Download A2 Activity 8 Response for Digesting Genes into Bite

A2 Activity 8
Response for Digesting Genes into Bite Size Pieces
A restriction digest is also known as DNA fragmentation. It uses a restriction
enzyme, which recognizes unique sequences in DNA, to selectively cut strands of
DNA into shorter segments, which are more suitable for analytical techniques.
Restriction digests are performed in Genetic fingerprinting techniques using
restriction fragment length polymorphisms and are used in many other DNA
manipulations.
Each restriction enzyme cuts DNA segments within a specific nucleotide sequence,
and always makes its incisions in the same way. These recognition sequences are
typically only four to twelve nucleotides long. Because there are only so many ways
to arrange the four nucleotides--A,C,G and T--into a four or eight or twelve nucleotide
sequence. Furthermore, restriction enzymes specific to hundreds of distinct
sequences have been identified. As a result, potential restriction sites appear in
almost any gene. So no matter the context in which a gene naturally appears, there
is probably a pair of restriction enzymes that can cut it out and which will produce
ends that enable the gene to be spliced into a plasmid; molecular biologists call this
‘cloning of the gene’.
Once the cuts are made with the restriction enzymes, the plasmid can then be
loaded into a gel for electrophoresis. In electrophoresis, the negatively charged DNA
is pulled through an agarose gel, causing the shortest fragments of the plasmid to
extend farther into the gel, and separating each cut segment from the other. A
marker is also loaded with the gel, indicating the amount of base pairs in each
segment of the plasmid. The new DNA fragment may then be extracted from the gel
by cutting it, and doing a gel purification. Through ligation of a vector which has also
been cut by a restriction enzyme, the fragment of interest can then be inserted into
this vector and a new synthetic plasmid is formed. The new plasmid is then
transformed into a biological system and replication occurs.
In order to tackle the second part of this activity you need to first get the DNA
sequence from the NCBI database. You can do this by following the link on the
NEBCUTTER website http://tools.neb.com/NEBcutter2/index.php. Once you have
opened the NEBCUTTER page using the above link, you need to click on the
‘Browse GenBank’ link. In the ‘search nucleotide for’ box at the top of the page,
enter the gene name Shh or sonic hedgehog. This will give you a whole list of the
Shh genes from different organisms and their related genes e.g. interacting partners
and ones with similar sequences to Shh, i.e. genes in the same gene family as Shh.
View the list and scroll down until you find the following sequence:NM_009170
Mus musculus sonic hedgehog (Shh), mRNA
gi|161484664|ref|NM_009170.3|[161484664]
Once located, the page containing all of this gene data by clicking on NM_009170,
scroll down the page to find the sequence (you have to go past the protein sequence
to find it).
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ORIGIN
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acaagctctc
61
ctcgagaccc
121 cagtctgggt
181 aagagaaaga
241 gagcgcaagg
301 cggggacagc
361 gttttctggt
421 gcagggggtt
481 ttattcccaa
541 caagaaactc
601 aggatgagga
661 atgccttggc
721 gctgggatga
781 acatcaccac
841 aagcaggttt
901 cagagaactc
961 tggagcaggg
1021 ctgacgacca
1081 gcgccaagaa
1141 ccgccgcgca
1201 gcgcgctctt
1261 gcggggaccg
1321 cgggcgcgta
1381 cgtgctacgc
1441 tggcgcacgc
1501 gcatccctgc
1561 actggtactc
1621 atcccttggg
1681 cggggcgggg
1741 taattataag
1801 gcagtccaaa
1861 tttatatatt
1921 tggctattta
1981 ccttaactag
2041 aaagattatt
2101 tcctgcgttt
2161 aagtgtaaac
2221 gtaaattaga
2281 taaaatatga
2341 tactgccttc
2401 aagactgtta
2461 ttttaacttc
2521 cccacgagga
2581 aactgagaag
2641 ttatagaaat
2701 aaaaaaaaaa
cagccttgct
aactccgatg
ggggatcgga
gccaggcagc
aggagcgcac
tcacaagtcc
gatccttgct
tggaaagagg
cgtagccgag
cgaacgattt
aaacacggga
catctctgtg
ggacggccat
gtccgaccgg
cgactgggtc
cgtggcggcc
cggcaccaag
gggccggctg
ggtcttctac
cctgctcttc
tgccagccgc
ccggctgctg
cgcgccgctc
tgtcatcgag
gctgctggcc
agcgcaatct
gcagctgctc
aatggcggtc
agcgactgcg
aataattcat
gtagactata
tttttgaaat
tttgtttcgt
tttgtgtctt
ttgtgaggcc
cagaaggcaa
taaaacctgc
tttttgagag
aaatatatta
ttggtttgta
acgcacacat
aaagaattta
tggagcctgt
taactgctgt
aaagcgtgcc
aaaaaaaaaa
accatttaaa
tgttccgtta
gacaagtccc
gccagaggga
acgcacacac
tcaggttccg
tcctcgctgc
cggcacccca
aagaccctag
aaggaactca
gcagaccggc
atgaaccagt
cattcagagg
gaccgcagca
tactatgaat
aaatccggcg
ctggtgaagg
ctgtacagcg
gtgatcgaga
gtggcgccgc
gtgcgccccg
cccgccgcgg
acggcgcacg
gagcacagct
gcgctggcac
gcaacggaag
taccacattg
aagtccagct
aaataaggaa
aataataata
aggaagcaaa
ttttcgttat
atgaatagat
ggataattta
aagcaacctg
acctccgcat
tccatggggg
atcaatacct
ttttaattta
tttgctttgt
atacactttt
ttagaaaata
agtttgtaca
aaatttacta
acacacaaaa
aaaaaaa
atcaggctct
ccagcgaccg
ctgcagcagc
acgaacgagc
ccgcgcgtac
cggacgagat
tggtgtgccc
aaaagctgac
gggccagcgg
cccccaatta
tgatgactca
ggcctggagt
agtctctaca
agtacggcat
ccaaagctca
gctgtttccc
acttacgtcc
acttcctcac
cgctggagcc
acaacgactc
ggcagcgcgt
tgcacagcgt
gcaccattct
gggcacaccg
ccgcccgcac
cgaggggcgc
gcacctggct
gaagcccgac
ctgatgggaa
ataatgataa
aaccccgggg
tgtcttatat
gttttaaaaa
ttattgtgtg
ctgaaagtct
tcctctcctg
tccacaaatt
aactgaatga
actattttcc
aaccgccact
ttttttgaca
atatttttta
gagaaaaaca
aaatgtattt
aaaaaaaaaa
ttttgtcttt
gcagcctgcc
ggcaggcaag
cgagcgagga
ccgctcgcgc
gctgctgctg
cgggctggcc
ccctttagcc
cagatatgaa
caaccccgac
gaggtgcaaa
gaagctgcga
ctatgagggt
gctggctcgc
catccactgt
gggatccgcc
cggagaccgc
cttcctggac
gcgcgagcgc
ggggcccacg
gtacgtggtg
gacgctgcga
catcaaccgg
ggccttcgcg
ggacggcggg
ggagccgact
gttggacagc
gggaccgggc
agcgcacgga
taataataat
agttctgttg
gggttgtttt
tatgaacgga
aactgtactc
atttttctac
ctatgctcct
atatttttat
catttcattt
aatgtaatag
ttgtcatgtt
gactggaaga
aaagtgcacc
aggatgtttt
ttgaatattt
aaaaaaaaaa
taattgctgt
atcgcagccc
gttatatagg
agggagagcc
acagacagcg
ctggccagat
tgtgggcccg
tacaagcagt
gggaagatca
atcatattta
gacaagttaa
gtgaccgagg
cgagcagtgg
ctggctgtgg
tctgtgaaag
accgtgcacc
gtgctggcgg
cgcgacgaag
ctgctgctca
cccgggccaa
gctgaacgcg
gaggaggagg
gtgctcgcct
cctttccgcc
ggcgggggca
gcgggcatcc
gagaccatgc
aaggggcggg
aggagacttt
aataagtagg
ttatgtttag
tctcctctcc
ccttcaagag
acagtgaggg
atgtcccttg
gctttcccgc
acacagaatt
tttgaaagtg
ccgtcttctg
cttggaaacc
actctgttat
tagcagcgag
tgcattaata
tgtaatagtt
aaaaaaaaaa
Copy this sequence into the sequence box on the NEBCUTTER site (or just paste
the Genebank number into the search box).
You will get back a chart of the enzymes that cut this sequence like the one below:This also shows an example of what a restriction digests would look like if you
digested the DNA with Bsu361 and Acu1 in the same reaction; you would get three
fragments as shown.
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< 1
><
2
><
3
>
At this point you need to go back to the NEB home page and use the enzyme finder
function on the NEB website http://www.neb.com/nebecomm/EnzymeFinder.asp.
Use this function to find the sequence required in the Shh gene to be a cut site; try it
for a number of these restriction enzymes e.g try XhoI, BamHI, SpeI, HincII. Look
through the whole sequence and try to find the critical sequence required for these
enzymes to cut. Once you have done this, check the length of the fragments and run
them on a virtual agarose gel. Follow this tutorial first:
http://learn.genetics.utah.edu/units/biotech/gel/ and this site to simulate running a gel
http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/virgel.html.
Then draw your own gel using your fragments from the different digests you have
done, remember to use a molecular weight marker, see
http://www.neb.com/nebecomm/products/productN3232.asp for a 1Kb molecular
weight marker.
Agarose gel electrophoresis
Electrophoresis can be used to separate nucleic acids by size. Since DNA in
negatively charged it will move towards the positive electrode. Gels can be made out
of a number of components that have a jelly like form, but agarose is the most
commonly used, it is a highly refined form of agar, which comes from seaweed. The
percentage gel (w/v) will vary depending on the size of the fragments you are trying
to resolve - see table below:Agarose (%)
0.5
0.7
1.0
1.2
1.5
Range of resolution of linear DNA fragments (Kb)
30 to 1
12 to 0.8
10 to 0.5
7 to 0.4
3 to 0.2
What gel concentration would you run your fragments on for maximum separation?
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Real life uses of restriction enzymes
How can we take genes from one organism and connect them with genes from
another organism?
Using restriction enzymes, we can cut DNA from each organism and then ligate (stick)
it together.
Why would we want to do this?
This is extremely useful if you want to have an organism make a protein it doesn't
normally make (e.g. bacteria making insulin).
If we take the human insulin gene, cut it out of the human genome using restriction
enzymes, ligate it into a plasmid with the ampicillin resistance gene, and transform
bacteria with this plasmid, then all the ampicillin resistant bacteria will be making
insulin that can be used to treat diabetes.
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