Download RECOMBINANT DNA

Standards:
 SC.BS. 2.1 Science, Technology and Society: Explain how scientific advancements and emerging
technology have influenced society.
RECOMBINANT DNA
Among the most significant developments in biology during the last 20 years is genetic engineering -- the
ability to manipulate DNA. One of the most common, and profitable, types of genetic engineering for
pharmaceutical companies is to insert a human gene into a bacterial plasmid. This joining of DNA from two
sources results in recombinant DNA. Once this recombinant DNA plasmid is taken in by a bacterial cell, the
bacterium will transcribe and translate the human gene, producing the human protein coded for in the gene.
Scientists can manipulate genetic material to produce many substances, a few of which are summarized in the
table below.
Recombinant substance
recombinant FSH
(follicle stimulating hormone)
recombinant HGH
(human growth hormone)
recombinant insulin
recombinant antihemophilia
factor (factor VIII)
recombinant albumin
recombinant interferon
FDA approved products
(examples)
Follistem ™
Humatrope®,
Nutropin®
Humulin®, Humalog®
Kogenate®,
Novoseven®
Albutein®
Alferon-N®
Treatment for
infertility
growth hormone deficiency
diabetes
bleeding episodes in
hemophiliacs
shock, patients taking dialysis
genital warts
The technology to produce these substances is called recombinant DNA technology. There are two major steps
involved:
1. Prepare the human gene to be inserted. This is done using a reverse transcription process involving
mRNA and will not be simulated.
2. Splice the human gene into the bacterial plasmid. This is simulated in this investigation, in which you
will splice either a human follicle stimulating hormone, human growth hormone, human antihemophilia
factor or human interferon gene into a bacterial plasmid.
Procedure:
The steps you will follow to insert a gene into the genetic system of a bacterium is as follows:
• construct a bacterial plasmid
• identify a restriction enzyme that will cut both the human DNA and the plasmid DNA
• cut the human DNA with the enzyme
• cut the plasmid with the enzyme
• form a recombinant plasmid
1. Obtain ONE strip of plasmid DNA and ONE strip of a human gene.
2. Genetic engineers use plasmids to introduce new genes into bacteria. The plasmid DNA is actually circular
and the two ends are normally connected. Tape together the two ends of the plasmid DNA molecule to form a
ring.
3. Genetic engineers use restriction enzymes as “scissors” to cut DNA sequences at specific locations. Each
restriction enzyme recognizes a specific sequence of bases, and cuts within that sequence. When DNA is cut,
the ends become “sticky”. They can bond to other sticky ends that have been cut with the same restriction
enzyme since they would have complementary nucleotide sequences. The following is a list of four restriction
enzymes, their recognition sites, and the sticky ends they create.
Name
Bam HI
Eco RI
Hin d III
Hpa II
Pme I
Recognition & Cutting Site
G |G A T C C
C C T A G| G
G |A A T T C
C T T A A |G
A|A G C T T
T T C G A|A
C|C G G
G G C|C
GTTT|AAAC
C A A A| T T T G
Sticky Ends
GATC
CTAG
AATT
TTAA
AGCT
TCGA
CG
GC
none
none
4. Mark ALL of the above restriction sites that are present on your piece of plasmid DNA.
Check below the recognition sites contained in the plasmid:
 Bam HI
 Eco RI
 Hin d III
 Hpa II
 Pme I
5. Imagine that the DNA from the human gene you selected is actually a portion of a much longer piece of
DNA. To cut this gene from the DNA molecule, you must use a restriction enzyme that will yield sticky ends
in the human gene that can bind with complementary sticky ends in the plasmid, without destroying the
coding region. Determine which ONE restriction enzyme will cut both ends of the human gene you selected
and also open the plasmid to yield complementary sticky ends.
Check below the human gene you Check below the restriction enzyme you have
selected:
selected to create a recombinant DNA plasmid:
 antihemophilia factor
 Bam HI
 follicle stimulating hormone
 Eco RI
 growth hormone
 Hin d III
 interferon
 Hpa II
 Pme I
6. Acting as the restriction enzyme, use the appropriate scissors to cut the human gene at the proper
restriction sites.
What sticky ends have you exposed in the gene?
Left : _________
Right: __________
7. Acting as the restriction enzyme, use the appropriate scissors to cut open the plasmid at the proper
restriction site.
What sticky ends have you exposed in the plasmid?
Left : _________
Right: __________
8. Complementary sticky ends of a gene and a plasmid are joined by enzymes called ligases. Using tape,
perform the function of a ligase by connecting the sticky ends of your human gene to the plasmid producing a
model of a circular recombinant DNA plasmid.
9. Lastly, label the inside of your recombinant plasmid with its engineer (you) and creation period and hand
it in.