JS 115- Introduction to STRs
I.
Pre class activities a.
Review Assignments and Schedules b.
Return and Review Exam 1
II. Learning Objectives
(C5 ) a.
Short Tandem Repeats
1. Biology of STRs
2. Fluorescence and Detection formats
3. Stutter
4. Statistics and Interpretation b.
Other markers: mtDNA and Y STRs

(Kuhl and Caskey 1993. Curr. Opin. in Genet. Dev. 3:404)
• Head to tail arrangements of sequence units ( 4bp),
• Common in genomes (thousands distributed)
• Polymorphic: vary in length by no. of and/or by content of repeats.
• Stably inherited on a human time scale (for most)
• Well studied b/c others are implicated in Human Diseases and therefore the subject of clinical studies.

5'
5'
occur by variation in
1) The number of repeats
*
GAAT GAAT GAAT GAAT GAAT
3'
GAAT GAAT GAAT
3'
5' and/or
2) The content of the repeats
GAAT GAAT GAAT GAT GAAT
3'

Fluorescent dye label
AATG AATG AATG
7 repeats
8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant
Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved from one another
Primer positions define PCR product size

Locus
Name
Chromosomal
Location
Repeat Motif
ISFH format
GenBank
Accession
Allele in
GenBank
Allele
Range
Number of
Alleles Seen
TAGA X14720
CSF1PO 5q33.3-34 12 6-16 15
CTTT
FGA 4q28 M64982 21 15-51.2
69
TCAT
TH01 11p15.5
D00269 9 3-14 20
GAAT
TPOX 2p23-pter M68651 11 6-13 10
[TCTG][TCTA]
VWA 12p12-pter M25858 18 10-24 28
[TCTG][TCTA]
D3S1358 3p Not available -9-20 20
AGAT
D5S818 5q21-31 G08446 11 7-16 10
GATA
D7S820 7q11.21-22 G08616
G08710
12 6-15 22
D8S1179 8
[TCTA][TCTG]
12 8-19 13
TATC
D13S317 13q22-31 G09017 13 5-15 14
GATA
D16S539 16q24-qter G07925 11 5-15 10
AGAA
D18S51 18q21.3
L18333 13 7-27 43
D21S11 21q21
Complex
[TCTA][TCTG]
AP000433 29 24-38 70

Fluorescent Emission Spectra for Dyes
Filters collect light in narrow range
Overlap is automatically calculated and subtracted using fluorescence “matrix” standards
5-FAM JOE NED ROX
100
80
60
40
20
0
520 540 560 580 600 620 640
WAVELENGTH (nm)
Laser excitation
(488, 514.5 nm) ABI 310 Filter Set F with color contributions between dyes

• 15 Markers Can Be amplified at once
• Sensitivities to levels less than
1 ng of DNA
• Ability to Handle Mixtures and Degraded Samples
• Different Fluorescent Dyes
Used to Distinguish STR
Alleles with Overlapping Size
Ranges

• Gel Electrophoresis
• Capillary Electrophoresis
• Microarrays (Nanogen)
• MALDITOF-MS (Sequenome)

anode
+
Buffer
Gel
Loading well cathode
-
Side view
Voltage
Gel lanes
Top view
+
-
DNA bands

Separation of DNA sequence length amplified products
-
Larger fragments
Smaller fragments
+

FMBIO II Detection of STR Alleles
DNA samples are loaded onto a polyacrylamide gel
Sample Separation
STR alleles separate during electrophoresis through the gel
Sample Detection (Post-
Electrophoresis)
505 nm scan to detect fluorescein-labels
585 nm scan to detect
TMR-labels

• 15 different STR loci may be typed on a single gel
• Scanned using a laser
• and filters to assist in detecting different colors (fluor tags)

CSF1PO
TPOX
Amelogenin
THO1
1 2 3 4 V S vWA

Laser
Capillary filled with polymer solution
Detection window
-
(cathode)
5-20 kV
(anode)
Inlet
Buffer
Outlet
Buffer
Data
Acquisition
Sample tray
Sample tray moves automatically beneath the cathode end of the capillary to deliver each sample in succession

Labeled DNA fragments
(PCR products)
Capillary or
Gel Lane
Sample Detection
CCD Panel
Size
Separation
Ar+
LASER
(488 nm)
Detection region
Fluorescence
Color
Separation
ABI Prism spectrograph

Electropherogram: ABI Prism 310 Genetic Analyzer

Going back to the gel electrophoresis, large
PCR fragments travel slower than small PCR fragments as electricity is applied.
Larger fragments
Smaller fragments

By the same token, smaller PCR fragments migrate through the capillary tube faster and thus are detected before the larger (slower) PCR fragments.

157
153
150
146
145 Laser - Camera

157
153
150
146
145
Laser - Camera

157
153
150
146
Laser - Camera

157
153
150
Laser - Camera

157
153
Laser - Camera

Smaller allelic fragments
Larger allelic fragments
NOTE: in an electropherogram,
-smaller DNA fragments (bottom of traditional gel) are on the left
- the larger fragments (top of the gel) are on the right.

The area under the peak is directly proportional to the intensity of the signal.

• Gels
– Advantages
• Fewer artifacts
• Generally less expensive
• Less sensitive to ambient temperature
– Disadvantages
• Not fully automated
• Need to pour and load gels
• Cannot easily reinject a sample
• CE
– Advantages
• Real time detection
• Better resolution of fragments and microvariants
• Fuly automated- no gel pouring or loading
• Can reinject samples
• Majority of crime labs are using CE
– Disadvantages
• Generally more artifacts
• More expensive
• Temperature sensitive

Locus 1 Locus 2 Locus 3
At each locus there are either one or two peaks. Two peaks at a locus site are called heterozygous while one peak is called homozygous.

Stutter is observed as a minor allele appearing one repeat unit smaller than the major STR allele. Some
STR loci are more prone to stutter than others.
Stutter becomes an issue in putative mixed samples where a decision must be made whether a band is due to stutter or from another DNA source.
General Rule » Do stutter validation studies

45
40
35
30
25
20
15
10
5
0
6 7 8 9 9.3
10
Number of repeats
TH01 Marker
Caucasians (N=427)
Blacks (N=414)
Hispanics (N=414)
*
Proc. Int. Sym. Hum. ID
(Promega) 1997, p. 34

1 in 10
Allele A has a frequency in a population of 1/10.
1 in 20
1 in 5
Allele B has a frequency in a population of 1/20.
Allele C has a frequency in a population of 1/5.
If all three alleles match in two samples then
1/10 x 1/20 x 1/5 = 1/1000

Co mbined D NA I ndex S ystem: http://www.fbi.gov/hq/lab/codis/index1.htm
• Used for linking serial crimes and unsolved cases with repeat offenders
• Launched October 1998
• Links all 50 states
• Requires >4 RFLP markers and/or 13 core STR markers
• As of September 2003
– Total number of profiles: 1,472,150
– Total Forensic profiles: 64,523
– Total Convicted Offender Profiles: 1,407,627
– 9,842 Investigations Aided through September 2003

•
Well characterized and studied (population, evolutionary, medical and forensic studies)
•
Uniparental maternal inheritance missing personsmat. lineage ref smpls
• Relatively small size (16kb) and high copy number –
good on low quantity/quality samples (hair, bone, teethancient/degraded)-(Think Peterson case)
•
Implicated in maternally inherited diseases : diabetes, deafness, hypertrophic cardiomyopathy and myopathy
•
Analysis by DNA sequencing- more complex than STR analysis
• mtDNA - many mitotypes are only found 1X. Some use counting method for statistics. Commonly found mitotypes are as frequent as 1 in 10.

•
Applications
– Forensic investigations (98% of violent crime by men)
– Biodefense- Male terrorist profiling
–
Genealogical and Evolutionary studies
•
Advantages to Human Identity Testing
– Male component isolated without differential extraction
–
Paternal lineages
– Some cases with no spermatazoa- use Y STRs
– Assess number of male donors/contributors
–
Same analysis as autosomal STRs
• Challenges
– Y STR kits not as abundant- now 12plexes available in 2003
– Some Y Haplogroups are common
–
Population specific haplotying needed for new markers

Summary 1
•
Review of DNA Function and Structure
• D eoxyribo N ucleic A cid : blueprints of life
3 main functions: RIM
» 1) Replication
» 2) Information Storage
» 3) Mutation for variation
• Central Dogma information flow--------------->
DNA------->RNA------>protein transcription translation
•
Function:
– RIM- Pacific RIM- Replication-Information-Mutation
– Information Storage- Phone Number analogy-Sequence
•
Structure:
– PBS- The only station Sierra and Gabriel can watch
– Asian Guys Can Teach: AGCT
– DNA is where its AT
– DNA velcro (David Letterman

• Review of PCR
– PCR is repeated rounds of template directed, DNA replication
• dNTPs added to 3’OH of a primer
• Components are template, primers, dNTPs, Mg++ and taq polymerase.
– Contamination prevention
• separation of pre and post PCR areas, use of dedicated equipment, aerosol pipette tips and controls, process one sample at a time, separate reference samples from evidence, avoid splashing, wear protective gear and reagent prep care.
–
PCR is useful on degraded DNA. Due to specificity of primers, will not amplify non human DNA
–
Pitfalls- inhibitors, primer binding site mutations (rare), contamination

Intro to STRs
– Head to tail arrangements 4 bp repeat units
– Polymorphic, Common, Stably Inherited, Implicated in
Diseases
–
Advantages- Discrete, Small- less prone to PA, Useful on highly degraded DNA, Ability to Multiplex , Provide powerful discrimination.
–
STR biological artifacts- stutter, adenylation, microvariants, null alleles, mutations
– Multiplexing STR loci provide powerful discrimination