Biology A
Techniques for Analyzing and Changing DNA
Probably the most important thing to know about a piece of DNA is “What is the sequence of
nucleotides, or bases that is present?” We won’t worry about how that is done, but if you want
to find out, you can go to http://www.pbs.org/wgbh/nova/body/sequence-DNA-for-yourself.html
and work through the animation. What you should know is that DNA is usually dealt with in
pieces much smaller than a whole chromosome – usually just a few hundred or thousand bases at
a time. So how can DNA be cut into manageable chunks?
Restriction Enzymes act like molecular scissors that can only cut DNA at a certain sequence of
bases. For example, EcoR1 will only cut where it finds GAATTC; it cuts between the G and the
first A on both strands like this. The unpaired bases or “sticky ends” will come in handy later.
There are lots of restriction enzymes, so knowing where each will cut and can allow cutting a
particular gene out of a chromosome. Many sequences for restriction enzymes occur at least
every few hundred bases in a random piece of DNA, so they can be used to cut an unknown
stretch into pieces that can be used for the techniques you read about in chapter 13.
For example, consider the piece of DNA below. Imagine that it is 4000 bases long. We know
that it can be cut at the positions marked by the restriction enzymes shown.
BamH1 EcoR1 Pst 1
Hind III
Pst 1
BamH1 Pst 1
Hind III
If you cut with BamH1, how many pieces would you get?
How big would each be?
Hind III?
Pst 1?
EcoR1?
How about Bam HI and EcoRI at the same time???
The number of pieces is pretty easy, the sizes less so, unless we are happy with “big” “small”
and “medium”. We are not happy, so gel electrophoresis can help.
The negatively charged DNA will move away from the negative electrode and shorter pieces will
move faster. If you add a sample of DNA pieces of known sizes, you can use them to compare
the unknown pieces,
Virtual gel electrophoresis:
http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/virgel.html
Use the “gel” to the left to sketch in the bands the result
from the digests of the chromosome on the last page.
Don’t forget to include the standard!
Now you can do some interesting things, like identify an individual by his or her unique DNA
sequences.
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DNA Fingerprinting
As you learned in chapter 13, there are regions of chromosomes with many differences in the
base sequences. These usually occur between genes because they are not changing any
important protein-building instructions. Cutting a sample of DNA from an individual with a
particular restriction enzyme will produce a unique pattern of bands when the DNA fragments
are separated by gel electrophoresis. Different people will show different patterns, but closely
related people will have a number of bands in common. A child’s pattern should be a mix of
what is present in his or her parents. Try this sweet little exercise:
Go to http://www.pbs.org/wgbh/nova/teachers/body/create-dna-fingerprint.html
Read through the introduction and click on “view”. Read the introductory story and follow the
instructions in the animation to create a DNA fingerprint of the sample and possible perpetrators.
Answer the following questions.
1. What does a restriction enzyme do to the DNA samples?
2. Should you cut all the samples you are comparing with the same restriction enzyme?
Explain.
3. There are a number of ways to make the colorless DNA visible after the gel has been run.
What is used in this investigation? Can you see the DNA directly or how do you detect
it?
4. So who committed the “crime”?
In reality, DNA fingerprinting, or as it is now more commonly called DNA profiling has become
a standard technique in identifying the perpetrators in real crime situations. CSI anyone? Here
are some real life examples.
Example 1: Here is an actual DNA comparison conducted in a case in England where defendant
2 was accused of a crime in which he admitted guilt. After DNA analysis, however, police
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realized he could not have been the culprit. After a mandatory DNA analysis of all males in the
town, the true criminal was found and convicted to life in prison.
In case you can’t read this, the DNA
standard is in lane 1, the technician who ran
the DNA put his DNA in lane 2, the rape
victim’s DNA is in lane 3, Defendant 1 is in
lane 4, Defendant 2 is in lane 5, another
standard is in lane 6 and DNA taken from
semen found in the rape victim is in lane 7.
Who is actually guilty?
Why might the technician’s DNA be
included?
Example 2.
Evidence collected at crime scene is in lanes 9 and 12.
The victim's DNA is in lane 4
The suspects' in 5 and 6
Remember evidence could have a mix of victim and
suspect DNA!
Who do you think is guilty?
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