Crime Scene Investigator PCR Basics™...Crime Scene Investigator PCR Basics Procedures Overview...

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