Simulation of wet labs

Digestion of Lambda DNAMapping of phage T7 DNADetection of Alu insertion in human DNA
Contributed by Brack Gillespie, Ashwaubenon High School, Ashwaubenon, WI, Rick Berken, East High School, Green Bay, WI, and Barbara Moffat, University of Waterloo, Ontario, Canada

These exercises were developed to be used along with electrophoresis labs, to prepare students for the lab and/or to allow extensions of the lab activity. They also could be used in place of the lab if time or equipment is not available.

1. Digestion of Lambda DNA

This is a standard lab activity to illustrate the basic features of restriction enzyme digestions. DNA isolated from bacteriophage Lambda is digested with common restriction enzymes – EcoRI, BamHI, HindIII – to demonstrate that enzymes with different recognition sites will yield different band patterns on a gel.

Activity extensions:

  • Use additional enzymes (provided for other case studies or generate your own enzyme files)
  • Simultaneous digestions with two or more enzymes
  • Mapping restriction sites along Lambda DNA (see part 2.a. below)

2. Mapping of phage T7 DNA

This activity has two parts: a) generating a restriction map of phage T7 DNA; and b) determining to which T7 gene(s) the probe binds.

a. Generating a T7 restriction map

Choose two of the enzymes in the T7 folder (NruI, BclI, StuI, BglII). Digest the T7 DNA with NruI, and also perform a double digest by cutting the DNA with NruI and then with one of the other enzymes. Based on the sizes of the fragments generated, determine the relative locations of the enzyme sites along the T7 DNA. Use the Options menu (on the Data Screen of Case It v7.0.4) to convert the gel fragments to mapping fragments, and then drag the fragments to solve the map.

b. Determine which genes are located in the probe binding region

After digesting the T7 DNA and running it on the gel, open the T7 probe and perform a Southern blot. The probe should bind to one fragment. Using the T7 map you created, determine where in the T7 DNA the fragment bound by the probe is located. What T7 genes are located in this region? How would you find this information?

3. Detection of Alu insertion in human DNA

This simulation is demonstrates the detection of the PV92 Alu insert by PCR amplification that is used in many biology classes. This lab allows students to isolate and analyze their own DNA, usually from cheek cells. The human genome contains a variety of transposable elements or “jumping genes” that increase genetic variability. The PV92 locus on chromosome 16 contains a Ya5 Alu insert in some individuals. Since the presence or absence of this insert is not associated with any known phenotype, it is suitable to use with students in a classroom setting. By using primers that bind just outside the insertion site, the presence of the Alu insert can be detected based on the size of the amplified DNA. More background information is available at the DNA Learning Center. There are three genotypes that can be detected: Alu insert on both chromosomes 16, i.e. homozygous for the insert (+/+), absence of the Alu insert on both chromosomes (homozygous -/-), and heterozygous (+/-).

DNA representing each of the three phenotypes is provided. Several primer sets that amplify part of the PV92 Alu locus are available through biology education companies. The primers provided here will amplify a fragment of 416 bp if the Alu insert is absent, and a fragment of 731 bp if the fragment is present.