Restriction Enzymes

Background Information:

One of the most important tools of genetic engineering is a group of special restriction enzymes These have the ability to cut DNA molecules at very precise sequences of 4 to 8 base pairs called recognition sites. These enzymes are the “molecular scissors” that allow genetic engineers to cut up DNA in a controlled way. These enzymes were discovered naturally in bacteria. Bacteria use these enzymes as a defense mechanism against the entry of foreign viral DNA. Restriction enzymes are named according to the species they were first isolated from. To date, there are over 400 restriction enzymes allowing genetic engineers the ability to isolate, sequence, and manipulate individual genes derived from any type of organism.

Recognitions Sites for Selected Restriction Enzymes

Enzyme / Source / Recognition Sites
EcoRI / Escherichia coli RY-13 / G / A A T T C
BamHI / Bacillus amyloliquefaciens H / G / G A T C C
HaeIII / Haemophilus aegyptius / G G / C C
HindIII / Haemophilus influenzae Rd / A / A G C T T
HpaI / Haemophilus parainfluenzae / G T T / A A C
HpaII / Haemophilus parainfluenzae / C C / G G
MboI / Moraxella bovis / / G A T C
NotI / Norcardia otitidis-caviarum / G C / G G C C G C
TaqI
SmaI / Thermus aquaticus
Serratia marcescans / T / C G A
C C C/ G G G
  1. Define what a restriction enzyme is
  1. What is a recognition site.
  1. The action of a specific sticky end restriction enzyme is illustrated below. Use the table on the prior page to:

a. Name the restriction enzymes used:______

b.  Name the organisms from which they were first isolated: ______

c.  State the base sequence for this restriction enzymes’ recognition sites: ______

d.  Identify whether they form sticky or blunt ends: ______

The sites at which the fragments of DNA are cut may result in overhanging “sticky ends” or non-overhanging “blunt ends”. Pieces may later be joined together using an enzyme called DNA ligase (molecular glue) in a process called ligation (gluing).

  1. Differentiate between a sticky end and a blunt end.

5. A genetic engineer wants to use the restriction enzyme BamHI to cut the DNA sequence below:

a. Consult the table above and state the recognition site for this enzyme: ______

b. Place a mark at each point where the restriction enzyme BamHI would cleave the DNA sequence below:

Section of a single strand of DNA of only 300 base pairs

10 20 30 40 50 60

AATGGGTACG/CACAGTGGAT/CCACGTAGTA/TGCGATGCGT/AGTGTTTATG/GAGAGAAGAA/

70 80 90 100 110 120

AACGCGTCGC/CTTTTATCGA/TGCTGTACGG/ATGCGGAAGT/GGCGATGAGG/ATCCATGCAA/

130 140 150 160 170 180

TCGCGGCCGA/TCGCGTAATA/TATCGTGGCT/GCGTTTATTA/TCGTGACTAG/TAGCAGTATG/

190 200 210 220 230 240

CGATGTGACT/GATGCTATGC/TGACTATGCT/ATGTTTTTAT/GCTGGATCCA/GCGTAAGCAT/

250 260 270 280 290 300

TTCGCTGCGT/GGATCCCATA/TCCTTATATG/CATATATTCT/TATACGGATC/GCGCACGTTT

c. State how many times the DNA was cut by the restriction enzymes: ______

d. State how many fragments of DNA were created by this action: ______

e. What were the lengths of each DNA fragment in base pairs: ______

______

______

______

Gel Electrophoresis

Background Information:

Gel electrophoresis is a method that separated large molecules (including nucleic acids or proteins) on the basis of size and electric charge. Such molecules possess a slight electric charge. In the case of DNA, DNA has a negative charge. To prepare DNA for gel electrophoresis, the DNA is often cut up into smaller pieces using restriction enzymes This will produce a range of DNA fragments of different lengths. During electrophoresis, molecules are forced to move through the pores of a gel called agarose gel, when the electrical current is applied. Active electrodes at each end of the gel provide the driving force. The electrical current from one electrode repels the molecules (the negative charge) while the other electrode (positive charge) pulls the fragments towards it. The frictional force of the agarose resists the flow of the molecules, separating them by size. The rate of migration through the gel is dependant on the strength of the electrical field, size and shape of the molecules, and on the strength and temperature of the buffer in which the molecules are moving through. In order to visualize the DNA fragments, staining is necessary. The stained separated molecules in each lane can be seen as a series of bands spread from one end of the gel to the other. The large fragments will be trapped quickly in the agarose and will not migrate as far as the shorter DNA fragments. Gel electrophoresis acts like a “molecular sieve”. The size of the fragments can be compared to DNA markers which are a mixture of DNA molecules of known molecular weights (sizes) which are also run through the gel.

1. Explain the purpose of gel electrophoresis:

2. Describe the two forces that control the speed at which fragments pass through the gel:

3. Explain why the smallest fragments travel through the gel the fastest:

4. The box below represents a gel during electrophoresis. You are to pretend that you have loaded and run your

gel. A DNA marker lane is given to you in Lane #1. In Lane #2, run undigested (uncut) DNA through it. In

Lane #3, run your digested (cut) DNA.

#1 #2 #3

(-)

300bp

250bp

200bp

150bp

100bp

50bp

25bp

(+)

Ligation

Background Information:

DNA fragments produced using restriction enzymes may be reassembled by a process known as ligation. Both the desired DNA fragment and the DNA that it will be inserted into must be cut with the same restriction enzyme. The pieces are then joined together using an enzyme known as DNA ligase. DNA ligase acts as “molecular glue”. DNA of different origins produced in this way is called recombinant DNA (because it is DNA that has been recombined from different sources). The combined techniques of using restriction enzymes and ligation are the basic tools of genetic engineering. If the DNA that is inserted in is from a different species of organisms, then the organism is said to be transgenic.

1. Explain the two main steps in the process of joining two DNA fragments together:

a. Annealing:

b.  DNA ligase:

c.  Why it necessary to use the same restriction enzyme for both fragments:

2. Explain why ligation is the reverse of the restriction enzyme process: