Overview - Boston University 3 PCR... · •Gel Electrophoresis –Set up PCR • Day 2: Wednesday...
Transcript of Overview - Boston University 3 PCR... · •Gel Electrophoresis –Set up PCR • Day 2: Wednesday...
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Overview
• Day 1: Tuesday – Introduction to DNA profiling
• How do we use DNA to solve crimes?
– Background• Polymerase Chain Reaction (PCR) • Gel Electrophoresis
– Set up PCR
• Day 2: Wednesday – Make and Run Agarose Gel – More Background
• Polymerase Chain Reaction (PCR) • Gel Electrophoresis
– Stain gel overnight
• Day 3: Thursday – Analysis and interpretation of results
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Overview
• Background ~30 min
– DNA profiling
– Polymerase Chain Reaction (PCR)
– Gel Electrophoresis
• Lab activity ~50 min
– PCR
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What is DNA profiling
• The use of molecular genetic methods to determine the exact genotype of a DNA sample in a way can distinguish one human being from another.
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How do crime scene investigators create a DNA profile?
• Step #1 Evidence is collected at the crime scene
• Biological sources: anything that has DNA!
Blood TissueSemen
UrineHair
Teeth Saliva Bone
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Step #2 DNA is Extracted
• Sources at scene
• Victim
• Suspects
Cell
Chromosome
DNA
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Step #3 DNA samples are processedSample Obtained
from Crime Scene
or Paternity
InvestigationBiology
DNA
Extraction
DNA
Quantification
PCR Amplification
of Multiple STR markers
Technology
Separation and Detection of
PCR Products
(STR Alleles)
Sample Genotype
Determination
GeneticsComparison of Sample
Genotype to Other Sample
Results
If match occurs, comparison of
DNA profile to population
databases
Generation of Case
Report with Probability
of Random Match
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How are suspects included or excluded from an investigation?
• Suspects are included in an investigation if their DNA profile matches with genotypes found at the crime scene
• Suspects can be excluded if their DNA profile does not match genotypes found at the crime scene
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What part of DNA is used?
• There are ~3 billion base pairs in the human genome
• >99.5% of human DNA sequences are the same in every person
• Small percentage of DNA is different allowing us to distinguish one individual from another
• Polymorphic DNA – “many forms”
• Do not control any traits, and have no function
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Variable Number Tandem Repeat (VNTR)
• DNA profiling uses repetitive ("repeat") sequences that vary a lot
• VNTR loci are very similar between closely related humans. (relatives)
• However, they are so variable that unrelated individuals are extremely unlikely to have the same VNTRs.
• Amplify loci and visualize to see differences
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Short Tandem Repeats (STR’s)
• Type of VNTR
– Variable number tandem repeat
• Unique from individual to individual and are “anonymous”
• Control no known trait or function STR region
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Example of an STR
• TH01 locus: Tyrosine hydroxylase intron 01
• Repeats of TCAT
• CCC TCAT TCAT TCAT TCAT TCAT TCATAAA
• This example has 6 TCAT repeats.
• There are more than 20 known TH01 alleles.
• Each individual inherits 1 allele from each parent.
• These make up the genotype. Ex. 6,2
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Example of an STR
• Ms. Smith’s TH01 locus for her two chromosomes is given below.
• What is her genotype?
• MOM’S CHROMOSOME
CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA
• DAD’S CHROMOSOME
CCC TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCATTCAT AAA
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Example of an STR
• Ms. Smith’s TH01 locus for her two chromosomes is given below.
• MOM’S CHROMOSOME– CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA
• DAD’S CHROMOSOME– CCC TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT
TCAT TCAT TCAT TCAT TCAT AAA
• What is her genotype?
• How can we visualize this difference?
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Polymerase Chain Reaction (PCR)
• Replication in a test tube
• Makes copies of a specific piece of DNA
• Amplify DNA so we can visualize it
• Allows you to look at one specific piece of DNA by making copies of *only* that piece of DNA
• Can amplify a very small amount of DNA
• PCR is like looking for a needle in a haystack, and then making a haystack out of the needle
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What do you need for PCR?
• What do you need for replication?
• 1
• 2
• 3
• 4
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The PCR ReactionWhat do you need?
•Template (the STR you want to amplify for the study)•Sequence-specific primers flanking the target sequence
•Nucleotides (dATP, dCTP, dGTP, dTTP)
•Magnesium chloride (enzyme cofactor)
•Buffer, containing salt
•Taq polymerase: Thermus aquaticus
5’
3’3’ 5’
3’5’
Forward primer
Reverse primer
Target sequence
5’
3’
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3 Steps of PCR
• Step #1 Denaturation
– Heat (94oC) to denature DNA template strands
• Step #2 Annealing
– Cool (52oC) to anneal primers to template
• Step #3 Extension
– Warm (72oC) to activate Taq polymerase, which
extends primers and replicates DNA
• Repeat 35 cycles
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Step #1 Denaturation
Heat causes DNA strands to separate
3’
5’
5’
3’
5’
3’
3’
5’
94oC
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Primers Anneal
• Primers bind to the template sequence
• Taqpolymerase recognizes 3’ end of primer + template strand
Primers anneal at 52oC
5’
3’
3’
5’
5’
3’
3’
5’
3’5’3’ 5’
3’ 5’
3’5’
Taq extends at 72oC
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What is happening in the PCR tube while in the thermocycler?
• PCR ANIMATION
• http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf
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Generation of Precise-length fragments
Cycle 1
Cycle 2
Cycle 3
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Food Fight !!!
• Crime Scene Evidence (CS)
– DNA isolated from a half eaten sandwich
• Suspects
– A: Dylan
– B: Sabrina
– C: Aristides
– D: Tarialas
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How to use a micropipette
• Press the plunger down to the first soft stop. You will feel it stop naturally
• Lower the tip into the loading dye and slowly let the plunger come back up and fill the pipet.
• Press down to the first soft stop to deliver the loading dye. If it all doesn't come out you can push down to the hard stop to give it one last chance to come out.
• Eject tip
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Set up PCR reactions1. Find the PCR tubes at your station.
2. Label them ‘CS’ for Crime Scene DNA, ‘A’ for Suspect A DNA, ‘B’ for Suspect B DNA, ‘C’ for Suspect C DNA, and ‘D’ for Suspect D DNA.
3. Keeping the tubes on ice, add 20 μl of Master Mix + blue primers to each tube.
4. Keeping the tubes on ice, add 20 µl of each DNA to the appropriately labeled tube.
5. USE A FRESH TIP EACH TIME!
6. Mix and put in thermal cycler
7. Cycle ~3 hours (OVERNIGHT)
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To visualize PCR products Crime Scene investigators use gel electrophoresis
(14)
(12)
(11)
(9)
(8)
(7)
(6)
(5)
(4)
(3)
(13)
(10)
TH01
alleles
Allele
ladderMother Father Child C Child D Child E
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Load Gel
• Obtain 5 PCR Reactions
– CS
– A
– B
– C
– D
• Add 10μl of Orange Loading Dye (LD) to each tube
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Load Gel
Lane Description
1 20 μl of CSI allele ladder
2 20 μl of Crime Scene PCR reaction
3 20 μl of Suspect A PCR reaction
4 20 μl of Suspect B PCR reaction
5 20 μl of Suspect C PCR reaction
6 20 μl of Suspect D PCR reaction
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Agarose Gel Electrophoresis
Individual 1’s DNA Individual 2’s DNA
Wells where DNA samples are loaded
Agarose Gel
A
B
C
D
E
A
B
C
D
B
D
E
C
A
B
D
C
A
+
--
Smaller fragments will move farther through the dense gel than larger fragments, so bands near the top are bigger!
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Agarose gel
• Agarose– gelatanous substance derived from algae– polymer of galactose
• Forms a matrix – Separate fragments of DNA – Smaller fragments travel farther than larger
• Higher percentage of agarose separates small molecules
• To make a gel– Grams of agarose/100ml of buffer
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Gel Electrophoresis• Place gel in gel box
• Pour buffer in box until gel wells are covered.
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Gel ElectrophoresisPlace 20ul of samples into appropriate wells
Set up electrophoresis chamber by putting top in place and connecting it to the power supply
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Gel Electrophoresis
• Gel runs from negative to positive
• DNA slightly negative
Gel running
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Analysis of Results
10
7
5
4
3
2
1
CS A B C D
genotype
5-2 7-4 5-2 7-2 10-3
AL: Allele ladder
CS: Crime Scene
A: Suspect A
B: Suspect B
C: Suspect C
D: Suspect D
AL
15
BX
P0
07
all
ele
s
• Who can’t be excluded?
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COmbined DNA Index System (CODIS)
• A federally maintained database used by law enforcement officials
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Real STR analysis
•Four different fluorescent tags have been used to identify 7 amplified loci•Allele ladders are indicated by arrows