Post on 27-Dec-2015
Laboratory #1:Laboratory #1: Forensic DNA Forensic DNA FingerprintingFingerprinting
Lab TimelineLab Timeline
Wheeler High SchoolWheeler High SchoolThe Center for Advanced Studies in Science, Math & TechnologyThe Center for Advanced Studies in Science, Math & Technology
Post-AP DNA/Genetics – Ms. Kelavkar
Laboratory Day 1Laboratory Day 1
Restriction Enzyme DigestRestriction Enzyme Digest
Post-AP DNA/Genetics – Kelavkar
Laboratory Day 2Laboratory Day 2
Gel Gel ElectrophoresElectrophoresisis
Post-AP DNA/Genetics – Kelavkar
Laboratory Day 3Laboratory Day 3
Destain gels, observe results & Destain gels, observe results & analyzeanalyze
Post-AP DNA/Genetics – Kelavkar
Laboratory Day 4Laboratory Day 4
Plasmid MappingPlasmid Mapping– How to guideHow to guide
Post-AP DNA/Genetics – Kelavkar
Day 1: Restriction Day 1: Restriction Enzyme DigestEnzyme Digest
Laboratory #1:Laboratory #1: Forensic DNA Forensic DNA FingerprintingFingerprinting
Wheeler High SchoolWheeler High SchoolThe Center for Advanced Studies in Science, Math & TechnologyThe Center for Advanced Studies in Science, Math & Technology
Post-AP DNA/Genetics – Kelavkar
DNA ReviewDNA Review
StructureStructure FunctionFunction
OCH2
O
P O
O
OBase
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
O
Phosphate
Phosphate
What are Restriction What are Restriction Enzymes?Enzymes?
Evolved by bacteria to protect against Evolved by bacteria to protect against viral infectionviral infection
““Molecular Scissors”Molecular Scissors”– Cut at specific sitesCut at specific sites
How was this evolutionary advantageous?How was this evolutionary advantageous?How might bacteria protect their own DNA from these How might bacteria protect their own DNA from these
restriction enzymes?restriction enzymes?
How are restriction How are restriction enzymes named?enzymes named? Restriction endonucleasesRestriction endonucleases
– EndoEndo = within = within– NucleaseNuclease = enzyme that cut’s nucleic = enzyme that cut’s nucleic
acidsacids
EcoRIEcoRI– Common restriction enzymeCommon restriction enzyme– Isolated from Isolated from Escherichia coliEscherichia coli
Thus Eco…Thus Eco… The RI specifies the a specific place the enzyme The RI specifies the a specific place the enzyme
is encodedis encoded
Blunt vs. Sticky EndsBlunt vs. Sticky Ends
Blunt end = Blunt end =
Sticky end = Sticky end =
Restriction SiteRestriction Site
Where the restriction enzyme cutsWhere the restriction enzyme cuts
If a specific restriction site occurs in If a specific restriction site occurs in more than one location on a DNA more than one location on a DNA molecule, cuts will be made at each of molecule, cuts will be made at each of those sites, giving multiple fragments.those sites, giving multiple fragments.
EcoRI– Eschericha coli– 5 prime overhang
Enzyme Site Enzyme Site RecognitionRecognition
• Each enzyme digests (cuts) DNA at a specific sequence = restriction site
• Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC)
Palindrome
Restriction site
Fragment 1 Fragment 2
Why use RE’s when Why use RE’s when conducting gel conducting gel electrophoresis?electrophoresis? Used to cut strands of Used to cut strands of
DNA at specific sitesDNA at specific sites
Cut DNA can be Cut DNA can be separated via gel separated via gel electrophoresis electrophoresis
Technicians use Technicians use RFLPRFLP (Restriction Fragment (Restriction Fragment Length Polymorphism) Length Polymorphism) to provide unique to provide unique banding patterns based banding patterns based on restriction siteson restriction sites
Today’s Lab Today’s Lab Considerations…Considerations…
1.1. Lab SafetyLab Safety
2.2. Lab GroupsLab Groups
3.3. Read protocol & conduct Read protocol & conduct experimentexperiment
4.4. Review QuestionsReview Questions
5.5. Clean-Up & Prep for next classClean-Up & Prep for next class
Day 2: Agarose Gel Day 2: Agarose Gel ElectrophoresisElectrophoresis
Laboratory #1:Laboratory #1: Forensic DNA Forensic DNA FingerprintingFingerprinting
Wheeler High SchoolWheeler High SchoolThe Center for Advanced Studies in Science, Math & TechnologyThe Center for Advanced Studies in Science, Math & Technology
Post-AP DNA/Genetics – Ms. Kelavkar
Agarose Gel Agarose Gel ElectrophoresisElectrophoresis Separates DNA fragments by sizeSeparates DNA fragments by size
Why do we load DNA on the
negative end of the chamber?
Running Your GelRunning Your Gel
Power Supply
–Agarose gel sieves Agarose gel sieves DNA fragments DNA fragments according to sizeaccording to size
–Small fragments Small fragments move farther than move farther than
large fragmentslarge fragments
Why does the density Why does the density of the gel matter?of the gel matter?
Staining GelStaining Gel
DNA is colorless, so we need to DNA is colorless, so we need to stain it to see the fragmentsstain it to see the fragments
We will use a 1x concentration of We will use a 1x concentration of Fast Blast DNA stain Fast Blast DNA stain
Overnight procedureOvernight procedure
Today’s Lab Today’s Lab Considerations…Considerations…
1.1. Lab SafetyLab Safety
2.2. Read protocol run gelsRead protocol run gels
3.3. Stain gels before the end of Stain gels before the end of class as directed class as directed
4.4. Review QuestionsReview Questions
Day 3: Visualization Day 3: Visualization of Gelof Gel
Laboratory #1:Laboratory #1: Forensic DNA Forensic DNA FingerprintingFingerprinting
Wheeler High SchoolWheeler High SchoolThe Center for Advanced Studies in Science, Math & TechnologyThe Center for Advanced Studies in Science, Math & Technology
Post-AP DNA/Genetics – Ms. Kelavkar
Trace your gel & take a digital Trace your gel & take a digital picturepicture– Use light box and plastic wrapUse light box and plastic wrap
Post-Lab analysis and Post-Lab analysis and interpretation (graphing) of interpretation (graphing) of resultsresults
Observing Your GelObserving Your Gel
RFLP – Interpreting RFLP – Interpreting ResultsResults
Allele 1
Allele 2
GAATTCGTTAAC
GAATTCGTTAAC
CTGCAGGAGCTC
CGGCAGGCGCTC
PstI EcoRI
1 2 3
3Fragment 1+2Different Base PairsNo restriction site
+
M A-1 A-2
Electrophoresis of restriction fragments
M: MarkerA-1: Allele 1 FragmentsA-2: Allele 2 Fragments
Molecular Weight Molecular Weight DeterminationDetermination
100
1,000
10,000
100,000
0 5 10 15 20 25 30
Distance, mm
Siz
e, b
ase
pai
rsB
A
Fingerprinting Standard Curve: Semi-logSize (bp)Size (bp) Distance Distance
(mm)(mm)
23,00023,000 11.0 11.0 9,4009,400 13.0 13.0
6,5006,500 15.0 15.0
4,4004,400 18.0 18.0
2,3002,300 23.0 23.0
2,0002,000 24.0 24.0
Today’s Lab Today’s Lab Considerations…Considerations… Obtain results/trace & pictureObtain results/trace & picture Post-Lab thought questionsPost-Lab thought questions Complete electrophoresis data Complete electrophoresis data
tabletable Graph dataGraph data Answer post-lab interpretation of Answer post-lab interpretation of
results questionsresults questions How to dry gelsHow to dry gels
Day 4: Drawing Day 4: Drawing Restriction MapsRestriction Maps
Laboratory #1:Laboratory #1: Forensic DNA Forensic DNA FingerprintingFingerprinting
Wheeler High SchoolWheeler High SchoolThe Center for Advanced Studies in Science, Math & TechnologyThe Center for Advanced Studies in Science, Math & Technology
Post-AP DNA/Genetics – Ms. Kelavkar
Reading Restriction Reading Restriction MapsMaps
OriOri (Origin of (Origin of replication)replication)
Beta lactamaseBeta lactamase – – allows for resistance allows for resistance to ampicillinto ampicillin
Lambda sequenceLambda sequence – – foreign DNA insertforeign DNA insert
Post-AP DNA/Genetics – Ms. Kelavkar
Mapping PlasmidsMapping Plasmids This requires logic!This requires logic!