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
- 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.
- 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.
- Have students use the sequences to determine the difference between
the normal gene and the mutant gene.
Discussion Questions
- 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.)
- 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.)
- 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.
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Resource Book Index: Stories from the Scientists
