Genetic Engineering Worksheets

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11 Δεκ 2012 (πριν από 4 χρόνια και 7 μήνες)

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Restriction Enzymes


Background Information
:

One of the
most important

tools of genetic engineering is a group of special
restriction enzymes

(
Restriction
endonucleases
). 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 sc
issors
” that allow genetic engineers to cut up
DNA
in a controlled way. F
irst isolated in 1970, these enzymes were discovered earlier in many bacteria.
The purified forms of these bacteri
al restriction enzymes are used today as tools to cut DNA. Restriction
enzymes are named according to the species they were first isolated from, followed by a number to
distinguish different enzymes isolated from the same organism.
It was observed that c
ertain bacteriophages
(viruses)
could not infect bacteria other than their usual hosts. It was found the reason for this was that
other potential hosts could destroy almost all of the phage DNA using restriction enzymes present naturally
in their cells; a
defense mechanism against the entry of foreign DNA.
By using this molecular tool kit, of
over 400 restriction enzymes recognizing about 100 recognition sites, genetic engineers can isolate, sequence,
and manipulate individual genes derived from any type o
f organism. 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

(gluin
g).


Recognitions Sites for Selected Restriction Enzymes

Enzyme

Source

Recognition Sites

Eco
RI

Escherichia coli

RY
-
13

G

/

A A T T C

Bam
HI

Bacillus amyloliquefaciens

H

G

/
G A T C C

Hae
III

Haemophilus aegyptius

G G

/

C C

Hind
III

Haemophilus influenzae

Rd

A

/

A G C T T

Hpa
I

Haemophilus parainfluenzae

G T T
/
A A C

Hpa
II

Haemophilus parainfluenzae

C C
/
G G

Mbo
I

Moraxella bovis

/
G A T C

Not
I

Norcardia otitidis
-
caviarum

G C
/
G G C C G C

Taq
I

Sma
I

Thermus aquaticus

Serratia marcescans

T
/
C G A

C C C
/ G G G


1.

Define what a restriction enzyme is





2.

What is a recognition site.




3.

Differentiate between a sticky end and a blunt end
.






4.

The action of a specific sticky end restriction enzyme is illustrated below. Use the table on the prior page to:

a.

Name the restriction enzyme
s

used:_______________________________________
_______________

b.

Name the organism
s

from which

they were

first isolated:

_____________________________
_
______

c.

State the base seque
nce for this restriction enzyme
s


recognition site
s
:

___________________
_____
__

d.

Identify whether they form sticky or blunt ends: ____________________________________________




5. A genetic engineer wants to use the restriction enzym
e
Bam
HI


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
Bam
HI would cleave the DNA sequence
below
:

Section of a

single strand of DNA of only 300 base pairs



10 20 30 40

50 60

AATGGGTAC
G/CACAGTGGAT/CCACGTAGTA/TGCGATGCGT/AGTGTTTATG/
GAGAGAAGAA/



70 80

90 100 110 1
20

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 s
ize, electric charge, and other physical properties. Such molecules possess a sligh
t 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. This is done by mixing DNA with restriction enzymes in controlled conditions for
about an hour. Called
r
estriction digestion
, it produces a range of DNA fragments of different lengths.
During electrophoresis, molecules are forced to move through the pores of a gel called
agarose
, when the
electrical current is applied. Active electrodes at each end of the g
el provide the driving force. The electrical
current from one electrode repels the molecules (the
anion

or negative charge)

while the other elect
r
ode
(cation or positive charge) pulls the fragments towards it
. The frictional force of the agarose resists t
he 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 ionic 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 wh
y 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 p
ieces 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 en
zyme process:


1.

If two pieces of DNA are cut by the
same restriction enzyme, they will
produce fragments with matching
sticky ends

2.

When two such matching sticky ends
come together, they can join by base
pairing. This process is called
annealing.
This can a
llow DNA
fragments from a different source,
perhaps a
plasmid
, to be joined to
the DNA fragment.

3.

The joined fragments will usually
form either a linear molecule or a
circular one, as shown here for a
plasmid. However, other
combinations of fragments can
o
ccur.

4.

The fragments of DNA are joined
together by the enzyme
DNA ligase
,
producing a molecule of
recombinant DNA
.