Laboratory 7: Restriction Fragment Length Polymorphism
Restriction Fragment Length Polymorphism (RFLP) is a technique, which
allows one to detect minor differences in DNA sequences between two individuals,
for such purposes as paternity cases or criminal cases. However, RFLP is not just
used in forensics, but it is also used to determine whether a person is carrying a
disease causing allele for a particular gene.
Specifically, when one is doing RFLP, one is going to subject several DNA
samples to restriction digestion with several different restriction enzymes (covered
last week). Usually the DNA that will be used is either genomic DNA obtained from a
blood sample etc., or copies of a specific gene obtained by PCR of the genomic DNA.
As one studies genomic DNA, one must realize that we are studying all of the
DNA an individual has, and, in effect, this DNA contains all the genes an individual
has. For each gene, we have two copies. One of the copies (alleles) comes from the
mother and the other comes from the father. In some cases, the DNA sequences of
both copies are the same, and thus the individuals are homozygotes. If the DNA
sequences of the copies are slightly different, then they are heterozygotes.
Oftentimes, the differences in DNA sequences affect the presence or absence of a
restriction enzyme recognition site. If this is the case, then when you cut with the
restriction enzyme, one copy will cut and the other one will not, leaving you a
specific banding pattern on your gel when you visualize your results. From this
banding pattern one can actually determine the genotype of the individual. By
determining the genotype of the individual in this manner, then you have data for
either disease diagnosis or forensics.
Let’s take a small example. Let's look at two people and the segments of DNA they
carry that contain this RFLP. Since Jack and Jill are both diploid organisms, they
have two copies of this gene. For simplicity in our analysis, we will follow one copy
at a time for each individual. Now, let’s start with copy one for each person. When
we examine one copy from Jack and one copy from Jill, we see that they are
completely identical in DNA sequence: Note, at each end there is an EcoRI site. If
one were to cut with the EcoRI enzyme, we would liberate a fragment that is rougly
2000 bp for each person.
Jack 1: -GAATTC---(1000 bp)---GCATGCATGCATGCATGCAT---(1000 bp)---GAATTCJill 1: -GAATTC---(1000bp)---GCATGCATGCATGCATGCAT---(1000 bp)---GAATTCWhen we examine their second copies of this gene, we see several things. First, for
Jack, the second copy is the same as the first. Thus, he is a homozygote. We further
see, that Jill’s second copy is different in sequence from Jack’s, as well as her first
copy. Specifically, we see that Jill’s copy has a third EcoRI site about 200 base pairs
from the first
Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)GAATTCJill 2: -GAATTC--(200 bp)-GAATTC--(800 kb)--GCATGCATGCATGCATGCAT--(1000
bp)-GAATTCTherefore, when Jack and Jill this copy cut, we will see different results. For
Jack, we will see a 2000 base pair band and for Jill, we will see 2 bands one 200 bp
and one 800 base pairs. Now, when we subject the DNA to RFLP analysis, we cannot
separate out the copies of the genes for each individual from each other. Therefore,
each individual’s DNA is cut in separate tubes. However, for each individual, each
copy of the gene will be cut in the same tube, and thus run in the same lane on the
gel. For Jack, we will only see the 2000 bp band because for each individual, after
cutting, we get a 2000 base pair band. For Jill, the situation is different. For copy 1,
after digestion she has a 1000 base pair band. For the second copy, she has a 200 bp
and an 800 bp band. On the gel for Jill’s sample you will see all three bands in her
lane. This allows you to determine the genotype for both individuals.
In this laboratory, we will use RFLP for forensic purposes to determine who
is the criminal who perpetrated our crime. Therefore, we have several DNA samples
we will subject to restriction digest analysis. One of these samples will be from the
crime scene and the others from the suspects. After analysis, we will compare the
results of the suspects to the crime scene. The perpetrator of the crime will have an
identical RFLP to the DNA at the crime scene.
Materials
1. Crime Scene DNA
2. Suspect 1 DNA
3. Suspect 2 DNA
4. Suspect 3 DNA
5. Suspect 4 DNA
6. Suspect 5 DNA
7. EcoRI/PstI enzyme mix
8. Agarose
9. 1 X TAE
10. 37 C water bath
11. Microfuge Tubes
Procedures:
A. Restriction Digestion of DNA
1. Obtain a tube of DNA from the crime scene, as well as a tube of DNA for each
suspect (a total of 5 suspects). Be sure to have tubes for five potential suspects.
2. Obtain a tube of EcoRI/PstI enzyme mix. This mix will contain the two restriction
enzymes EcoRI and PstI.
3. Obtain 6 empty microfuge tubes. Label the first tube CS (crime scene). Label the
subsequent tubes in order S1-S5.
4. Pipet 10 ul of each DNA solution to your corresponding labeled tube.
5. Pipet 10 ul of enzyme mix to each of your labeled tubes. Within the mix, there
are the appropriate buffers for the enzymes to function properly.
6. Tightly cap your tube and flick the tube to mix the components.
7. Place your samples in the microfuge and quickspin them for 10 seconds to bring
your enzyme/DNA mix to the bottom of the tube.
8. Incubate your samples at 37 C for 45 minutes
B. Pouring and Running The Gel
1. Prepare a 40 mL solution of 1% agarose in 1X TAE in a flask and melt the agarose
into solution in the microwave.
2. When your solution is cool to the touch, pour your gel into the casting tray.
3. Once your gel has solidified, place the gel into the gel box, and prepare for your
gel run
4. Add 5 ul of DNA dye to each of your reaction tubes (after the 45 minute
incubation is finished).
5. Cap each tube. Mix the components by flicking the tube. Then, quick spin the
components to the bottom of the tube as before.
6. Obtain a tube of DNA ladder
7. Add DNA samples to each well in the appropriate order
Lane 1: 10 ul ladder
Lane 2: 20 ul Crime scene
Lane 3 20 ul Suspect 1
Lane 4: 20 ul Suspect 2
Lane 5: 20 ul Suspect 3
Lane 6: 20 ul Suspect 4
Lane 7: 20 ul Suspect 5
8. Run your gel at 110 V for approximately 45 minutes (or until the lead dye is at
least 2/3 down the gel).
9. When your gel run is complete, stain your gels