Download BioRad #166-0007EDU: Forensic DNA Fingerprinting Checklist PREP

BioRad #166-0007EDU: Forensic DNA Fingerprinting Checklist
PREP
Name:_________________________________Date:_______________
Technicians working in forensic labs are often asked to do DNA profiling (fingerprinting) to analyze
evidence in law enforcement cases and other applications. Restriction Fragment Length Polymorphism
(RFLP) has been the workhorse of forensic DNA profiling for many years. First described by English
geneticist Alec Jeffries in 1985, RFLP analysis provides a unique banding pattern based on the restriction
sites present in an individual’s DNA sequence.
Restriction enzymes (endonucleases) are natural defense mechanisms of bacteria against invading
bacteria. Restriction enzymes act like molecular scissors, making cuts at specific sequence of base pairs
(palindromes) that it recognizes. Bacteria’s own DNA is protected by methyl groups at sites that would be cut
(restriction sites). Restriction endonucleases are named for the bacteria from which they were isolated. For
example, EcoRI was isolated from Escherichia coli. Endonucleases slide along the DNA helix until it
recognizes specific base pairs that signal it to stop. The enzyme then cuts the DNA at that site – called a
restriction site.
The two restriction enzymes used in this lab are EcoRI and PstI (Providencia stuartii). The
palindromic sequence recognized by EcoRI is GAATTC and the sequence recognized by PstI is CTGCAG.
Both cut between the G and A.
After the DNA is cut, fragments of varying sizes are produced and can now be separated using
agarose gel electrophoresis. Electrophoresis means to carry with electricity. DNA has a negative charge
due to the phosphates in its backbone and will be drawn toward the positive pole (anode) when placed in an
electric field. The matrix of agarose acts as a molecular sieve through which smaller DNA fragments can
move more easily than larger ones. Smaller DNA fragments will travel farther than larger ones. When loading
a gel, load DNA on the negative end (black) and it will run to red (positive end).
This kit contains plasmid DNA. One sample represents DNA obtained at the crime scene. The five
other samples are the possible suspects. Your job is to determine which suspect committed the crime.
Other DNA Fingerprinting Scenarios:
1) Food identification (endangered species identification): To determine the purity of ground beef.
Hamburger has been shown to often be a mixture of pork and other non-beef meats. Also, making
sure the fish served in sushi is not whales or dolphins.
2) Accused and convicted felons set free because of DNA typing.
3) Identifying human remains.
4) Proving paternity.
5) Determining the relatedness of humans: DNA typing has shown that the 5,000 year old “Iceman”
found in a melting glacier is most closely related to modern Europeans.
6) Identifying organisms that cause disease: Using DNA technology, scientists in Nicaragua and
Ecuador were able to detect tuberculosis and identify the dengue virus.
Princeton Satellite Site
Spring 2010
K. McKone
BioRad #166-0007EDU: Forensic DNA Fingerprinting Checklist
PREP
Name:_________________________________Date:_______________
I. Rehydrate DNA samples. Designed for 8 groups.
Needed: 6 DNA sample vials (in kit)
6-colored microtubes to match vial
1-Sterile water (in kit) – placed in
One microtube
_______1) Distribute the 6 DNA sample vials w/ matching color microtube. Label colored
microtube w/ code abbreviation found in chart below.
_____ 2) Place 1,000µL of sterile water in a microtube. This tube will be used by each person rehydrating
samples.
_______3) Add 200 µL of sterile water to each lyophilized (freeze-dried) DNA samples. Write the
name of student assigned to DNA sample in boxes below.
_______4) Replace stopper and shake vigorously (vortex if possible but keep finger on the
stopper so it won’t be a popper). CRITICAL to dissolve all powder; some may be stuck
in the stopper.
_______5) Allow samples to rehydrate @ room temp. for 15 minutes or water bath @ 37oC for
10 minutes.
_______6) Transfer to color-coded 1.5 mL microtube & return your prepared solution to teacher.
Green
Blue
Orange
Purple(Violet) Pink(Red)
Yellow
Crime Scene DNA
(CS)
Student:
Suspect 1
(S1)
Suspect 2
(S2)
Suspect 3
(S3)
Suspect 5
(S5)
Suspect 4
(S4)
Rehydrated DNA samples contain buffer, pH 8.0, NaCl, MgCl2.
II. GET YOUR MOLECULAR SCISSORS READY! Rehydrate lyophilized EcoRI/PstI enzyme mix
(Restriction Enzymes)
________7) ONLY ONE PERSON DOES THIS! Student: _________________
Add 750 µL sterile water and swirl to resuspend enzymes. Allow it to rehydrate on ice for 5
minutes . KEEP on ice, but do not freeze, once hydrated.
REHYDRATED ENZYMES should be used w/in 12 HOURS.
READY TO BEGIN THE FORENSIC LAB!
Princeton Satellite Site
Spring 2010
K. McKone