Restriction Enzymes - Word Activity
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Restriction Enzymes

Classroom Activities

Access Excellence Classic Collection


How Restriction Enzyme, Probes and RFLP's Work

Modified by Access Excellence from the National Institute for Deafness and Other Communication Disorders Hereditary Hearing Impairment Resource Registry at the Boys Town National Research Hospital
    555 N. 30th St.
    Omaha, NE 68131
    (800) 320-1171

Abstract

A simulation activity that uses letters and words to model the action of restriction nucleases on DNA molecules.

Materials

Sentence to represent a DNA molecule:
In the early 1970's, scientists discovered that bacteria had enzymes that would attack foreign DNA and cut the DNA up into little pieces.

Restriction enzyme

TH1--this mythical restriction nuclease recognizes the sequence "the." When it encounters it's recognition sequence, it cuts between the letters "h" and "e" in the sequence.

Sequence specific probe:

A probe which binds specifically to the sequence "DNA." This probe will not bind to other sequences of letters in our DNA molecule.

Activity

Cleavage and electrophoresis of DNA molecule

Let's assume the sentence above represents a length of DNA isolated from a certain person. Let's also assume that we have an enzyme--called TH1--with the recognition sequence "the," and that this cuts between "h" and "e" in the sequence 'the'.

What would happen if our DNA molecule were treated with the TH1 enzyme? The TH1 restriction enzyme cuts the sentence into three pieces. Remember that when cleaved DNA molecules are run out on a gel, the larger pieces will progress through the gel more slowly than the smaller pieces. Therefore, the larger pieces will remain closer to the top of the gel while the smaller pieces progress closer to the bottom of the gel. Below is an example of the results of TH1 cleavage of our DNA molecule:

**Top of Gel**

e early 1970's, scientists discovered that bacteria had enzymes that would attack foreign DNA and cut th (86 letters)

e DNA up into little pieces (22 letters)

In th (4 letters)

**Bottom of Gel**

As you can see, treatment of the DNA molecule with the TH1 enzyme produces three fragments ranging in size from 86 to 4 letters and which separate from each other as they are run out on a gel.

Sequence Specific Probe

Although the DNA of two individuals of the same species is very similar, it does differ slightly from individual to individual. These slight DNA variations are called "genetic polymorphisms." DNA analysis can involve identification of the locations of genetic polymorphisms. This identification makes use of "probes"--molecules which bind specifically to short sequences of bases. If that short sequence happens to be polymorphic between two individuals, than the binding pattern of the probe will also be different for the two individuals. Let's see what will happen if we use a probe specific to the word "DNA," and what happens when that probe is used to analyze two polymorphisms of our DNA sentence. If we apply a solution of the "DNA" probe to the gel, it will only attach to fragments containing the sequence "DNA." Therefore, it will only bind to two of the fragments on the gel. If the probe was selected to be detectable, for example by attaching it to a radioactive molecule, it will be possible to detect its presence on the gel. (In the case of a radioactive probe, a piece of undeveloped film is laid over the gel. The radioactive probe will expose the film in its immediate area, thus producing a spot or spots on the film which correspond to the location of the probe on the gel.) What would our gel look like if we probed it with a "DNA" specific probe?

**Top of Gel**

e early 1970's, scientists discovered that bacteria had enzymes that would attack foreign DNA and cut th (86 letters)

e DNA up into little pieces (22 letters)

**Bottom of Gel**

Each DNA fragment detected in this way is called a Restriction Length Fragment Polymorphism or RFLP. Using this technique, we can say that this person's DNA has RFLPs of 22 and 86 bases when we cut with the TH1 enzyme and probe with a "DNA" specific probe.

Polymorphisms and RFLP analysis: Genes come in pairs. Each member of the pair is called an allele. Often alleles are very similar, but not identical--they are polymorphic. Let's say the allele to this gene above looks like this:

In the early 1970's, there were repeats of bacterial enzymes that would attack foreign DNA, cutting them into little pieces.

Using the same Th1 restriction enzyme, "DNA" probe and electrophoresis, the fragments on the resulting gel would look like this:

**Top of Gel**

ere were reports of bacterial enzymes that would attack foreign DNA, cutting th (64 letters)

em into little pieces (18 letters)

e early 1970's th (13 letters)

in th (4 letters)

**Bottom of Gel**

This allele has four fragments. These fragments are different lengths than the fragments produced by treatment of the original sentence. In addition, only the RFLP of 64 bases will bond to the "DNA" specific probe.

A person containing two different alleles for any given gene is considered to be "heterozygous" for that particular gene. A person containing two identical alleles for any given gene is considered to be "homozygous" for that gene.

What would the RFLP pattern look like if a person were heterozygous for the gene that we have been discussing? What if the person were homozygous?

Extension Activity

Come up with two similar sentences. Create a restriction enzyme that will cut the two sentences. Decide what probe you will use to analyze the resulting gel. Show the RFLP pattern for individuals who are homozygous for your gene. What about for heterozygous individuals?

Definitions

  1. Restriction Enzyme- an enzyme capable of cleaving a DNA molecule at a site determined by a specific sequence of bases known as a recognition sequence.

  2. Genetic probes- is a piece of DNA (15 to 50 bases long) that matches the message you are trying to find. This probe is also labeled with a radioactive chemical (P32) so it can be seen on special paper called a blot.

  3. RFLP (pronounced "rif-lip"): The initials stand for Restriction Fragment Length Polymorphism. These are different (polymorphic) pieces of DNA (length) that are produced by cutting the DNA with restriction enzymes and probing them with radioactive labeled DNA.


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