Bio H – Molecular genetics
Human DNA (Containing Gene for Growth Hormone)
C C C T G T A T A A G C T T A T G G C T A C A G G C T C C CG G A C G A A G C T T A
G G G A C A T A T T C G A A T A C C G A T GT C C G A G G GC C T G C T T C G A A T
Human DNA (Containing Gene for Growth Hormone)
C C C T G T A T A A G C T T A T G G C T A C A G G C T C C CG G A C G A A G C T T A
G G G A C A T A T T C G A A T A C C G A T GT C C G A G G GC C T G C T T C G A A T
Human DNA (Containing Gene for Growth Hormone)
C C C T G T A T A A G C T T A T G G C T A C A G G C T C C CG G A C G A A G C T T A
G G G A C A T A T T C G A A T A C C G A T GT C C G A G G GC C T G C T T C G A A T
Bio H – Molecular genetics
Plasmid DNA (Bacterial DNA)
GGATCCTGACACGGAACGTCAAGCTTCCC
CCTAGGACTGTGCCTTGCAGTTCGAAGGG
Plasmid DNA (Bacterial DNA)
GGATCCTGACACGGAACGTCAAGCTTCCC
CCTAGGACTGTGCCTTGCAGTTCGAAGGG
Plasmid DNA (Bacterial DNA)
GGATCCTGACACGGAACGTCAAGCTTCCC
CCTAGGACTGTGCCTTGCAGTTCGAAGGG
Bio H – Molecular genetics
Cloning a Paper Gene
As we discussed in the notes, if we want to get a bacterial cell to copy or translate a human gene for us, we need to cut out the human
gene and insert it into a circular piece of DNA from the bacteria called a plasmid. The plasmid should contain all of the DNA that the
bacteria needs to turn the human gene on and off (a bacterial gene promoter) AND a second gene called a selectable marker.
Selectable markers are genes that make it easy to differentiate between the bacteria that took up our human gene and those that didn’t.
The most common selectable marker is a gene that gives the bacteria resistance to antibiotics. When we grow the bacteria, we can add
antibiotics to the petri dish and only the bacteria with the selectable marker and the human gene will grow. All unsuccessful bacteria
will be killed by the antibiotic. Once we’ve made our plasmid containing the human gene and the selectable marker, we will
transform the bacteria. This just means we will mix the DNA and bacteria together and treat the bacterial cells in a particular way to
increase the chances that they will take up the DNA. If the bacteria picks up the plasmid, it will express both the human gene AND
the selectable marker gene.
Your job is going to be to find an appropriate place to cut both your plasmid and human genes so that they can be linked together. BE
CAREFUL – you can not disrupt the sequence of either the human gene OR the selectable marker, in this case, the ampicillin
resistance gene.
Normally plasmids are about 3 – 5 kb (kilobases) and genes, while they vary greatly, are on average 1.5 kb. We’ve shortened the
sequences significantly for this assignment to simplify things a little bit.
Step 1: Cut out both sequences in their entirety. Tape the ends of the PLASMID DNA together to make a circle. (remember plasmids
are not linear, they are circular)
Step 2: Now we need know where we can cut the plasmid. The ampicillin resistance gene is represented by the sequence: GACACG.
Find this sequence in the plasmid and highlight it. Do not cut anything yet.
Step 3: We also need to know where our gene starts and stops in the human DNA. We know that the mRNA for the human gene has
the following sequence: G A A U A C C G A U G U C C G A G G G C C U G C. Find the corresponding sequence in the piece of
human DNA and highlight it.
Bio H – Molecular genetics
Step 4: Now that the plasmid DNA and the human gene DNA have been determined, the next step is to cut each one with a restriction
enzyme. The human gene must be cut out of the human DNA and the plasmid must be cut open before the human gene can be
inserted. The scissors for doing the cuts are restriction enzymes. Below is a short list of a few of the enzymes available. Study the list
carefully and select the enzyme that would be appropriate for cutting out the growth hormone gene from the human DNA and for
cutting the plasmid open. If some "unwanted" bases are included in the human piece of DNA, no harm done.
Step 5: Once you have selected your enzyme, you have identified the sites at which the two DNA's will be cut. Using your 3-D paper
plasmid and the human DNA found on the separate sheet, mark IN PENCIL the sites at which the enzyme will cut. When you are sure
the marks are in the correct positions, cut the ends with scissors. This will produce uneven ends which have unpaired bases. Study
these ends and decide how the human DNA can be joined to that of the bacterial plasmid. Tape the pieces together. You now have
"recombinant DNA."
Bio H – Molecular genetics
Discussion Questions:
1. Why is it better to use the same restriction enzyme to cut both the plasmid and the human gene?
2. What is the sequence of the restriction site that you used? What is the name of the corresponding enzyme?
3. Why is it that the bacteria, a species from a completely different kingdom, can still transcribe and translate a human gene?
4. How are we differentiating between the bacteria that were successfully transformed and those that were not?
5. What is the advantage of cloning genes in bacteria rather than an organism more similar to us?