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Determination of Phylogenetic Relationships Using Restriction Analysis

By David S. Ely

Type of Entry:

  • Project

Type of Activity:

  • Hands-on
  • Inquiry lab

Target Audience:

  • Advanced/AP Biology
  • Genetics, Biotechnology

Background information:

A series of very exciting and innovative laboratory activities dealing with evolutionary molecular biology has been conducted over the past two years. The general idea was to determine evolutionary relationships among several strains of mice by examining some genes in their mitochondrial DNA. The idea and materials for these labs came from an evolutionary biologist at the University of Vermont. My contact there graciously provided the DNA necessary for our molecular biology experiments. Rodent (mouse) mitochondrial DNA was isolated and amplified using polymerase chain reaction (PCR) techniques.

Description of Activity

It took several weeks of molecular biology lab activities using amplified DNA to establish phylogenetic relationships among the mice. Pieces of mitochondrial DNA genes from several strains of mice were amplified. Teams of students were given samples of mitochondrial DNA from several sources. Over several weeks, each of these samples was treated with a variety of restriction enzymes. Some enzymes had short recognition sites while others were six-base cutters. The samples were digested employing a normal DNA digestion protocol. A sample (DNA), a specific restriction enzyme, and the enzyme's appropriate buffer were loaded into micro tubes and incubated for DNA digestion. A water bath was used for the digests and incubated for 45 minutes. The digests were then combined with loading and marking dyes. These mixtures, and a lane of known DNA fragment standards, were run out on 1% agarose electrophoretic gels. The time necessary for electrophoresis was variable depending upon student schedule. Some DNA fragments are extremely small so it was necessary to treat the gels with ethidium bromide instead of methylene blue. Gels were exposed to an ultraviolet transilluminator to analyze and establish DNA fragment profiles and photographs were taken. Over a series of labs, different enzymes were used to look for variation in fragment patterns.


Students applied various mathematical analyses to interpret their data. By using the resulting DNA fragment patterns from several enzymes students established a possible evolutionary relationship among the mice. Because they used a variety of enzymes, information about the sequence of nucleotides in mitochondrial DNA was ascertained. Identification of both highly conserved sequences and highly variable sequences were noted.

The students presented their data and findings using a laboratory write-up research format and presented the findings to other sutdents via a poster session.

This activity has been used in the Advanced Placement Biology classes and Applied Genetics.

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