Crime Scene Investigator PCR Basics™
Target Audience
• The Crime Scene Investigator PCR Basics™ Kit is intended to be an introduction to the polymerase chain reaction (PCR)
• Students will have a much better appreciation of the kit if they have some understanding of DNA structure and function
Workshop Time Line • Introduction to DNA profiling
• Set up PCR reactions
• Electrophorese PCR products
• Analysis and interpretation of results
What is DNA profiling?
DNA profiling is the use of molecular genetic methods to determine the exact genotype of a DNA sample in a way that can basically distinguish one human being from another
The unique genotype of each sample is called a DNA profile.
How do crime scene investigators create a DNA profile?
1. Evidence is collected at the crime scene:
Blood TissueSemen
UrineHair
Teeth Saliva Bone
2. DNA is extracted from sources at the crime scene and from victim and suspects
How do crime scene investigators create a DNA profile?
Since humans are 99.9% identical where do crime scene investigators look for differences in DNA profiles?
4. Crime Scene Investigators search in areas of the genome that are unique from individual to individual and are “anonymous” (control no known trait or function)
The areas examined are Short Tandem Repeats or STR’s
STR region
Example of an STR: TH01 The TH01 locus contains repeats of TCAT.
CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA
This example has 6 TCAT repeats.
There are more than 20 known TH01 alleles.
Each individual inherits 1 allele from each parent.
Determining genotypes for individuals using STRs
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 TCATTCAT TCAT TCAT TCAT TCAT TCAT TCAT AAA
To determine the genotype (DNA profile) Crime Scene Investigators make billions of copies of the target sequence using PCR Starting DNA
Template
5’
5’
3’
3’
Target DNA
What’s the point of PCR?
• PCR, or the polymerase chain reaction, makes copies of a specific piece of DNA
• PCR allows you to look at one specific piece of DNA by making copies of *only* that piece of DNA
• PCR is like looking for a needle in a haystack, and then making a haystack out of the needle
GENE
Polymerase Chain Reaction (PCR)
PCRPolymerase Chain Reaction
metodo enzimatico di sintesi che permette l’amplificazione in vitro di
specifiche sequenze di DNA
Kary Mullis
…while driving in his Honda Civic, Mullis had the flash of insight that…
Kary Mullis developed technique (1983)
…two short DNA segments,
oligonucleotideprimers, could
direct the amplification of a
segment of DNA!!!
PCR exploits certain features of DNA replication
-DNA polymerase can be directed to synthesize a specific region of DNA
-PCR results in the amplification of the specified region
What’s needed
>YLR175W Chr 12
ATGTCAAAGGAGGATTTCGTTATTAAGCCTGAAGCTGCAGGTGCTTCCACTGACACTAGT
GAATGGCCATTACTTTTGAAGAATTTTGATAAGTTATTAGTCAGAAGTGGTCATTATACC
CCTATCCCAGCTGGTAGTTCACCACTAAAGAGAGACCTGAAATCATATATCAGCTCTGGT
GTCATTAATCTAGATAAACCTTCCAACCCATCATCGCACGAAGTTGTAGCTTGGATCAAA
GTCGAAACTGAGAAGGAAGAAGTGAAAGAGGATGACAGCAAAAAGGAAAAGAAAGAGAAG
AAGGACAAGAAGGAAAAGAAGGAAAAGAAAGAAAAGAAGGACAAGAAGGAAAAGAAAGAG
AAAAAGGAGAAGAAAAGAAAGTCTGAAGACGGTGATTCTGAGGAAAAGAAATCTAAGAAA
TCTAAGAAATGA
The template DNA(complex genomic, viral, and plasmid templates)
>YLR175W Chr 12
5’ATGTCAAAGGAGGATTTCGTTATTAAGCCTGAAGCTGCAGGTGCTTCCACTGACACTA
GTGAATGGCCATTACTTTTGAAGAATTTTGATAAGTTATTAGTCAGAAGTGGTCATTATA
CCCCTATCCCAGCTGGTAGTTCACCACTAAAGAGAGACCTGAAATCATATATCAGCTCTG
GTGTCATTAATCTAGATAAACCTTCCAACCCATCATCGCACGAAGTTGTAGCTTGGATCA
AAGTCGAAACTGAGAAGGAAGAAGTGAAAGAGGATGACAGCAAAAAGGAAAAGAAAGAGA
AGAAGGACAAGAAGGAAAAGAAGGAAAAGAAAGAAAAGAAGGACAAGAAGGAAAAGAAAG
AGAAAAAGGAGAAGAAAAGAAAGTCTGAAGACGGTGATTCTGAGGAAAAGAAATCTAAGA
AATCTAAGAAATGA-3’
OLIGO DOWN
OLIGO UP
…short oligonucleotide primersspecific for a DNA sequence, with the ability to hybridize to the opposite
strands of template
oligonucleotides features:
-Tm= 69,5+0,41 (%GC) Tm’= Tm- (500/nt tot)
-%GC (50-60%)
-3’ end stability
-lenght (18-24nt)
5’-ATGTCAAAGGAGGATTTCGT-3’
3’-TCCACGAAGGTGACTGTGAT-5’
OLIGO UP
OLIGO DOWN
5’-ATGTCAAAGGAGGATTTCGTTATTAAGCCTGAAGCTGCAGGTGCTTCCACTGACACTA-3’
3’-TACAGTTTCCTCCTAAAGCAATAATTCGGACTTCGACGTCCACGAAGGTGACTGTGAT-5’dsDNA
…thermostable DNA polymerase (such as Taq or Pfupolymerase)…
1kb/min
1kb/min
Extension timesActivitiesError rateDNA polymerase
5’-3’ polymerase activity8 x 10 -6Taq
5’-3’ polymerase activity3’-5’ proofreading activity1.3 x 10 -6PfuTurbo
OLIGO UP
OLIGO DOWN
5’
5’
3’
3’
> Filamento
5’-ATGTCAAGGAGGATTTCGTTATTAAGCCTGAAGCTGCAGGTGCTTCCACTGACACTAGT
GAATGGCCATTACTTTTGAAGAATTTTGATAAGTTATTAGTCAGAAGTGGTCATTATACC
CCTATCCCAGCTGGTAGTTCACCACTAAAGAGAGACCTGAAATCATATATCAGCTCTGGT
GTCATTAATCTACATAAACCTTCCAACCCATCATCGCACGAAGTTGTAGCTTGGATCAAA
GTCGAAACTGAGAAGGAAGAAGTGAAAGAGGATGACAGCAAAAAGGAAAAGAAAGAGAAG
AAGGACAAGAAGGAAAAGAAGGAAAAGAAAGAAAAGAAGGACAAGAAGGAAAAGAAAGAG
AAAAAGGAGAAGAAAAGAAAGTCTGAAGACGGTGATTCTGAGGAAAAGAAATCTAAGAA-3’
oligonucleotides design:
… 4dNTP substrates (meaning dATP, dGTP, dCTP, dTTP):
…a machine that can change the incubation temperature of the reaction tube automatically
Polymerase Chain Reaction- the early years-
95°C
50°C
72°CPolymerase Chain Reaction
- today-
30-40 cycles
Three basic steps repeated a number of times
– DENATURATION 95°C ≤ 1 min• heat solution to separate template strands
– ANNEALING 50-55°C ≤ 1 min• cool solution to allow primers to basepair
– SYNTHESIS 68-72°C ≤ 1 min• DNA synthesis by DNA Pol• primers extended and dNTPs incorporated
dsDNA
DENATURATION
ANNEALING
SYNTHESIS
I cycle…
dsDNA
..II cycle
DENATURATION
ANNEALING
SYNTHESIS
…additional cycles
(2n-2n)xn= cycles numberx=starting template DNA
Experimental procedure
In a sterile 0,5 ml microfuge tube, mix in the following order
2-3µg of DNA
amplification bufferMgCl2primer upprimer downtemplate DNA (10-100ng)dNTPpolymerase enzimeH2O to a final volume
Powerful technique
Polymerase Chain Reaction
Carried out in vitro (in lab)
Extremely simple technique
Contaminations
Advantages Disadvantages
Possible aspecifity
PCR applications
Es. Huntington desease, mutazione nel gene IT15
In modo analogo si possono rivelare tutte le mutazioni costituite da cambiamenti di lunghezza del gene coinvolto (inserzioni, delezioni, espansioni).
Rivelazione di mutazioni che variano la dimensione dei geni
APPLICAZIONI
DNA profiling is used to determine which suspect can not be excluded from suspicion.
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
Crime Scene Investigator PCR Basics™ProceduresOverview
LaboratoryQuick Guide
Set up PCR reactions 1. Find the PCR tubes at your station.
Label them ‘CS’ for Crime Scene DNA, ‘A’ for Suspect A DNA, ‘B’ for Suspect BDNA, ‘C’ for Suspect C DNA, and ‘D’ for Suspect D DNA.
2. Keeping the tubes on ice, add 20 µl of Master Mix + blue primers to each tube.
3. Keeping the tubes on ice, add 20 µl of each DNA to the appropriately labeled tube.
4. USE A FRESH TIP EACH TIME!
5. Mix and put in thermal cycler
6. Cycle ~3 hours
The PCR Reaction
What do you need?
What is needed for PCR?
• Template (containing 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
5’3’5’
3’ 3’ 5’3’5’
Forward primer
Reverse primer
Target sequence
What is happening in the PCR tube while in the thermocycler? PCR Animation
http://www.bio-rad.com/flash/07-0335/07-0335_PCR.html
The PCR Reaction
How does it work?
Heat (94oC) to denature DNA strands
Cool (52oC) to anneal primers to template
Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA
Repeat 35 cycles
Denaturing Template DNA
Heat causes DNA strands to separate
3’
5’
5’
3’
Denaturation of DNA at 94oC
5’
3’
3’
5’
Annealing Primers
Primers bind to the template sequence
Taq polymerase recognizes 3’ end of primer + template strand
Primers anneal at 52oC5’
3’
3’
5’
5’
3’
3’
5’
3’5’3’ 5’
3’ 5’3’5’
Taq extends at 72oC
STR DNA is replicated
Repeat denaturing, annealing, and extending 35 cycles
Cycle 1
Cycle 2
Cycle 3The exact-length target product is made in the third cycle
To visualize PCR products Crime Scene investigators use gel electrophoresis
(14)
(12)
(11)
(9)
(8)
(7)
(6)
(5)
(4)
(3)
(13)
(10)
TH01 alleles
Alleleladder Mother Father Child C Child D Child E
ElectrophoresePCR products 1. Add 10 ul of Orange G Loading Dye to
each PCR tube and mix
2. Set up gel and electrophoresis equipment
3. Load 20 ul of CSI allele ladder to Lane 1
4. Load 20 ul of your PCR reactions in lanes 2 to 6
5. Electrophorese samples
6. Stain gel with Fast Blast DNA Stain
7. Analyze results
Using the digital micropipet
Add 10ul of loading dye to each microtube
AgaroseElectrophoresis
Place gel in gel box
Pour buffer in box until gel wells are covered.
Elettroforesi di Acidi NucleiciElettroforesi di Acidi NucleiciSouthernSouthern e e NorthernNorthern BlotBlot
ELETTROFORESI SU GELELETTROFORESI SU GEL
Permette la separazione di frammenti di DNA/RNA Permette la separazione di frammenti di DNA/RNA da una miscela complessada una miscela complessa
EE’’ una una tecnica fondamentaletecnica fondamentale per:per:
••ll’’analisianalisi (elettroforesi(elettroforesi analiticaanalitica))••la la purificazionepurificazione degli acidi nucleici (elettroforesi degli acidi nucleici (elettroforesi preparativapreparativa))
-- ++
RNA e DNA sono RNA e DNA sono carichi negativamentecarichi negativamente
catodocatodo anodoanodo
Effetto Effetto ““setacciosetaccio”” in un gel uniformein un gel uniforme
--
++
ELETTROFORESI IN GEL DI ELETTROFORESI IN GEL DI AGAROSIOAGAROSIO
LL’’agarosioagarosio èè un un polisaccaridepolisaccaridecostituito da residui alternati di costituito da residui alternati di DD--galattosgalattosiioo e e 3,63,6--anidroanidro--LL--galattosgalattosiioo..
ELETTROFORESI IN GEL DI ELETTROFORESI IN GEL DI AGAROSIOAGAROSIO
Consente la separazioni di molecole di Consente la separazioni di molecole di grandigrandi dimensioni: 0.1 dimensioni: 0.1 -- 20 kb20 kb
Concentrazione Concentrazione
di agarosiodi agarosio
RisoluzioneRisoluzione0.8% : 0.5 0.8% : 0.5 –– 20 kb20 kb
2% : 0.1 2% : 0.1 –– 3 kb3 kb
Come si prepara un gel dCome si prepara un gel d’’agarosioagarosio
Alla soluzione dAlla soluzione d’’agarosioagarosiosi aggiunge si aggiunge EtidioEtidio BromuroBromuro((EtBrEtBr))
Si scioglie lSi scioglie l’’agarosioagarosio ininpolvere in una soluzione polvere in una soluzione tampone a 100tampone a 100°°CC
100V 0,2A
tampone elettroforetico
generatore di corrente
Polo +
Polo -
gel di agarosio 1.2%
scatola elettroforetica
ELETTROFORESI IN GEL DI ELETTROFORESI IN GEL DI AGAROSIOAGAROSIO
100V 0,2A
Polo +
Polo -
LoadingLoading dyedye aiuta il caricamento del campione nel pozzetto del gel. Contienaiuta il caricamento del campione nel pozzetto del gel. Contiene e glicerologlicerolo, , Blu di Blu di bromofenolobromofenolo e e Blu di Blu di xilencianoloxilencianolo che migrano nel gel a velocitche migrano nel gel a velocitàà diversa.diversa.
ELETTROFORESI IN GEL DI ELETTROFORESI IN GEL DI AGAROSIOAGAROSIO
100V 0,2A
Polo +
Polo -
ON
LoadingLoading dyedye aiuta il caricamento del campione nel pozzetto del gel. Contienaiuta il caricamento del campione nel pozzetto del gel. Contiene e glicerologlicerolo, , Blu di Blu di bromofenolobromofenolo e e Blu di Blu di xilencianoloxilencianolo che migrano nel gel a velocitche migrano nel gel a velocitàà diversa.diversa.
ELETTROFORESI IN GEL DI ELETTROFORESI IN GEL DI AGAROSIOAGAROSIO
100V 0,2A
Polo +
Polo -
ON
LoadingLoading dyedye aiuta il caricamento del campione nel pozzetto del gel. Contienaiuta il caricamento del campione nel pozzetto del gel. Contiene e glicerologlicerolo, , Blu di Blu di bromofenolobromofenolo e e Blu di Blu di xilencianoloxilencianolo che migrano nel gel a velocitche migrano nel gel a velocitàà diversa.diversa.
ELETTROFORESI IN GEL DI ELETTROFORESI IN GEL DI AGAROSIOAGAROSIO
EtidioEtidio Bromuro (Bromuro (EtBrEtBr))::
colorante fluorescentecolorante fluorescenteassorbe la luce assorbe la luce UVUV a a 300 300 nmnmdando colore giallodando colore giallo--arancioarancio
EE’’ utile sia per utile sia per visualizzarevisualizzare, , sia per sia per quantificarequantificare il il campione: lcampione: l’’intensitintensitàà della della fluorescenza fluorescenza èè, infatti, , infatti, proporzionale alla quantitproporzionale alla quantitààdel campione.del campione.
Per frammenti Per frammenti linearilineari di DNA di DNA e/o RNA e/o RNA la distanza di la distanza di migrazione migrazione èè inversamente inversamente proporzionaleproporzionale alle dimensioni alle dimensioni della molecola (ovvero alla della molecola (ovvero alla sua lunghezza in basi)sua lunghezza in basi)
Conoscendo la distanza di Conoscendo la distanza di migrazione di frammenti di migrazione di frammenti di dimensione nota (M) posso dimensione nota (M) posso ““interpolareinterpolare”” la lunghezza la lunghezza delle molecole A e Bdelle molecole A e B
Dai Geni ai Genomi-EdiSES-
A = 2.5 A = 2.5 kbkbB = 6 B = 6 kbkb
Non tutte le molecole di DNA sono lineari (Non tutte le molecole di DNA sono lineari (eses.: .: plasmidiplasmidi batterici)batterici)
Sebbene le due forme abbiano le Sebbene le due forme abbiano le stessestesse dimensioni, esse migreranno dimensioni, esse migreranno in maniera differentein maniera differente
plasmide circolare “rilassato”
nick
plasmidesuperavvolto
migra più lentamente rispetto ad un DNA
lineare o superavvolto della stessa massa
pBR322
pBR322pBR322 pBR322pBR322dopo digestionedopo digestione
plasmide“linearizzato”
Place 20ul of samples into appropriate wells
Set up electrophoresis chamber by putting top in place and connecting it to the power supply
Gel running
AgaroseElectrophoresisRunning
Agarose gel sievesDNA fragments according to size– Small fragments
move farther than large fragments
Use a 3% gel to separate small fragment sizes
Milestones in Forensic DNA analysis
1985 Alec Jeffries develops RFLP
1990 PCR analysis using single locus STR begins
1992 FBI initiates STR work
1994 DNA Identification Act: provides funding for national DNA database
1995 OJ Simpson trial focuses public attention on DNA evidence
1998 FBI starts CODIS database; Swissair disaster – all remains identified using STR DNA profiling
2001 World Trade Center disaster in NYC – many remains identified using a combination of DNA profiling approaches
2004 Indian Ocean tsunami; Interpol and other world agencies use DNA profiling to identify victims
Today Trace your Genetic Genealogy; commercially available packages can trace paternal/maternalancestry
DNA Testing Today
GeneTree.com & Ancestry.comprovide DNA tests from $99-$200 to trace genealogy
Genealogical Analysis Uses:DNA sequencing (mtDNA)
Single Nucleotide Polymorphism testing (SNPs)
Short Tandem Repeat testing (STRs)
Crime scene investigators use techniques that are fast, cost effective, and have a high Power of Discrimination
The Power of Discrimination is the ability of a test to distinguish between different samples (genotypes)
slow fast
low
high
mtDNAmtDNA Blood group Blood group typingtyping
RFLP RFLP analysisanalysis
STR STR analysisanalysis
Speed of Analysis
Pow
er o
f D
iscr
imin
atio
n
Statistics of Chance: M&M Locus
6 Possible Alleles:•Green•Red•Yellow•Blue•Brown•Orange
Probabilities• One allele from each parent means 2
copies of gene/locus
1
6
1
6X =
1
36Frequency of any M&M genotype
Probabilities
Mike & Ikes: 5 alleles
Jolly Rancher: 5 alleles
1
6
1
6
1
5
1
5
1
5
1
5
1
36
1
25
1
25
Locus
M&M6 alleles
Jolly Rancher5 alleles
Mike & Ikes5 alleles
x =
x =
x =
1
22,500=Chance an individual has a given genotype
1
506,250,000=Chance 2 people have the same genotype
Who can’t we exclude from the pool of suspects?
Real-WorldProbabilities •Forensics labs use 13 different loci
with multiple alleles
•Allele frequencies DO NOT follow mathematical principles - allele frequencies vary by population.
•These 13 loci allow for discrimination of any two people in the world (with the exception of identical twins), living or dead.
•Probability of a random match when all 13 loci typed: ~1 in 3 trillion.
Butler et al 2003 J Forensic Sci. www.cstl.nist.gov/biotech/strbase/pub_pres/Butler2003a.pdf
.00211
.014.002.00810
.150.151.1149
.096.194.0848
.279.421.1907
.214.124.2326
.004.0025
Latinos
n=140
African American
n=258
Caucasians
n=302
Allele
TH01 Published Allele Frequencies by Population
CODISCOmbined DNA Index System
A federally maintained database used by law enforcement officials
13 loci guarantees high power of discrimination
Real STR analysis
Four different fluorescent tags have been used to identify 7 amplified loci
Allele ladders are indicated by arrows
Analysis of Results:
Who can’t be excluded?
10754321
CS A B C D
genotype
5-2 7-4 5-2 7-2 10-3
AL: Allele ladderCS: Crime SceneA: Suspect AB: Suspect BC: Suspect CD: Suspect D
AL
15
BXP
007
alle
les
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