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Activity 7: DNA Screening

Objective

Students will examine diagrams of DNA sequencing gels to identify allele mutations in cystic fibrosis patients.

Background Information

Determining the nucleotide sequence of a single gene was once enormously difficult. However, restriction enzymes can cut the very long DNA found in cells and viruses into discrete, reproducible fragments with unique sequences. This helps scientists quickly identify known sequences within larger fragments.

One of the most commonly used techniques for determining the nucleotide sequence of a DNA fragment is the Sanger method, named after its developer, Frederick Sanger. This method involves synthesizing DNA strands complementary to the strands of DNA being sequenced, and then separating the new DNA strands by a process called gel electrophoresis.

First a mixture containing a strand of the DNA fragment is divided into four portions. Each portion contains all the ingredients needed for the synthesis of complementary strands. In addition, each portion contains a modified form of one of the four different nucleotide bases. Although each mixture contains both the modified and unmodified forms of one nucleotide base, only one form will be incorporated into the synthesized strand at any point in the sequence. When one of the modified bases is incorporated, synthesis of the strand stops. This creates strands of various lengths for each base.

The strands in each mixture are then separated. Gel electrophoresis separates the strands on the basis of their rate of movement under the influence of an electric field. Smaller fragments move through a gel faster than larger fragments. This forms a band pattern down the length of the gel. The position of a band across the width of a gel indicates the base nucleotide within that fragment. Thus, the sequence of the bands down the length of the gel is determined by the sequence of the bases within the DNA fragment. The sequence of the original DNA strand is then deduced from the complementary sequence of the new strand.



Materials

For each group:

Preparation

Duplicate Handout 6 to distribute to students.

Instructions

  1. Explain to the students that the two diagrams on the Handout represent sequencing gels that were obtained from human DNA. Point out that Diagram A comes from a person who is homozygous for a normal allele of the cystic fibrosis gene, while Diagram B comes from a person who is homozygous for a mutant allele of the cystic fibrosis gene.
  2. Point out that students can determine the corresponding base for each band by the band's position in the gel. If a band appears in the left-most column of the gel, it corresponds to guanine. A band in the second column from the left corresponds to adenine. A band in the third column corresponds to thymine. And a band in the righthand column corresponds to cytosine. Also explain that the sequence of the bands from top to bottom indicates the sequence of the bases in the gene. Have students determine the nucleotide sequence indicated by the bands in each diagram, and record their answers on Handout 6 in the space provided. They should also determine the complementary sequences, and write the sequences in the space provided.
  3. Have students use the sequences to determine the difference between the normal gene and the mutant gene.

Discussion Questions

  1. Are the sequences shown in the gels the same as the sequences from the original DNA? (No.) Why or why not? (The sequences in the gels are com plementary to the seguences of the original DNA.)
  2. What is the difference between the two genes? (The nucleotide seguence in Diagram B has three extra bases at the beginning, and is missing a three base series, TCT, that is contain within the base sequence in Diagram A.) What could account for the difference? (The extra three bases at the beginning of the seguence in Diagram B could be due to a longer fragment cut. The missing three-base seguence could account for the difference between the mutant allele and the normal allele.)
  3. How might this type of procedure be useful for determining the function of certain nucleotide sequences? (Answers will vary.)

Extension

Have students model the replication of DNA strands using plastic beads that snap-together (also called pop beads). Sort the beads into four single-color piles. Cut the tips off of some of the beads in each pile. Explain each color represents a different nucleotide base. The beads with tips represent unmodified bases and the beads without tips represent modified bases. Have students put together DNA fragments by having them randomly pick out beads from each pile. When the students chose a bead that does not have a tip, the fragment is complete. Help students see that this process is similar to that of using modified nucleotide bases for DNA sequencing.



Links:



Resource Book Index: Stories from the Scientists


Winding Your Way Through DNA Resource Book Index


Winding Your Way Through DNA Lectures Index


About Biotech Index


 
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