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Transcript of JS 190- Introduction to STRs I.Pre class activities a.Review Assignments and Schedules b.Exam 2...
JS 190- Introduction to STRs
I. Pre class activitiesa. Review Assignments and Schedulesb. Exam 2 moved to Weds 11 April
II. Learning Objectives
a. Short Tandem Repeats 1. Biology of STRs2. Fluorescence and Detection formats3. Stutter4. Statistics and Interpretation
b. Other markers: mtDNA and Y STRs
Short Tandem Repeats:a subgroup of tandem repeats
(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.
STR Polymorphisms occur by variation in
1) The number of repeats
GAAT GAAT GAAT5' 3'
GAAT GAAT GAAT GAAT GAAT5' 3'
and/or 2) The content of the repeats
GAAT GAAT GAAT GAT GAAT5' 3'
*
Short Tandem Repeats (STRs)
the repeat region is variable between samples while the flanking regions where PCR primers bind are constant
AATG
7 repeats
8 repeats
AATG AATG
Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved from one another
Primer positions define PCR product size
Fluorescent dye label
Information on 13 CODIS STRs
D18S51
18q21.3 AGAA L18333 13 7-27 43
Locus Name
Chromosomal Location
Repeat MotifISFH format
GenBank Accession
Allele in GenBank
Allele Range
Number of Alleles Seen
CSF1PO
5q33.3-34 TAGA X14720 12 6-16 15
FGA
4q28 CTTT M64982 21 15-51.2 69
TH01
11p15.5 TCAT D00269 9 3-14 20
TPOX
2p23-pter GAAT M68651 11 6-13 10
VWA
12p12-pter [TCTG][TCTA] M25858 18 10-24 28
D3S1358
3p [TCTG][TCTA] Not available -- 9-20 20
D5S818
5q21-31 AGAT G08446 11 7-16 10
D7S820
7q11.21-22 GATA G08616 12 6-15 22
D8S1179
8 [TCTA][TCTG] G08710 12 8-19 13
D13S317
13q22-31 TATC G09017 13 5-15 14
D16S539
16q24-qter GATA G07925 11 5-15 10
D21S11 21q21 Complex [TCTA][TCTG]
AP000433 29 24-38 70
520 540 560 580 600 620 640WAVELENGTH (nm)
100
80
60
40
20
0
5-FAM JOE NED ROX
Laser excitation(488, 514.5 nm)Laser excitation(488, 514.5 nm)
No
rmal
ized
Flu
ore
sce
nt
Inte
ns
ity
Fluorescent Emission Spectra for DyesFilters collect light in narrow range
Overlap is automatically calculated and subtracted using fluorescence “matrix” standards
ABI 310 Filter Set F with color contributions between dyes
Multiplex PCR• 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
Detection Formats
• Gel Electrophoresis
• Capillary Electrophoresis
• Microarrays (Nanogen)
• MALDITOF-MS (Sequenome)
Gel Electrophoresis System
-
Voltage
Gel
Loading well
+anode cathode
Side view Top view
Gel lanes
DNA bands
Buffer
+
-
Separation of DNA sequence length amplified products
-
+
Smallerfragments
Largerfragments
FMBIO II Detection of STR AllelesDNA samples are
loaded onto a polyacrylamide gel
STR alleles separate during electrophoresis through the gel
Sample Separation
Sample Detection (Post-Electrophoresis)
505 nm scan to detect fluorescein-labels
585 nm scan to detect TMR-labels
Example of STR test result
• 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)
1 432 V S
CSF1PO
TPOX
Amelogenin
THO1
vWA
Capillary Electrophoresis SystemLaser
InletBuffer
Capillary filled with polymer solution
5-20 kV- +
OutletBuffer
Sample tray
Detection window
(cathode) (anode)
Data AcquisitionSample tray moves
automatically beneath the cathode end of the capillary to deliver each sample in succession
Sample Detection
CCD Panel
ColorSeparation
Ar+ LASER (488 nm)
Fluorescence ABI Prism spectrograph
Capillary or Gel Lane
Size Separation
Labeled DNA fragments (PCR products)
Detection region
Principles of CE Sample Separation
and Detection
Results are interpreted and printed
Electropherogram:Electropherogram: ABI Prism 310 Genetic Analyzer
STR Peaks - What do They Represent?
Going back to the gel electrophoresis, large PCR fragments travel slower than small PCR fragments as electricity is applied.
Ele
ctri
cal C
urr
en
t
Smallerfragments
Largerfragments
What STR Peaks Show
By the same token, smaller PCR fragments migrate through the capillary tube faster and thus are detected before the larger (slower) PCR fragments.
Laser - Camera145
146
150
153
157
145
Laser - Camera146
150
153
157
Laser - Camera
146
150
153
157
Laser - Camera
150
153
157
Laser - Camera
153
157
STR Peaks - What do They Represent?
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.
Larger allelicfragments
Smaller allelicfragments
STR Peaks - What do They Represent?
The area under the peak is directly proportional to the intensity of the signal.
Comparison of Gels vs CE
• 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
Heterozygous versus Homozygousin SINGLE SOURCE samples
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.
Heterozygous Heterozygous Homozygous
STR - Mixture and StutterStutter 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
STR Allele Frequencies
0
5
10
15
20
25
30
35
40
45
6 7 8 9 9.3 10
Caucasians (N=427)
Blacks (N=414)
Hispanics (N=414)
TH01 Marker
*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34
Number of repeats
Fre
qu
ency
Probability Analysis - The Product Rule
1 in 10
1 in 20
1 in 5
If all three alleles match in two samples then1/10 x 1/20 x 1/5 = 1/1000
Allele A has a frequency in a population of 1/10.
Allele B has a frequency in a population of 1/20.
Allele C has a frequency in a population of 1/5.
FBI’s CODIS DNA DatabaseCombined DNA Index System: http://www.fbi.gov/about-us/lab/codis/ndis-statisticsUsed 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 February 2012
– Total number of profiles: 10,560,300– Total Forensic profiles: 417,200– Total Hits: 173,500– 166,700Investigations Aided
Why mtDNA SNPs?
• Well characterized and studied (population, evolutionary, medical and forensic studies)
• Uniparental maternal inheritance missing persons-mat. lineage ref smpls
• Relatively small size (16kb) and high copy number – good on low quantity/quality samples (hair, bone, teeth- ancient/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.
Why Y?• 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
Review of STRs
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