Fragment Samples

Lab 6 Part B. Restriction Site Mapping of a Polymorphic Mitochondrial DNA (mtDNA)

Mitochondrial DNA is DNA that is located in the mitochondria, organelle that converts chemical energy from food into ATP. MtDNA is a small portion of DNA found in cells, but since this evolves faster than nuclear DNA it has become important in phylogenetics and evolutionary biology. This DNA is inherited from the mother. This DNA is highly conserved and has a slow mutation rate, but polymorphisms can occur. A polymorphism is the presence of genetic variation within a population, upon which natural selection can occur. Today students will map a polymorphic mtDNA. Section 1:

You have isolated some mitochondrial DNA (mtDNA) from Walleye (Sander vitreous). The mtDNA was digested with four restriction endonucleases Apa Ⅰ, Bcl Ⅱ, Cla Ⅰ, and Dra Ⅰ. The fragments obtained (sizes in kilobase pairs) with each enzyme were as follows:

Apa Ⅰ 11.5, 3.0, 2.0

Bcl Ⅱ 1 cut only – fragment size 16.5

Cla Ⅰ 11.3, 5.2

Dra Ⅰ 13.0, 3.5

You wish to map these restriction sites on the molecule, so you conduct a series of double digests, which give the following sized fragments:

Apa I/Bcl II 10.0, 3.0, 2.0, 1.5

Apa I/Cla Ⅰ 7.5, 4.0, 2.0, 1.8, 1.2

Apa I/Dra I 9.5, 3.0, 2.0, 1.5, 0.5

Bcl II/Cla I 6.0, 5.3, 5.2

Bcl II/Dra I 13.0, 3.0, 0.5

Cla I/Dra I 5.5, 5.2, 3.5, 2.3

Draw a linear restriction site map for this walleye mtDNA that is opened at the single Bcl Ⅱ restriction site. Show all maps neatly.

Section 2:

A polymorphism is now found using Dra Ⅰ such that the new fragment pattern for this enzyme becomes: 8.0, 5.0, 3.5. What has happened? You conduct two more double digests in order to map this variable site, giving results as follows:

Cla I/Dra I 5.2, 5.0, 3.5, 2.3, 0.5

Apa I/Dra I 5.0, 4.5, 3.0, 2.0, 1.5, 0.5 D

raw a map of these new double digests and show the variable site with an *. Draw a picture of a gel showing all fragment samples used in this section.

Section 3:

You examine another individual with Dra Ⅰ revealing the following fragment results: 9.0, 5.0, 3.5. You think that this is a bit odd. So, you conduct a few more digests, getting the following results:

Apa Ⅰ 11.5, 4.0, 2.0

Cla Ⅰ 11.3, 6.2

Where is this phenomenon affecting the mtDNA molecule? Indicate the portion of the molecule that is affected. What fragments would you expect to result from a Cla Ⅰ/Dra Ⅰ double digest on this individual?

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