Laboratory #1: Forensic DNA Fingerprinting Lab Timeline Wheeler High School The Center for Advanced...

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


Gel Gel ElectrophoresElectrophoresisis



Destain gels, observe results & Destain gels, observe results & analyzeanalyze



Plasmid MappingPlasmid Mapping– How to guideHow to guide


Day 1: Restriction Day 1: Restriction Enzyme DigestEnzyme Digest




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




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




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




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


Mapping PlasmidsMapping Plasmids This requires logic!This requires logic!

Laboratory #1: Forensic DNA Fingerprinting Lab Timeline Wheeler High School The Center for Advanced...

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