Transcript Activity 4.5 Forensic DNA Fingerprinting

DNA Structure
and Analysis
Activity 4.5: Forensic
DNA Fingerprinting
Activity 4.5: Forensic DNA Fingerprinting
 Research Question:
– Which suspects can be eliminated from the investigation?
 Objectives:
– Digest DNA samples from the crime scene and the five
suspects with EcoRI and PstI restriction enzymes
– Load and run samples on a 1% agarose TAE gel
– Generate a standard curve based on lambda HindIII DNA
standard
– Determine the length of all DNA fragments by comparison to
the standard curve
– Determine if the DNA from the crime scene matches the DNA
from any of the suspects
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Safety Reminders
 Follow all laboratory safety procedures
– Appropriate PPE should be worn at all times
– Attention should be given when using electricity in the
presence of liquids. If there are leaks in equipment,
notify teacher immediately
– Turn off power before removing lid and ensure that lid
is in place before turning power on
– If ethidium bromide or SYBR® Safe stain is used,
follow appropriate safety precautions
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Skills to Master
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Perform a restriction digest (Activity 4.4)
Perform agarose gel electrophoresis (Activity 4.3)
Analyze an agarose gel (Activity 4.4)
Generate standard curve using a DNA standard
(Activity 4.4)
 Determine DNA fragment sizes using standard
curve (Activity 4.4)
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Student Workstation Materials
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Setting Up a Restriction Digest
 Play video: Restriction Digestion of DNA
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Running an Agarose Gel
 Play video: Agarose Gel Electrophoresis
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Protocol Highlights/Tips
 When setting up restriction
digests use fresh tips each time
to prevent contamination
 Tubes can be incubated in a
water bath, dry bath, or at room
temperature overnight
– If incubating overnight, it is helpful to
incubate for a short while at 37ºC
first, then let come to room
temperature overnight
– If left too long, some enzymes
exhibit STAR activity, which means
they will cut in places that are not a
complete match. So digesting longer
than recommended times could give
suboptimal results
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Protocol Highlights/Tips
 Once the gel is stained and imaged, measure the
distance that the lambda HindIII standard bands
moved from the well
 Record all information into the notebook using a
table similar to Activity 4.4
 Measure the bands in the other lanes and record
in the table
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Protocol Highlights/Tips
 Graph the lambda HindIII control,
using distance traveled as the X axis
and DNA base pairs as the Y axis
 This can be done on semilog graph
paper or with Microsoft Excel
– If Microsoft Excel is used, adding a
trend line to the linear part of the
curve and displaying the equation
allows one to mathematically solve for
y given x
 Convert all of the distance
measurements into base pairs
using the graph
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Lambda HindII Standard
Summary
 Make sure to:
– Record all steps in your notebook
– Ensure all lane contents are
recorded in an organized manner in
your notebook
– Measure bands using a ruler that
measures using millimeters
– Measure to the leading edge of the
band as it travels away from the
well
– Compare crime scene bands to all
other suspects
– Who can be eliminated?
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