Part 1 – Using restriction enzymes to cut DNA

stubbornnessuglyΒιοτεχνολογία

12 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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Name: ___________________________________________ Period: ______ Date: _______________


LAB



Using

Restriction Enzymes

Introduction

Genetic engineering is possible because of special enzymes that cut DNA. These
enzymes are called
restriction enzymes
, or restriction endonucleases. Restriction
enzymes are proteins produced by bacteria to prevent or restrict invasion by foreign
DNA. They act as
DNA scissors
, cutting the foreign DNA into pieces so that it
cannot function.


For this activity, you will u
se the following restriction enzymes:


Eco
RI

5’
-
GAATTC
-
3’

Sma
I

5’
-
CCCGGG
-
3’


3’
-
CTTAAG
-
5’

3’
-
GGGCCC
-
5’



Hind
III

5’
-
AAGCTT
-
3’


3’
-
TTCGAA
-
5’

Procedure

Part 1


Using restriction enzymes to cut DNA

1.

Cut the DNA sequence strips along their borders. These strips represent double stranded
DNA molecules.

2.

Cut strip 1 using the restriction enzyme
Eco
RI.

Remember to locate the sequence where it
cuts!
Label each piece
Eco
RI
. What types of ends result from
a cut with
Eco
RI
?

3.

Simulate the activity of
Sma
I by cutting strip 2.
Label each piece
Sma
I
.
What types of
ends result from
a cut with
Sma
I
?

4.

Simulate the activity of
Hind
III by cutting strip 3.
Label each piece
Hind
III
.
What types
of ends result from
a cu
t with
Hind
III
?

5.

Cut strip 4 using the restriction enzyme
Eco
RI.
Label each piece
Eco
RI
.
What types of
ends result from
a cut with
Eco
RI
?

Part 2


Matching correct DNA fragments

6.

Pick up the “front end” of the segment you cut using
Eco
RI (strip 4) and the “
back end” of
the segment cut with
Hind
III (strip 3). If you try to combine these pieces of DNA, are the
base sequences where the
E
co
RI and
Hind
III sticky ends overlap
complementary
?
Explain your response.

7.

Put down the
Hind
III

strip and pick up the “back end” of strip 1, which was cut using
Eco
RI. Do you think these fragments of DNA would join in nature? Explain your
response.

8.

Imagine that you cut
a sample of your own DNA

with
Eco
RI. Do you think that the single
-
stranded “st
icky ends” of your own resulting DNA fragments would be
complementary
to
the single
-
stranded ends of the fragments from strip 1 & strip 4? Explain your response.

9.

How would the fragments of DNA cut by the same rest
r
iction enzymes be put together
(what woul
d reform the bonds between the sugars and phosphates)?

Part 3


Interpreting Restriction Maps


The picture you see is a
restriction map for YIP5
, a
5,541
-
base
-
pair plasmid.
A
plasmid

is a small ring
of bacterial DNA
. When scientists study a DNA
molecule,

one of the first things they do is figure out
where many of these restriction sites are. They then
create a restriction “map,” showing the locations of
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Eco
RI cuts after the 1
st

base pair
out of 5,541;
Hind
III cuts after the 32
nd

base pair, etc.



10.

If
o
nly

the restriction enzymes
Eco
RI and
Eag
I were put into a test tube with this plasmid,
how many fragments of DNA would be produced? (Hint: think about it like a rubberband
you are cutting twice…)


11.

What would the
length in base pairs

be for the shorter of
these fragments? (HINT: Look at
the cuts you made!)



Fragment 1: 942


1 = ________________ bp (base pairs)


12.

If the restriction enzymes
Hind
III and
Apa
I

were put into a test tube with this plasmid, how
many fragments of DNA would be produced?


13.

What would the
length in base pairs

be for the shorter of these fragments? (see the cuts
made!)


14.

If the restriction enzymes
Hind
III,
Apa
I, and
Pvu
II

were put into a test tube with this
plasmid, how many fragments of DNA would be produced?


15.

Can you determine how long
each

fragment would be?