Carlos Franco                                                                                                                                                        Return to Biology Menu

Summer 2001

Living Environment

  

How do restriction enzymes work?

 

Objective:

1.      Student will learn how restriction enzymes work.

2.      Students will understand the importance of restriction enzymes in studying DNA

 

Standards addressed: 4 and 7

 

Motivation:

 Analyze the following strand of DNA after exposure with restriction enzyme A, B, and C.

 

Viral DNA

Restriction

 Enzyme

Product after enzyme Exposure

5’ATGCCCTGCATGGCAA3’

    I I I I I I I I I I I I I I I I I I I

3’TACGGGACGTACCGTT5’

 

A

5’ATGCCCTG     CATGGCAA3’

    I I I I I I I I I      I I I I I I I I I

3’TACGGGAC    GTACCGTT5’

 

5’ATGCCCTGCATGGCAA3’

    I I I I I I I I I I I I I I I I I I I

3’TACGGGACGTACCGTT5’

 

B

5’ATGCCCTGCAT         GGCAA3’

    I I I I I I I I I I I I  I I I    I I I I I I

3’TACGGGACGTACC         GTT5’

 

5’ATGCCCTGCATGGCAA3’

    I I I I I I I I I I I I I I I I I I I

3’TACGGGACGTACCGTT5’

 

 

C

5’ATGCCCTGCATG       GCAA3’ I

    I I I I I I I I I I  I I I   I I I I I I I I

3’TACGGGACG        TACCGTT5’

 

 

Development:

Q. There are three classes of cut due to restriction enzyme exposure.   Based on your analysis of the products after enzyme exposure can you describe this cuts?

 Answer: Blunt cuts (enzyme A); 5’ overhangs (enzyme B);  and 3’ overhangs (enzyme C)

 

Q. Analyze the following DNA strand and note the sequence:

 

5’-A T G C C C T G C A T G G C A A -3’

      I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I  I                    This fragment is exposed to a

3-T A C G G G A C G T A C C G T T -5’                 restriction enzyme and a piece of DNA is added.

 

5’-A T G C C C T G  C T G C C  C A T G G C A A -3’    What type of cut was done

      I  I  I  I  I  I  I  I  I  I  I   I  I  I  I  I  I  I  I  I   I  I  I  I           on the original strand to insert the added piece?

3-  T A C G G G A C G A C G C  G T A C C G T T -5’     

 

*I have highlighted the inserted piece.  Allow students to determine the inserted sequence by analyzing the  original strand with the recombinant strand.   

       

-         Restriction enzymes cut at specific sites along a DNA strand.  Why do you think molecular biologists want to be sure where exactly a restriction enzyme cuts?

 

Study the following table that indicates the sequence a particular enzyme must recognize before cutting:

 

Restriction enzyme name

Specific sites of action

Rsa I

5’-GT¯AC-3’

3’-CA­TG-5”

 

ScaI

5’-AGT¯ACT-3’

3’- TCA­TGA-5’

 

SmaI

5’-CCC¯GGG- 3’

3’-GGG­CCC-5’

 

 

Q. Based on the information provided by this table determine in how many pieces will the following stand be cut if exposed to Rsa I ? Draw each piece

 

5’-AGGTGGACTGTGTCTGCCTGAGTACTAAGGTGTACCGTGGTGCCCGGG-3’

     I I I I I I I I I I I  I I I I I I I I I  I I I I I I I I I I I I I  I I I I  I I I I  I I I  I I I  I I I  I I I

3’-TCCACCTGACACAGACGGACTCATGATTCCACATGGCACCACGGGCCC-5’

  

Q. Based on the information provided by this table determine in how many pieces will the following stand be cut if exposed SmaI? Draw each piece

 

5’-AGGTGGACTGTGTCTGCCTGAGTACTAAGGTGTACCGTGGTGCCCGGG-3’

     I I I I I I I I I I I  I I I I I I I I I  I I I I I I I I I I I I I  I I I I  I I I I  I I I  I I I  I I I  I I I

3’-TCCACCTGACACAGACGGACTCATGATTCCACATGGCACCACGGGCCC-5’

 

Q. Based on the information provided by this table determine in how many pieces will the following stand be cut if exposed SmaI? Draw each piece

 

5’-AGGTGGACTGTGTCTGCCTGAGTACTAAGGTGTACCGTGGTGCCCGGG-3’

     I I I I I I I I I I I  I I I I I I I I I  I I I I I I I I I I I I I  I I I I  I I I I  I I I  I I I  I I I  I I I

3’-TCCACCTGACACAGACGGACTCATGATTCCACATGGCACCACGGGCCC-5’

Q. As you can see restriction enzymes cut at specific sites.  How do you see restriction enzymes being important in studying DNA? Proteins? Recombinant DNA?

Hint: DNA- RNA- Protein

 

Summary:

Write three important five important facts about restriction enzymes

  

Homework:

Find an articles on the Internet where restriction enzymes  were used to study, analyze and solve a problem.