Forensic DNA Fingerprinting Kit Instruction - Amherst College

46 Pages·2012·14.38 MB·English

ETxpMlorer
Forensic DNA Fingerprinting Kit
Instruction Manual
Catalog #1 66-0007EDU
explorer.bio-rad.com
The kit is shipped at room temperature. Open immediately upon
arrival and store reagent bag at—20°C within 3 weeks of receipt.
Duplication of any part of this document is permitted for classroom use only.
Please visit explorer.bio-racLcomto access our selection of language translations for
Biotechnology Explorer kit curriculum.
For technical service call your local Bio-Rad office, or in the US, call 1-800-4BIORAD (1-800-424-6723)
Can DNAevidencesolvehuman problems?
DNAfingerprinting is now used routinelytosolvecrimes. In recentyears, newsstories have
reported howmiriisculeamountsofDNAhavebeen usedto identifyindividualsinvolved in
incidentseven manyyearsinthe past, aswell asexonerate innocentpeoplefrom incrimi
nation.
The powerof DNAasatoolforindividual identificationcapturesstudents’ imaginations.
Thisactivityprovidesin-depth instructionabouthowrestrictionenzymescleave DNA, how
electrophoresisasusedtoseparateandvisualize DNAfragments and howthesetechniques
can becombinedtoobtain a DNAfingerprint. Principlesofrestriction analysis, plasmid
mappingand DNAfragmentsizedeterminationcan alsobedocumentedwiththis kit.
Openthe doorto rich discussionsaboutscientific, ethical, and legal implicationsof DNA
profiling DNAfingerpnnting is used in medicalandforensicprocedures aswell as in
paternitydeterminationstodiscerngeneticrelationshipsbetweenindMdualsatthemolecular
level. This kitallowsstudentstoplaythe role ofaforensicscientistand makeapositive
ID—thatis, tosimulate using real DNAas evidenceandfigureoutforthemselves: “Who
done it?”
Inthisactivity, studentsanalyzesixdifferentsamplesofplasmid DNA. Onesamplecollected
from ahypothetical“crime scene”andfivesamplesobtainedfrom “suspects”aredigested
withtwo restriction enzymes. The resulting DNAfragmentsareseparated andvisualized in
agarosegels using Bio-Rad’sFast BlastTM DNA stainingsolution. Based onthe restriction
fragmentpatterns, studentscomparetheevidenceand matchoneofthesuspects’ DNAto
thesamplecollected atthecrimescene.
Asanalternativetotheclassicalhumanforensicapplicationsforthiskit, haveyourstudents
imaginetheyarehightech pathologists investigatinganoutbreakofan aggressiveinfectious
diseasethathasneverbeenseen before.TheCentersforDiseaseControland Prevention
suspectsthatanewstrainofbacteriahasarisenthatnotonlyisthecauseofthenewdisease,
butalsohasacquiredmultipleresistanceplasmidsfromsomeotherbacterialstrains. Their
jobistodevelopa DNAdiagnostictoolforidentifyingtheculpritplasmids. Theydecideto
use restrictionenzymeanalysisand“DNAelectrophoresisfingerpnnting”to identifyand
distinguish differentsuspectplasmidsandtracktheirspreadthroughtheenvironment DNA
fromtheculturesofanumberofstricken patientshasbeen isolated. Haveyourstudents
identifythe newkillerbug beforethepathogen getsoutintothegeneralpopulation and
startsatrueepidemic!
We strivetocontinuallyimproveourBiotechnology Explorerkitsandcurricula. Please
shareyourstories, commentsandsuggestions!
Youcandownloadthiscomplete instructionmanualonthe Internet. Visit usontheWebat
explorerbio-racLcomorcall usinthe USat I-800-4BIORAD (1-800-424-6723).
Thiscurriculum wasdeveloped in collaborationwith Len Poli and RussJanigianofthe SF.
Base BiotechnologyProgram in San Francisco California and PeggySkinnerofthe Bush
School in Seattle, Washington. We’d liketothankthemfortheirinvaluableguidanceand
contributionstothiscurrkulum.
Ron Mardigian
BiotechnologyExplorerProgram
Bio-Rad Life ScienceGroup
2000Alfred Nobel Drive
Hercules, California94547

Visualization of DNA Fragments
1. When the electrophoresis run iscomplete,
turn off the power and remove the top of
thechamber. Carefully removethegel and
trayfrom thegel box. Be careful the gel
—
is very slippery. Slide the gel into the
staining tray.
2. You havetwooptionsforstainingyourgel:
Quickstaining (requires 12—15 minutes)
a. Add 120 ml of lOOx Fast Blast DNA
stain into a staining tray (2 gels per
tray).
b. Stain the gels for 2 minutes with
gentle agitation. Save the used stain
forfuture use.
c. Transfer the gels into a large
washing container and rinse with
warm (40—55°C) tap water for
approximately 10 seconds.
d. Destain bywashing twice in warmtap
waterfor 5 minutes each with gentle
shaking for best results.
e. Record results.
f. Trim away any unloaded lanes. U— U
g. Air-drythe gel on gel supportfilm and
tapethedried gel intoyourlaboratory
notebook.
Overnight staining
a. Add 120 ml of lx Fast Blast DNA
stain to the staining tray (2 gels per
tray).
b. Let the gels stain overnight, with
gentle shaking for best results. No
destaining is required.
c. Pour off the stain into a waste
beaker.
d. Record results.
e. Trim away any unloaded lanes.
f. Air-drythe gel on gel supportfilm and
tapethe dried gel intoyourlaboratory
notebook.
StudentManual
22
Student Manual
Pre-Lab Introduction to DNA Fingerprinting
You areaboutto perform aprocedureknown as DNAfingerprinting. Thedataobtained
mayallowyoutodetermine ifthesamplesof DNAthatyouwillbe providedwith arefromthe
sameindMdualorfrom differentindividuals. Forthisexperiment itisnecessaryto reviewthe
structureof DNA molecules.
DNAconsistsofaseriesofnitrogenousbase moleculesheldtogetherbyweakhydrogen
bonds. These base pairsare in turn bondedtoasugar-phosphatebackbone. Thefour
nitrogenousbasesareadenine, thymine,guanine, andcytosine (A, T, G, and C).
Rememberthe base-pairing rule isA Tand G C. Refertothefigure belowofa DNA
- -
molecule.
TheStructureofDNA
The schematics above representaverysmall section of DNAfrom three different
individuals. In this representation of DNAthe symbol system is asfollows:
Backbone:
S= Fivecarbon sugarmolecule known asdeoxyribose
p= Phosphate group
DNA NucleotideBases:
A=adenine C =cytosine G guanine T=thymine
Analysisofthethree DNAsamplesabove (see nextpage) mighthelp usdetect
similaritiesand differencesin samplesof DNAfrom differentpeople.
StudentManual
23
Pre-Lab Focus Questions: Introduction to DNA Fingerprinting
Consideration What isthestructureofDNA?
1 Comparethe“backbone”ofthesugar-phosphate arrangementinthesidechains ofall
threefigures. Arethereanydifferences?
2. Intheabovefigure, doallthreesamplescontainthe same bases? Describeyour
observations.
a Arethe bases paired in an identical mannerin allthreesamples? Describethe pattern
ofthe base pairbonding.
4. Inyourattempttoanalyze DNAsamplesfromthreedifferentindividuals, whatcondusions
canyoumakeaboutthesimilaritiesanddifferencesofthe DNAsamples?
5. Whatwillyou need tocompare betweenthese DNAsamplestodetermineiftheyare
identicalornon-identical?
StudentManual
24
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___________ ______________
4 DNAfragmentsizecanbeexpressedasthenumberofbasepairsinthefragment. Indicate
thesizeofthefragments[mentionanydiscrepancyyoumaydetect].
a) Thesmallerfragmentis base pairs (bp).
b) Whatisthe length ofthe longerfragment?
5 ConsiderthetwosamplesofDNAshown below-singlestrandsareshownforsimplicity:
Sample#1
CAGTGATCTCGAATTCGCTAGTAACGTT
Sample#2
TCATGAATTCCTGGAATCAGCAAATGCA
Ifboth samples aretreatedwiththe restriction enzyme EcoRl [recognition sequence
GAATTC]then indicatethe numberoffragments and thesizeofeachfragmentfrom each
sampleofDNA.
Sample# I Sample#2
#offragments: #offragments:_________
Listfragmentsize in order: largest —>smallest
Sample# I Sample#2
StudentManual
26
Post-Lab: Analysis of Results
Ifthe overnightstaining protocol wasusedto stain gels, record yourresults and drygelsas
described in the gel staining procedures in Lesson 2 page 38.
Attachthe plasticsheettracingofthe banding patternsfromthe DNAelectrophoresis
below.
Tracingofetectrophoresisgel
Attachthe driedgel showingthe banding patternsfromthe DNAelectrophoresis
below.
<0
Dnedelectrophoresisgel I Z
StudentManual 0
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