BIO304: Forensic DNA Fingerprinting Lab
DNA Fingerprinting, method of identification that compares fragments of DNA.
It is sometimes called DNA typing. With the exception of identical twins, the complete
DNA of each individual is unique.
A DNA fingerprint is constructed by first extracting a DNA sample from body
tissue or fluid such as hair, blood, or saliva. The sample is then segmented using
enzymes, and the segments are arranged by size using a process called electrophoresis.
The segments are typically marked with radioactive probes and exposed on X-ray film,
where they form a characteristic pattern of black bars—the DNA fingerprint. If the DNA
fingerprints produced from two different samples match, the two samples probably came
from the same person.
DNA fingerprinting was first developed as an identification technique in 1985.
Originally used to detect the presence of genetic diseases, DNA fingerprinting soon came
to be used in criminal investigations and forensic science. The first criminal conviction
based on DNA evidence in the United States occurred in 1988. In criminal investigations,
DNA fingerprints derived from evidence collected at the crime scene are compared to the
DNA fingerprints of suspects. The DNA evidence can implicate or exonerate a suspect.
Various methods utilized in DNA fingerprinting:
Restriction Fragment Length Polymorphism (RFLP)
RFLP is a technique for analyzing the variable lengths of DNA fragments that result from
digesting a DNA sample with a special kind of enzyme. This enzyme, a restriction
endonuclease, cuts DNA at a specific sequence pattern know as a restriction
endonuclease recognition site. The presence or absence of certain recognition sites in a
DNA sample generates variable lengths of DNA fragments, which are separated using gel
electrophoresis. They are then hybridized with DNA probes that bind to a complementary
DNA sequence in the sample. RFLP is one of the original applications of DNA analysis
to forensic investigation. With the development of newer, more efficient DNA-analysis
techniques, RFLP is not used as much as it once was because it requires relatively large
amounts of DNA. In addition, samples degraded by environmental factors, such as dirt or
mold, do not work well with RFLP.
PCR Analysis
PCR (polymerase chain reaction) is used to make millions of exact copies of DNA from a
biological sample. DNA amplification with PCR allows DNA analysis on biological
samples as small as a few skin cells. With RFLP, DNA samples would have to be about
the size of a quarter. The ability of PCR to amplify such tiny quantities of DNA enables
even highly degraded samples to be analyzed. Great care, however, must be taken to
prevent contamination with other biological materials during the identifying, collecting,
and preserving of a sample.
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STR Analysis
Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within
nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile
from another. The Federal Bureau of Investigation (FBI) uses a standard set of 13
specific STR regions for CODIS. CODIS is a software program that operates local, state,
and national databases of DNA profiles from convicted offenders, unsolved crime scene
evidence, and missing persons. The odds that two individuals will have the same 13-loci
DNA profile is about one in one billion.
Mitochondrial DNA Analysis
Mitochondrial DNA analysis (mtDNA) can be used to examine the DNA from samples
that cannot be analyzed by RFLP or STR. Nuclear DNA must be extracted from samples
for use in RFLP, PCR, and STR; however, mtDNA analysis uses DNA extracted from
another cellular organelle called a mitochondrion. While older biological samples that
lack nucleated cellular material, such as hair, bones, and teeth, cannot be analyzed with
STR and RFLP, they can be analyzed with mtDNA. In the investigation of cases that
have gone unsolved for many years, mtDNA is extremely valuable. All mothers have the
same mitochondrial DNA as their daughters. This is because the mitochondria of each
new embryo comes from the mother's egg cell. The father's sperm contributes only
nuclear DNA. Comparing the mtDNA profile of unidentified remains with the profile of a
potential maternal relative can be an important technique in missing person
investigations.
In today’s lab we will be performing a modified RFLP analysis of DNA collected at a
crime scene and comparing that with DNA isolated from various suspects.
Materials
Pre digested DNA samples:
Crime Scene (CS)
Suspect 1 (S1)
Suspect 2 (S2)
Suspect 3 (S3)
Suspect 4 (S4)
Suspect 5 (S5)
Electrophoresis gel box
100X Fast Blast DNA Stain
sample loading dye (LD)
200l pipet tips (yellow tips)
P20 pipettor
P200 pipettor
1% agarose gel
1X TAE Buffer
HindIII size marker (M)
Power supply
Procedure:
1. Add 5l of sample loading dye (LD) to each DNA sample. Be sure to use a clean pipet
tip for each sample.
2. Place the 1% agarose gel into the gel electrophoresis box and very carefully remove
the comb from the gel by pulling straight up.
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3. Pour enough 1X TAE buffer into the electrophoresis chamber so that your gel is
completely submerged.
4. Using a separate pipet tip for each sample, load you digested DNA samples into the gel
as follows:
NOTE: Gels are typically read from left to right.
Lane 1:
Lane 2:
Lane 3:
Lane 4:
Lane 5:
Lane 6:
Lane 7:
10l HindIII size marker
20l CS DNA
20l S1 DNA
20l S2 DNA
20l S3 DNA
20l S4 DNA
20l S5 DNA
5. Secure the lid on the gel box.
6. Turn on the power supply, set it for 100V and electrophorese samples for 30-40
minutes.
7. When the electrophoresis is complete, turn off the power supply and remove the lid
from the gel box. Carefully remove the gel tray from the electrophoresis chamber and
slide your gel into a staining tray which has your groups initials. Be careful, as the gel is
very slippery.
8. Pour enough of the 100X Fast Blast Stain into the staining trays so that your gel is
submerged.
9. Stain gels for 2-3 minutes.
10. Pour off the stain and add tap water (again, be sure that your gel is submerged). Rinse
the gel for ~10 seconds.
11. Pour off rinse water, and again submerge your gel in tap water. Gently rock or shake
your gel for ~5 minutes.
12. Remove rinse water and repeat rinse with clean tap water.
13. Pour off water and examine the stained gel for expected DNA bands.
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