Download MCDB 1041 3/15/13 Working with DNA and Biotechnology Part I

MCDB 1041 3/15/13 Working with DNA and Biotechnology Part I: Working with DNA You work in a clinic doing prenatal testing and genetic counseling. You use PCR analysis combined with
restriction enzyme digests to determine whether fetuses are affected by cystic fibrosis, caused by a mutation on
both copies of chromosome 7, in the cystic fibrosis (CF) gene. Below is a region of DNA (from the middle of
the CF gene). Sequence of normal CF gene:
5’ ACGCCGCTACGT TAGACTTCGCTACAAGACGG 3’
3’ TGCGGCG ATGCAATCTGAAGCGATGTTCTGCC 5’
Sequence of mutant CF gene
5’ ACGCCGCTACGT TAGAATTCGCTACAAGACGG 3’
3’ TGCGGCG ATGCAATCTTAAGCGATGTTCTGCC 5’
1. Circle the mutation in the sequence above.
One of the restriction enzymes (R.E.) you can use cuts at the following sequence, at the stars: G*AATTC
CTTAA*G
2. Mark on the sequence(s) above where this R.E. will cut the DNA.
You have been charged with doing DNA analysis on three recently born babies. You determine that one child
is normal (no mutations in the cystic fibrosis gene on chromosome 7), one is a carrier (one mutation), and one
has cystic fibrosis (a mutation on each chromosome)—see the diagram below.
The line in the diagram indicates the site of the mutation within the CF gene that you just found in questions 8
and 9 above. The CF gene DNA is isolated from cells from the fetus’ cells by PCR, and is 10 KB in length.
The restriction enzyme shown above cuts the mutant CF gene into two pieces (7 kb and 3 kb). You cut your
samples of DNA from each fetus, and then run the DNA on the gel.
Ch. 7
Ch. 7
Ch. 7
normal
Ch. 7
Ch. 7
Cystic fibrosis
Ch. 7
carrier
3. What would you expect to see when you run the gel? Draw the bands each child has. Make sure to indicate
the correct intensity of each band (ie, more DNA, darker band).
Child A is normal.
Child B has cystic fibrosis
Child C is a carrier
A
B
C
12 KB
11 KB
10 KB
9 KB
6 KB
3 KB
1 KB
4. Below is a pedigree showing inheritance of an autosomal recessive disease. Carriers are marked. The gene
being analyzed is 15 KB in length normally. The mutation is a deletion of 2 KB. For each individual, imagine
you have done PCR to amplify just this gene sequence from their DNA. For each numbered individual, draw in
the band(s) of DNA on the gel that would result from the PCR.
1
2
1
__
2
3
4
5
ladder
20
15
3
4
5
10
8
6
+
5. Let’s say you are testing newborns to see if they have or are carriers of an X-linked recessive disease,
hemophilia (due to a mutation in “coagulation factor VIII” –also called “F8”-- which results in failure of blood
to clot properly).
a. Using the white boards: make up a pedigree for three generations that shows unaffected, affected and
carrier individuals in a pedigree for hemophilia. You can copy your pedigree into the space below, so you can
refer back to it later.
b. Say that the F8 gene is 15 KB in length. If you wanted to analyze whether individuals in a family had
or carried hemophilia before they showed any symptoms, what kind of mutations would you be able to assay
using just PCR and gel electrophoresis. What kind of mutations could you assay using PCR, restriction enzyme
digests and gel electrophoresis?
c. Imagine that the mutation you are assaying that leads to hemophilia is a single base change that
removes a restriction enzyme site. If that site is normally at the 5 KB point of the gene, indicate what results
would be expected for unaffected, affected and carrier individuals by drawing a gel and drawing the pattern on
the gel expected for your analysis of each individual in the pedigree.
Part II Biotechnology: Genetically Modified Foods What is a genetically modified food? The U.S. is one of the primary producers of GMO foods in the world. The creation of GMOs involves
using recombinant DNA technology to place genes from one organism into another of a different species to
confer a useful trait. For example, the company Monsanto developed a pest-resistant potato plant by
incorporating a gene from a soil bacterium into the genome of a potato plant: this gene produces a compound
that kills the Colorado Potato Beetle. These potatoes are commercially grown in the U.S. The pesticide that
used to be sprayed on the potatoes to fight the beetle is no longer necessary. How do you generate the
recombinant DNA that is used to make a GMO?
M C D B 1041 A ctivity 8: Biotechn
Let’s take the creation of a product called “golden rice” as an example. Vitamin A deficiency is a problem in the developing world, especially for pregnant women and Part I: Genetically Modified Foods
children. Nearly 400 million people in the world are at A
risk of a vitamin A deficiency, hich can lead to especially
Vitamin
deficiency
is a problem
in thewdeveloping
world,
blindness and an increase in the severity of infections i
n y
oung c
hildren. F
oods l
ike c
arrots, s
weet 400 million people in the world are at risk of a vitamin A deficiency, w
potatoes, and spinach contain β-­‐carotene (precursor to invitamin A), but hese foods re not children.
always Foods like carr
increase
the severity
of tinfections
in ayoung
available in the developing world. By adding a E-carotene
gene called p
hytoene s
ynthase (
psy) f
rom the dare
affodil (precursor to vitamin A), but these foods
not always ava
plant, plus a promoter region that determines where the gene will be expressed, β-­‐carotene will accumulate in the rice grain, and be converted Imagine
into Vitamin before eing harvested. rice that would contain Vi
your A
goal
is to bgenerate
engineered
phytoene synthase (psy) from the daffodil plant, plus a promoter region
To make golden rice, we first need to generate expressed,
recombinant DNA. Twill
o do accumulate
this, we use acterial E-carotene
inthe thebrice
grain.system described in class. We need to put different pieces of DNA together and then amplify them, which is why we use this technique as opposed to just PCR. The
picture below represents a piece of double-stranded DNA from da
phytoene synthase gene (psy), as well as additional sequences of DNA
The picture at right represents a piece of double-­‐ stranded DNA from daffodil. This DNA E P
B H
B
includes the daffodil phytoene synthase gene (psy), as well as additional sequences of DNA. Each line represent 1 Kilobase (1KB). Each line represent 1 Kilobase (1KB). You can amplify this 12 KB seq
using PCR.
This DNA sequence can be cut by 4 different restriction enzymes E=Eco RI This DNA sequence can be cut by 4 different restriction enzymes (deno
P=Pst1 of the nucleotide sequences that occur at two of these restriction enzym
B=BglII the REs.
H=HindIII). Letter on the DNA
Restriction Enzyme Recognition
Name of
Below is a list of the nucleotide sequences that sequence
occur at two of these restriction sites, along with Enzyme
and cuttingenzyme sequence
the actual names of the REs. The psy gene is bE
etween the E and H restriction G* A Aenzyme T T C sites.
Eco RI
Letter on the DNA
Restriction Enzyme Recognition
Name of Restriction
C T T A A *G
sequence
and cutting sequence
Enzyme
P
C T G C A*G
Pst I
E
G* A A T T C
Eco RI
G *A C G T C
C T T A A *G
H
C T G C A*G
Hind
III of DNA shown below, find the restriction enzyme
1. In the
strand
G *A C G T C
TATAAGATTGCGATGCCCTGCAGCTATTCGGCTGCCTAAAA
1. In the strand of DNA shown below, find the restriction enzyme sites. ATATTCTAACGCTACGGGACGTCGATAAGCCGACGGATTTT
TATAAGATTGCGATGCCCTGCAGCTATTCGGCTGCCTAAAATCGGCCCCTAAGAATTCTTATCG
ATATTCTAACGCTACGGGACGTCGATAAGCCGACGGATTTTAGCCGGGGATTCTTAAGAATAGC
If this sequence (above) of DNA were cut with both enzymes, how ma
If this sequence (above) of DNA were cut with both
E and
H restriction
enzymes,
many
pieces would be
These
restriction
enzymes
producehow
³sticky
ends´
created?
²where DNA nucleotides are not bound to their
pair. Thus, they can be easily hooked up to
another piece that has the complementary
unbound nucleotides:
2. You search a database and learn that the psy gene you want to isolate begins at 2 KB and ends at 6 KB in the
piece of double stranded DNA in the diagram above. If you cut the DNA with a variety of different restriction
enzymes (shown below with the letter of the restriction enzyme that was used to cut the DNA): which band on
the gel below contains your gene of interest? (write the lane and the band size)
2. Find the band (circle it) on the gel below that contains that should contain the psy gene of interest.
E
P
B
H
H+ E
B+P
Ladder(DNA of
known sizes for
referen ce)
12 KB
11 KB
10 KB
M C D B 1041 A ctivity 8: B iotechnology
9 KB
Pa r t I : G enetically M odified F oods
6 KB
Vitamin A deficiency is a problem in the developing world, especially for pregnan
3 KB
400 million people in the world are at risk of a vitamin
A deficiency, which can le
increase in the severity of infections in young children. Foods like carrots, sweet
1 KB
E-carotene (precursor to vitamin A), but these foods are not always available in th
Imagine
your
goal
toband generate
engineered
that
would
3. You cut the daffodil psy is
DNA out of the gel and purify rice
the DNA from the gel. contain
Now you wVitamin
ant to add A. B
phytoene
synthase
(psy)
the
plant,
plus
a gel
promoter
region
that determ
3. another You cut pthe
daffodil
psy
out
the
geldaffodil
and
purify the
specialized
iece of DNA to DNA
this pband
iece: from
the ofphyotene desaturase (DNA
crt 1) from
gene the
from ausing
soil baacterium . Tkit.
hese expressed,
E-carotene
in the
grain.
You
want
to put
the
daffodil
gene
What
two genes together will apsy
llow fwill
or tinto
he accumulate
rthe
ice plasmid
seed to cbelow.
ontain beta rice
crestriction
arotene. enzymes should you use to cut
the plasmid? Why?
Thecrt1 picture
represents a piece of double-stranded DNA from daffodil. This
The gene is rbelow
epresented Bgl II below, with letters to indicate the location of restriction enzyme sites (the Phosphomannose
phytoene
synthase
ast twell
as additional
sequences of DNA.
lines no longer represent 1gene
KB, so (psy),
just look he location of the RE sites). HindaIII
isomerase
100 bases
Eco RI
selectable marker
100 bases
E
P
B Pst I H
B B
P
B
E
150 bases
Bgl II
ori
Crt gene. sequence xtends from (1KB).
P to E sites.You can amplify this 12 KB sequence from
Each
line Coding represent
1 eKilobase
4. using
How many
pieces of DNA will result from cutting this circular plasmid?
PCR.
5. If Your
ultimate
goal
is to
generate
rice
that
expresses
psy
gene
in the
of the
rice,bwhere
you w
ant these two genes tcan
o be hbe
ooked in by
sequence, and put from
irestriction
nto tdaffodil
he bacterial pseed
lasmid shown elow, by E, P
This
DNA
sequence
cut
4 the
different
enzymes
(denoted
it with willthe
provide
b-carotene
those
it. that
back from
the beginning,
outline
the steps you
should take
the nucleotide
crt1 gene in fto
ront of eating
the psy gStarting
ene: occur
of
sequences
at two
of these
restriction
enzyme
sites, alo
toa. make
this
happen. [you
may find
it useful
tose return
toothe
map
you
made in class on Wednesday to get
W
hat r
estriction e
nzymes s
hould y
ou u
t
o c
ut ut t
he c
rt g
ene? the REs.
started]
b. What restriction enzymes should you then use to cut the plasmid (you want to put BOTH genes into the Letter on the DNA
Restriction Enzyme Recognition
Name of Restriction
You
have: to be replicated as a unit) plasmid sequence
and
cutting
sequence
Enzyme
‡ Daffodil DNA
E
G* A A T T C
Eco RI
‡ PCR primers that flank the PSY gene
C T T A A *G
‡ Any of
the above Restriction enzymes.
9:/$..$
P
T G called
C A*G
Pst factors
I
‡ A fragment of DNA containingCa promoter
GLU that is only bound by transcription
in the
%&'()&'*+,,'(-$
.('*-"+(-$
G
*A
C
G
T
C
endosperm (basically the seed of the
plant, which is the rice):
4#,5$...$
(-/-01+2/-$*+"3-"$
B P
E
H
60'$7.$
‡ Bacterial plasmid (see diagram above)
%(1$8$
GLU
9:/$..$ shown below, find the restriction
In the strand of DNA
enzyme sites.
‡ 1.
Bacteria
‡ Rice seeds
TATAAGATTGCGATGCCCTGCAGCTATTCGGCTGCCTAAAATCGGCCC
ATATTCTAACGCTACGGGACGTCGATAAGCCGACGGATTTTAGCCGG
!"#$
If this sequence (above) of DNA were cut with both enzymes, how many pieces w
These restriction enzymes produce ³sticky ends´
²where DNA nucleotides are not bound to their
pair. Thus, they can be easily hooked up to
another piece that has the complementary
unbound nucleotides:
4. Once you cut and piece all your DNA sequences back together, you’ll have a plasmid that contains the crt and psy genes, and you can put this plasmid into bacteria to replicate. Later, when you want to isolate the DNA from the bacteria, you’ll need to know what size the DNA is. If the plasmid is 3.5 KB in size, the psy DNA is 4 KB, and the crt1 is 1 KB, draw a gel below showing the plasmid WITH the two pieces of DNA, and in a separate lane, the result of cutting the plasmid with the enzymes HindIII and Pst1. A
C +P uncut cBut with H
12
9 KB
KB 11 KB
8 KB
10
7 6 6 KB
4 3 KB
3 1 KB
1 9 KB
Getting more copies of the recombinant DNA: The bacteria now replicate in culture, and the scientist selects the bacterial colonies that contain the recombinant DNA. 5. You might have noticed that instead of an antibiotic resistance selectable marker on the plasmid there is a phosphomannose isomerase selectable marker. Why might you want to use the phosphomannose isomerase selectable marker instead of the antibiotic resistance gene normally present in these plasmids? Creating the genetically modified plant In making transgenic plants, it is relatively easy to get the recombinant piece of DNA into the plant. The plasmid containing the recombinant DNA is incorporated into the genome of a bacterium called Agrobacteria. These bacteria naturally infect plant seeds. If the Agrobacteria are made to contain the gene of interest, as described above, then when the bacteria infects the plant, it transfers in this recombinant DNA. If the bacterial infection doesn't work, there is another technique in which the recombinant DNA is essentially injected into the plant seeds (called the "biolistic" method). 6. Your ultimate goal is to generate rice that expresses the psy gene from daffodil in the seed of the rice, where it will provide b-­‐carotene to those eating it. Starting back from the beginning, outline the steps you should take to make a plasmid that contains the psy gene from daffodils. In 2000, rice that contained daffodil phytoene synthase (psy) and bacterial phyotene desaturase (crt 1) was introduced to the world. This rice is called Golden Rice. Its introduction was met with praise from some groups and opposition from other groups (like Greenpeace). Members of Greenpeace are opposed to all genetically modified foods. China and India are starting to farm Golden Rice and researchers are now trying to figure out to add vitamins A and E, iron, and zinc into bananas, cassava, rice, and sorghum. Interestingly, 12 years later, the use of golden rice is still minimal. A lot of this has to do with politics. If you are interested, here are several links where you can read more about golden rice: Pro: http://www.goldenrice.org/
Con: http://www.i-sis.org.uk/rice.php
Right here in Boulder county, people have also debated the use of farm lands to grow genetically modified
crops (beets in this case). The originally proposed ban was NOT upheld this past December, so such crops can
still be grown in Boulder county.
See these links for some interesting articles in the Boulder Daily Camera:
Pro: http://www.dailycamera.com/ci_18912267
Con: http://www.commondreams.org/view/2011/05/08-2
MCDB 1041 Activity 7: to turn in to your LA as a group
Your names:
1. Please copy here (from Part 1 #5), the pedigree and gel you created to identify people who were carriers or
had hemophilia
2. You are in the grocery store in the US and you see a sale on golden rice. Presuming it tastes the same as
regular rice, would you buy it and eat it? If you were taking a trip to a developing country and it was legal
to do so, would you bring golden rice seed to the community?
Why or why not? Please consider both science and politics!