JS 115 Introduction to STRs

1
JS 115- Introduction to STRs

• Pre class activities
• Review Assignments and Schedules
• Return and Review Exam 1
• II. Learning Objectives (C5)
• Short Tandem Repeats
• 1. Biology of STRs
• 2. Fluorescence and Detection formats
• 3. Stutter
• 4. Statistics and Interpretation
• Other markers mtDNA and Y STRs

2
Short Tandem Repeats a subgroup of tandem
repeats (Kuhl and Caskey 1993. Curr. Opin. in
Genet. Dev. 3404)

• 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.

3
(No Transcript)
4
Short Tandem Repeats (STRs)
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
5
Information on 13 CODIS STRs
D18S51  
18q21.3
AGAA
L18333
13
7-27
43
6
Fluorescent Emission Spectra for DyesFilters
collect light in narrow rangeOverlap is
automatically calculated and subtracted using
fluorescence matrix standards
ABI 310 Filter Set F with color contributions
between dyes
7
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

8
Detection Formats

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

9
Gel Electrophoresis System
10
Separation of DNA sequence length amplified
products
Larger fragments
Smaller fragments

11
FMBIO II Detection of STR Alleles
12
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)

13
1
4
3
2
V
S
14
Capillary Electrophoresis System
Sample tray moves automatically beneath the
cathode end of the capillary to deliver each
sample in succession
15
Principles of CE Sample Separation and Detection
Labeled DNA fragments (PCR products)
Capillary or Gel Lane
16
Results are interpreted and printed
Electropherogram ABI Prism 310 Genetic Analyzer
17
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.
Larger fragments
Smaller fragments
18
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.
19
157
153
150
146
Laser - Camera
145
20
157
153
150
Laser - Camera
146
145
21
157
153
Laser - Camera
150
146
22
157
Laser - Camera
153
150
23
157
Laser - Camera
153
24
STR Peaks - What do They Represent?
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.
25
STR Peaks - What do They Represent?
The area under the peak is directly proportional
to the intensity of the signal.
26
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

27
Heterozygous versus Homozygousin SINGLE SOURCE
samples
Locus 1
Locus 2
Locus 3
Heterozygous
Heterozygous
Homozygous
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.
28
STR - Mixture and Stutter
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
29
STR Allele Frequencies
TH01 Marker
Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34
30
Probability Analysis - The Product Rule
1 in 10
Allele A has a frequency in a population of 1/10.
1 in 20
Allele B has a frequency in a population of 1/20.
1 in 5
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
31
FBIs CODIS DNA Database

• Combined DNA Index System http//www.fbi.gov/hq/l
  ab/codis/index1.htm
• Used for linking serial crimes and unsolved cases
  with repeat offenders
• Launched October 1998
• Links all 50 states
• Requires gt4 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

32
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.

33
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

34
Summary 1

• Review of DNA Function and Structure
• DeoxyriboNucleic Acid blueprints of life
• 3 main functions RIM
• 1) Replication
• 2) Information Storage
• 3) Mutation for variation
• Central Dogma
• information flow---------------gt
• DNA-------gtRNA------gtprotein
• 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

35
Review of PCR

• Review of PCR
• PCR is repeated rounds of template directed, DNA
  replication
• dNTPs added to 3OH 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

36
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