Taq Beads Protocol: 0.2-ml PCR Tubes with Taq DNA Polymerase Beads

!PCR of Vrs1 Gene from Two-Row and Six-Row Samples

1. Obtain your DNA in the 2.0 ml microcentrifuge tube. Begin to thaw it out. Obtain a PCR tube with a Taq Polymerase bead at the bottom and label it like the other tubes with your OWB #, class period, date, and initials (you can just use your initials instead of your full name since the tube is small).
2. While you are waiting, put crushed ice in your cup.
3. Make sure the bead is at the bottom of the tube. Your instructor will add 24 µl of the Vrs1 primer mix to your PCR tubes.
4. Add 1 µl of your DNA template to your PCR tube.
5. Vortex the tube until the bead fully dissolves and the solution is clear.
6. Tap the PCR tube on the table to get all of the solution down to the bottom of the tube.
7. Store tubes on ice until instructed to transfer your tubes to the thermal cycler.
8. Give your tube of DNA back to the instructor for storage at –20°C. 

Restriction Digest of the rs1 Gene from Two-Row and Six-Row Plants

Materials:

Vrs1 PCR Product (DNA)                    
New England Biolabs (NEB) Buffer 4
NciI Restriction Enzyme
Molecular-Grade Water
Micropipettes, Gel Box
Tips, Parafilm, Loading Dye
0.2-ml PCR Tubes
1.5-ml Centrifuge Tube
37°C Waterbath or Thermal Cycler
Cup, Ice, Markers

Day 1: Restriction digestion of PCR product

1. Obtain a cup with crushed ice. Get your Vrs1 PCR product tube from your instructor and place on ice.
2. Obtain a new, empty 0.2 ml PCR tube and label it like the Vrs1 PCR tube (your OWB #, class period, date, and initials), but add the word 'Digest' to the side.
3. Add 20 µl of the Vrs1 PCR product to the new tube. Keep the remaining tube on ice.
4. Take the tube to your instructor to have 5 µl of the reaction mix added.
5. Store tubes on ice until instructed to transfer your tubes to be incubated at 37°C for 1 h.
   

Day 2: Vrs1 restriction product electrophoresis

1. ​Obtain your digest product from the instructor.
2. On a piece of wax paper (parafilm), combine 3 µl of loading dye with 10 µl of digest product. Draw up and dispense the mixture with your pipette three to four times to mix it. Your product should be a blue color once mixed.
3. Take your wax paper over to the gel electrophoresis. Add 10 µl of the blue DNA/dye mixture to the correct gel well indicated below (5 ml for uncut).
   

Gel Map

4. ​​Repeat Steps 1-3 for each sample you want to load onto the electrophoresis gel.
5. Be sure to use a new tip for each sample! Don't worry about getting the micropipette tip point down into the gel well. Hold the tip over the gel well you are targeting and dispense the DNA. The loading dye causes it to sink down into the well.
6. When all samples are loaded, run the gel electrophoresis at 70 volts for 1 hour 15 minutes. These conditions are optimal for the resolution of the DNA fragments; however, they can be adjusted to complete the run within a class period (e.g., 80 volts for 50 minutes).
7. Gently remove the gels and photograph for later analysis.
   

Day Three: Analyzing the data

Calculate the size of each fragment of the Vrs1 (two-row) allele after restriction digestion (# of fragments and size of each)

Number: ________

Size of fragments (from largest to smallest): _____________

Calculate the size of fragment of the vrs1 (six-row) allele after restriction digestion (# of fragments and size of each)

Number: ________

Size of fragments (from largest to smallest): ______________

Researchers can estimate the size of fragments by comparing them to a lane (called the ladder) with fragments of known sizes. Looking at the picture of your gel, what are the sizes of the restriction digestion fragments in the two-row and six-row samples. Use the table below to fill in the sizes of the fragments measured on the gel.

 
Discussion Questions

1. Does the data support your hypotheses?
   
   Answer: Dependent on classroom results.
   
   
2. Explain and give reasons why you may see unexpected banding in your samples.
   
   Answer: Restriction enzyme did not cut completely (partial digest), genotypes were mixed up, a heterozygote will produce all combinations of Vrs1 and vrs1 fragments.​
   

​​Overview​​Experiment 1​​Experiment 3
​iTAG Instructor’s Planning Resources
​​AppendixGlossary​​