DNA Fingerprinting and DNA Isolation

I- DNA Fingerprinting
Human genome is made of more than 3 million nucleotides (see figure). Less than 2% of the
genome is made of proteins encoding genes, coding DNA. Genes determine our phenotype
(skin color, height). We are very similar by the coding DNA.
Our difference is located in the rest of our genome, non- coding DNA.
Non-coding DNA is made with repetitive sequences (repeats) of
nucleotides of different lengths. They are used to make DNA
fingerprinting (DNA typing, DNA profile). The repeats and their
lengths are different between individuals, restriction fragment length
polymorphism (RFLP).
DNA fingerprinting was developed by Alec Jeffrey in 1985. This
method was first used to solve crimes. Today, it is also used to
determine ancestry, paternity, victims identity after natural (hurricane Katrina) or man-made
disasters (9/11, wars).
DNA fingerprinting requires many steps: Samples collection, extraction and fragmentation of
DNA, separation of DNA fragments, staining and comparison of DNA collected from
different samples.
1- Collection of tissue samples, extraction and fragmentation of DNA. DNA is collected
from different sources. If the goal is to determine who is the crime perpetrator, DNA
is collected from the crime scene and from the suspects (like in todays experiment).
DNA can be collected from any part of a living organism (saliva, semen, hair).
If the amount of DNA collected from the crime scene is not enough to be analyzed, it
is amplified using a technique called: Polymerase chain reaction (PCR).
DNA is cut in fragments with chemical scissors called restriction enzymes
(restriction endonucleases). Hundreds of restriction enzymes are available (EcoRI,
EcoRII, Hind III, TAQ I.). Restriction enzymes are different by their binding and
cutting sites on DNA. They are used to study fragments length difference between
individuals, EcoRI will be used in this lab.
EcoRI recognition
----G A A T T C-------C T T A A G----

Results of the restriction

----G
A A T T C-------C T T A A
G----

EcoRI recognition
2- Gel electrophoresis is used to separate DNA fragments. EcoRI cuts DNA in different
length fragments. Fragments are sorted out by size using an agarose gel

electrophoresis. Negatively charged DNA fragments (because of their phosphate

groups) are loaded in the gel wells located near the negative pole of the
electrophoresis apparatus and are attracted to its opposite positive pole. Shorter DNA
fragments will migrate quicker and farther than the longer fragments.
3- Gel containing DNA fragments is stained and patterns of different DNAs are
compared. DNA fragments are stained by
specific dye, like fast blue. In our
experiment, specific SYBR DNA safe
dye is added to the buffer used to make
the gels. No additional stain is used after
running the gel.

Fingerprinting experiment
Label one of each 6 colored tubes as follows: CS (crime scene), S1 (suspect 1), S2 (suspect
2)S5 (suspect 5)
Mix 10 l of each DNA sample (CS, S1, S2, S3, S4, S5) with 10 l restriction enzyme (use
vortex). CS is the DNA collected from crime scene and S1 to S5 are DNAs collected from
suspect 1 to 5. Use microcentrifuge to settle the liquid at the bottom of the tube. Incubate at
370 C for 45 minutes in a water bath. Make sure that the bottom of the tubes is in the water.

While you are waiting for the restriction enzyme to cut DNA samples in fragments, make the
gels:
- 0.5 g agarose in 50 ml buffer 1 X Tris-Acetate-EDTA (TAE) in a 250 ml flask, use
microwave (set it at medium, swirl every 30 seconds until agarose is dissolved. Keep an eye
on it, if you don't, the gel will overflow and you have to make a new one and clean the
microwave.
- Dissolved agarose is sticky; please clean up!!
- Do not dispose the agarose excess in the sink or let it solidify in the beaker.
- After agarose is completely melted, wait until it cools down to 650 C before pouring it in the
casting tray with both extremities taped (dont forget to put the comb in one side of the
casting tray before pouring the gel!!!). It should be solid and ready to use after 10-20 minutes.

- Transfer the gel in the electrophoresis apparatus (after removing the tape and the comb) that
is connected to the power supply.
- Cover the gel with the buffer 0.25X TAE (notice that we use another buffer concentration
to run the gel) in the electrophoresis apparatus.
- After 45 minutes incubation of the mixture DNA-restriction enzyme, add 5 l loading dye
(LD) to each tube (CS, S1, S2, S3, S4, S5) and mix (use vortex and then microcentrifuge).
Loading dye is used to stain the solution (not DNA) to make the gel loading and the
electrophoresis progress easy to see. Do not use any pipette with a maximum volume over 20
l
- Load the gel starting from the left well. The wells should be next to the negative pole (black
pole) of the electrophoresis apparatus
- The first well will be loaded with 10 l of DNA marker (M) or fragments ladder, the
second with 20 l DNA from crime scene (CS), the third with 20 l from S1, the
seventh well with 20 l DNA from S5.
- Run the gel for 20 minutes at 200 V
- DNA can be stained with fast blast DNA staining solution. In this experiment we are using
SYBR safe DNA stain. It was already added to the buffer you used to make your gels. Use
the gel imager to visualize the bands on gels; follow the instructions on the machine. A copy
of the gel image can be saved on a flash drive or sent to your email address.

Below is the result that you are expected to get after you run your gel:
M

CS

S1

S2

S3 S4 S5
Wells

+
II- DNA Isolation
DNA can be isolated from any living organism (bacteria, animal, plant). In this experiment
we use banana.
Isolation of DNA follows 4 steps:
1- Breaking cells: In a blender, mix 50 ml distilled water with a banana
2- Removal of lipids by a detergent: Add few drops of dishwashing liquid to the
previous mixture and blend. Use cheesecloth to filter the mixture in a beaker kept on
ice.
3- Deproteinization: In a cold test tube, mix 2 ml (4%) meat tenderizer to 4 ml of the
filtrate. Meat tenderizer strips proteins (histones and non-histones) associated to
DNA.
4- Precipitation: After 10 min, slowly add 6 ml of cold 95% ethanol to the tube. Youll
see a whitish substance precipitate. The whitish substance (DNA) can be spooled
around a cold glass rod.