CLONING PAPER PLASMIDS
INTRODUCTION
The "Cloning Paper Plasmid" activity can help you see how genes may be
manipulated for genetic research, namely, gene cloning/genetic engineering.
This activity will help you conceptualize the mechanics involved in cutting and
ligating DNAs into a plasmid vector with "sticky ends" of complementary DNA
base pairs.
BACKGROUND INFORMATION:
In this activity, you will clone the DNA molecule to make a vaccine. Four steps
are required to carry out "DNA cloning".
1. DNA isolation: Technically DNA can be isolated from any organism with
DNA.
2. Restriction endonuclease digestion: "Restriction enzymes" recognize and
cut specific sequences of DNA base pairs creating DNA fragments.
a. DNA restriction enzymes recognize short, specific palindrome (that
is, it reads the same backward and forward) sequences of DNA
bases and make breaks in the sugar-phosphate backbone of the
DNA in the region of the recognized sequence.
b. Some restriction enzymes make staggered cuts in the opposite
strands creating complementary, single stranded “sticky” ends;
other restriction enzymes make a cut across both strands creating
DNA fragments with "blunt" ends.
c. These "restriction enzymes" revolutionized molecular biology by
allowing DNA to be cut at known sites with discrete DNA ends.
There are more than 180 commercially available restriction
enzymes for cutting DNA at specific sites.
3. DNA ligation: DNA ligase is an enzyme that recombines DNA fragments
created with restriction enzymes by forming phosphodiester bonds
between two DNA molecules, joining them into a single molecule - thus,
"recombination".
4. Cell transformation: The vector DNA (plasmid) containing the "new" genes
must now be inserted into host cells. The cells are made porous to DNA
by a number of techniques and become transformed as they take up the
recombinant DNA molecule. A group of genetically identical cells, all
containing the same recombinant DNA molecule are called clones. The
unique gene recombination now may be replicated and expressed by
these cells.
You will simulate recombinant DNA techniques and make a polyvalent vaccine
paper model. The term polyvalent refers to the ability of a single virus to impart
immunity to another, unrelated virus. Immunity is developed against foreign
antigens which are molecules that are not recognized as "self". For viruses,
antigens are often the protein coat. If the gene for the major antigen of one virus
is spliced or cloned into a second non-virulent virus and that antigen is expressed
(the protein is made) then this recombinant virus will immunize a host against
both. To date several antigens have already been inserted into Vaccinia and
proved effective in producing antibodies and immunity in animals.
The Vaccinia virus (a weakened poxvirus similar to the smallpox virus) has been
used to virtually eradicate smallpox from the world. In the early 1980's, it was
suggested that a recombinant Vaccinia virus joined with gene coding for an
antigen from another disease organism would have many advantages as a live
vaccine with the ability to replicate.
ACTIVITY: Cloning Paper Plasmid
PURPOSE:
To apply your knowledge of the "genetic code" in the making of a
RECOMBINANT vaccine against smallpox and hepatitis B
 To visually demonstrate recombination of DNA in the making of
vaccines
 To understand how genes can be inserted into another DNA "recombine"
 To conceptualize "restriction enzymes" and recognition of specific
sites
MATERIALS:
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DNA to be combined: carry other DNA. This DNA will allow the
recombinant DNA molecule (that you will make) to be duplicated in
the bacterial host, Escherichia coli.
VACCINIA VIRUS: Containing genes which code for surface
proteins. These proteins will act as antigens against which
antibodies can be formed by the person immunized. (Smallpox
virus is so closely related to Vaccinia that Vaccinia virus is what is
given in the traditional smallpox vaccination.) This DNA must be
kept intact so it can serve as a vector in a mammalian host.
HEPATITIS B VIRUS: Containing the gene for the major surface
antigen (HBsAg). By inserting this gene into the vaccinia virus DNA,
immunity may be elicited for both smallpox and hepatitis.
DNA LIGASE: The enzyme that joins or "ligates" pieces of DNA
together (use Scotch tape).
RESTRICTION ENDONUCLEASES: Used to cut DNA at specific
sites to make the pieces we need. Use scissors to "NICK" double
stranded DNA on both strands. After being nicked, the strands are
held together only by weak hydrogen bonds between
complementary A-T and G-C pairs, fall apart and the DNA is
broken. These enzymes are found in nature and used by bacteria
to cut up and destroy "foreign" DNA. The unique ability to cut DNA
only at specific nucleotide sequences has made these enzymes the
cornerstone of the field of genetic engineering.
RESTRICTION ENDONUCLEASES: DNA SITES WHERE "NICKS" ARE MADE
While over 100 are available, we will be working with four fairly common
Restriction Enzymes.
Where does it
exist naturally?
DNA
Sequence
Recognized
Ends of Cleaved
Molecule
Human and
Animal Intestines
5'GAATTC
3'CTTAAG
G AATTC
CTTAA G
Bacillus
Soil Saprophyte
amyloliquefaciens (Decomposer)
5'GGATCC
3'CCTAGG
G GATCC
CCTAG G
Restriction
Enzyme
Source
EcoRI
Escherichia coli
BamHI
HindIII
Haemophilus
influenzae
SstI
Streptomyces
stanford
Upper respiratory
tract in humans &
causes meningitis
5'AAGCTT
3'TTCGAA
Soil decomposers
5’GAGCTC
3’CTCGAG
A AGCTT
TTCGA A
GAGCT C
C TCGAG
In this exercise, you will be given three strips of paper representing a plasmid
DNA (pBR322), a gene from Vaccinia and a gene from Hepatitis.
PROCEDURE
1. Pick up a sheet of paper with a one-stranded sequence of the pBR322
DNA on it. Write the complementary strand using base pair rules. When
you are done, circularize it into a small plasmid by using tape to connect
the free ends. Plasmids are circular, double-stranded extra chromosomal
DNA molecules that contain specialized genes and have the ability to be
replicated in bacteria.
2. Isolate (cut out) the Vaccinia DNA fragment. Examine the DNA sequence
for restriction enzymes that can be used to cut the vector pBR322.
3. Identify the restriction endonuclease used to generate the Vaccinia virus
DNA fragment. Cut the vector, pBR322 with the same enzyme. BE SURE
TO PAY ATTENTION TO WHERE THE ENZYME ACTUALLY NICKS
THE STRANDS OF DNA TO GENERATE STICKY ENDS. Make sure the
"sticky ends" of the two DNAs are complementary A-T, C-G.
4. Ligate (scotch tape) the Vaccinia DNA and the Plasmid pBr322 vector.
You NOW have a recombinant DNA plasmid that codes for the surface
proteins of the virus.
5. Isolate (cut out) the Hepatitis B DNA fragment. You will now insert the
hepatitis B DNA into Vaccinia virus DNA segment of your first recombinant
DNA molecule.
6. Identify a restriction enzyme that can be used to cut out the HBsAg gene
and insert it into your recombinant DNA molecule (step 4). REMEMBER:
IF THERE ARE TWO SITES ON A DNA MOLECULE RECOGNIZED BY
A SPECIFIC RESTRICTION ENZYME, IT WILL CUT BOTH.
7. Ligate (Scotch tape) the Hepatitis B DNA fragment to the Vaccinia virus
DNA. You NOW have a large circular molecule with DNA from three
different sources. COMPARE your paper model with other students'.
Discuss any differences.
Post-Lab Analysis
Answer the following questions in your lab manual:
1. How can this recombinant DNA molecule be used to generate a recombinant
vaccine?
2. What is the next step necessary in order for this new gene combination to be
expressed?
3. Draw a flow-chart (a procedure using pictures) describing the your answer to
number two.