FORENSICDNAANALYSIS

ShortTandemRepeats(STR)
p
( )
Dr. Ahmed KASSAB
Forensic Biology Department
NAUSS

FORENSICDNAANALYSIS

RFLP
STR

SNP

Forensiccases

matchingsuspectwith
t hi
t ith
evidence

Paternitytesting
identifying father
identifyingfather

Historicalinvestigations
Missingpersons
i
investigations
ti ti
Massdisasters putting
piecesbacktogether

Mili
DNA d

MilitaryDNAdogtag
ConvictedfelonDNA
databases

RepeatedDNASequences

DNA is found in the cells in our body
DNAisfoundinthecellsinourbody.

Nucleus
(B i off the
(Brain
th cell)
ll)

Mitochondria
Mi
h di
(more later)

RepeatedDNASequences

RepeatedDNA
Repeated
DNA
sequencesare
classifiedas:
SatelliteDNA
MinisatelliteDNA
Mi i t llit DNA
MicrosatelliteDNA

RepeatedDNASequences

E k
EukaryoticGenomesfullofrepeatedDNA
ti G
f ll f
t d DNA
Repeat Unit

Satellite DNA
Minisatellite DNA or
Variable Number of Tandem
R
Repeats
(VNTR)
Microsatellite DNA or
Short Tandem Repeats (STR)

100 300s bp

# Repeats
???

10 100s bp

7 - 80

2 6 bp

5 - >40

Satellite DNA
SatelliteDNA
Consistsofhighnumber
ofrepetitions,usually
between1000and100
000copiesofthemotif.
Thelengthofthemotif
The length of the motif
100and300basepairs.
y
y
Usuallyonlyoccursata
fewloci.

-
)
(
-)



(

Minisatellites
Consist
Consistofshorter
of shorter
motifs
10and60basepairs.

Lessrepetitivethan
satellites(740).
Occuratmanyloci.

-)


(

( -)

Microsatellites
Microsatellites= Short Tandem Repeats
( STR)

Microsatellites
Have
Haveveryshortmotifs
very short motifs
mostcommonly
between2and6base
pairs.
Havethelowestdegree
ofrepetition
Occuratmany,many
l
loci.

(-)


(5 - >40)

FunctionsofRepeatingSequencesofDNA






(


)

Somearetranscribed
andtranslatedsuchas
prolinerichplant
proteins.
Protectchromosome
endsfromdegradation.

FunctionsofRepeatingSequencesofDNA





Enhance
Enhanceorreducethe
or reduce the
transcriptionratesof
neighboringgenes.
Associatedwithgenetic
diseasesinhumans.
FragileXsyndromein
humansdueto
chromosomal instability
chromosomalinstability
duetorepeating
sequences.

Selfish DNA
SelfishDNA
RepeatingDNAsequencesmaybe
Repeating
DNA sequences maybe Selfish
SelfishDNA
DNA
withnofunction.
Selfish DNA has three properties:
SelfishDNAhasthreeproperties:
itspreadsbyformingadditionalcopiesofitself
within a genome
withinagenome.
itmakesnospecificcontributiontothe
phenotype.
phenotype
organismhasnowaytoremoveit.

Microsatellites
Short
S
o Tandem
a de Repeats
epea s ( S
STR))

Characteristics of Microsatellites
CharacteristicsofMicrosatellites
Common
Commonineukaryotic
in eukaryotic

genomesbutarerareor
absentinprokaryotes.

Polymorphismsaredue
todifferencesinthe

numberofrepeatsofa
motif.

CharacteristicsofMicrosatellites
(
(Continued
)
Flankedbyuniquesequencesthat
areconservedacrossaspecies.
Allhumansforexamplewill
havethesamesequencesof
DNAoneithersideofthe
microsatellite(unless
mutation).
Oftenmorethantwoalleles
presentatalocus.Inhumans,
morethan80alleleshavebeen
d
documentedatonelocus.
d
l

CharacteristicsofMicrosatellites
(
(Continued)
d)
Microsatellitesare
c osa e es a e

codominantsoifthetwo

allelesofanindividualare

diff
differenttheycanbothbe
h
b hb

identified.
PCR
EasilyamplifiedbyPCR
Easily amplified by PCR
andvariableinall
populationsofagiven
species.

ExamplesofMicrosatelliteSequences
Examples
of Microsatellite Sequences

1baserepeat

GTAAAAAAAAAAAAAAAGGAT

2basesrepeated
2
bases repeated GTCACACACACACACACAGGAT
(dinucleotide repeat)
3basesrepeated
(trinucleotide repeat)

GTCACCACCACCACCACCACGGAT

4basesrepeat
(tetranucleotide repeat)

GTCAGACAGACAGACAGAGGAT

TypesOfMicrosatelliteSequences

Dinucleotide,
Trinucleotide,
Tetranucleotide,
Pentanucleotide,
Hexanucleotide.

TypesOfMicrosatelliteSequences

Simple repeats: identical
length and sequence
sequence.
Compound repeats: two
or more adjacent simple
repeats.
Complex repeats: repeat
blocks varying in unit
length and sequence

FormationandEvolutionof
Microsatellites
ll
Thehighpolymorphism
ofmicrosatelliteloci
f i
t llit l i
resultsfromhigh
mutation rates
mutationrates.

EvolutionofMicrosatellites
F
FactorsAffectingtheMutationalProcess
Aff i
h M
i
lP
Repeatlengthandbase
Repeat
length and base
compositionaffectthe
mutationrate.
Dinucleotides(2bp
motif)mutatefaster
than trinucleotides (3 bp
thantrinucleotides(3bp
motif).
SequenceswithhighAT
contentmutatefaster
thansequenceswith
high GC content
highGCcontent.

.
)

(

AT

GC

MicrosatellitesinForensics

WhySTRsarePreferredGeneticMarkers

STR

Rapidprocessing.
Abundantthroughoutthe
genome.
Highlyvariablewithin
populations.
l ti
Smallsizerangeallowsmultiplex
development.
Discrete alleles allow digital
Discreteallelesallowdigital
recordofdata.
Allelicladderssimplify
interpretation.
PCRbasedwhichallowsuseof
smallamountsofDNAmaterial.
p
p
Smallproductsizecompatible
withdegradedDNA.

PCR

Degraded DNA and Microsatellites

DegradedDNAandMicrosatellites
Microsatellitesareused
foranalysisoftheshort
piecesofDNA.
Byamplifyingthe
microsatelliteusingPCR
i
lli
i PCR
aslittleas50picograms
of DNA have been
ofDNAhavebeen
successfullyanalyzed.

STR

(
STR

( )

Degraded DNA and Microsatellites

DegradedDNAandMicrosatellites
DNADegradedwhen
eg aded e
exposedtoinfluencesof
ultravioletlight
decay
humidity
oryearsofstoragein
preservatives.


Collection

Specimen Storage

Specimen Storage

Extraction

DNA
Quantitation

Multiplex PCR Amplification

Extraction

Quantitation

Genotyping

Interpretation
of Results

STR Typing
Male: 13,14-15,16-12,13-10,13-15,16

DNA Database

Interpretation of Results

Database
Storage & Searching

LociUsedinMicrosatelliteAnalysis
STR

Systemswhichcoamplify

multiplelociaremostoften

usedinforensiccases.

Themorelocithatare
The more loci that are
studied,themore

Short
Tandem
Repeats
informationthatcanbe

gained from a particular
gainedfromaparticular

sampleofDNA.
TheUnitedKingdomusesa

multiplex reaction of 6 or 7
multiplexreactionof6or7


differentlociinforensics.

Short Tandem Repeats (STR)

ShortTandemRepeats(STR)
L
Locus
or L
Loci:i
Refers to the location on the chromosome.

Allele:
Refers to the type of DNA.
For STRs,

the allele will be the number of repeats.

CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC

Short Tandem Repeats (STR)

ShortTandemRepeats(STR)
D5S818

2 alleles
ll l per llocus

Short Tandem Repeats

ShortTandemRepeats
Example

Locus:

D5S818

Alleles:

7,9

Paternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATCC
Maternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC

13CODISLoci
STR Marker

Chrom osom e

Repeat Sequence

Repeat units

TPOX
CSF1PO
D5S818
D13S317
D16S539
TH01
D7S820

2
5
5
13
16
11
7

AATG
AGAT
AGAT
TATC
GATA
AATG*
GATA*

6 - 14
6 - 15
7 -15
7 - 15
5 8 - 15
5,
3, 5 - 12
6 - 14

D18S51

18

AGAA*

8 - 27

FGA

CTTT**

15 - 30

VWA
D3S1358
D8S1179
D21S11

12
3
8
21

TCTA**
TCTA**
TATC**
TCTA***

11 - 22
9 119,
11 20
8 - 19
24 - 38

* Simple repeats with non-consensus

non consensus alleles
** Compound repeats with non-consensus alleles
*** Complex repeats

Other Alleles

10.3

8.3, 9.3, 10.3, 13.3

13.2, 14.2, 15.2
17 2 19.2
17.2,
19 2
16.2 -30.2
22.3, 34.2, 46.2
15.2
15 2 16.2
15.2,
16 2
24.2 - 35.2

See Appendix I in text

13CODISLoci
TPOX
D3S1358
D8S1179

D5S818
FGA
CSF1PO

TH01
VWA

D7S820

AMEL
D13S317

D16S539

D18S51

D21S11

AMEL

ShortTandemRepeats
p
LociNomenclature
3
5

3
3
5

Intergenic DNA
Gene 1

Gene 2

Gene 1

Gene 2
Introns

5
3

5
5
3

ShortTandemRepeats
p
Loci Nomenclature
LociNomenclature

Introns: STR name based on gene.

TH01 -

located in the 1st intron (01) of gene for

tyrosine hydroxylase

Intergenic DNA: based on chromosome

D5S818

D = DNA
5 = chromosome 5
S = single copy in genome
818 = 818th locus described on Ch 5

ShortTandemRepeats
p
Allele Nomenclature
AlleleNomenclature

Allele named after # of repeat sequences

Are multiple choices which is correct?

TCAT, CATT, ATTC, TGAA, GAAT, AATG

ShortTandemRepeats
p
Allele Nomenclature
AlleleNomenclature
International Society of Forensic Haemogenetics
(ISFH) issued
i
d guidelines
id li
iin 1994 and
d 1997:
1997

Intron STRs - use coding strand

Intergenic DNA use strand first described in
literature of database entry (Genbank)
First motif that can define the repeat is used

Grandfathers.

ShortTandemRepeats
p
D5S818

2 alleles
ll l per llocus


Collection

Specimen Storage

Specimen Storage

Extraction

DNA
Quantitation

Multiplex PCR Amplification

Extraction

Quantitation

Genotyping

Interpretation
of Results

STR Typing
Male: 13,14-15,16-12,13-10,13-15,16

DNA Database

Interpretation of Results

Database
Storage & Searching

PCR Hood

The Thermal Cycler

Amplifies DNA

ShortTandemRepeats
p
BasicStepsinAnalysis
Extraction:
Separates DNA from sample

Amplification
p
or PCR:
Amplifies small portions of DNA (STR regions)

S
Separation:
ti
Separates amplified fragments according to size.

FMBIO

Separates and Measures Amplified DNA

ABI 310 Genetic Analyzer

Separates and Measures Amplified DNA

ShortTandemRepeats
Capillary
y Electrophoresis
Samplewillhaveoneortwopeaksateachloci.

ShortTandemRepeats
Comparetoaladder thathasallpeaksat
eachloci.

ShortTandemRepeats
ShortTandemRepeats
p
DNA Profiles are compared
p
TPOX CSF1PO
D8S1179

D5S818

Blood stain

7,9

10,13

7,15

8,8

Suspect 1

8,9

10,10

9,10

11,12

Suspect 2

10 11
10,11

9 13
9,13

8 14
8,14

9 12
9,12

Suspect 3

7,9

10,13

7,15

8,8