Short Tandem Repeat Markers

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Short Tandem Repeat Markers

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Review of Repeated DNA Tandems

• Satellite DNA refers to long repeat units of
  hundreds to thousands of bp. Often found
  surrounding the centromere.
• Minisatellite or VNTR have a core repeat unit of
  10-100 bp (D1S80)
• Microsatellite or STR have repeat units 2-6 bp.

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Short Tandem Repeats (STR)

• Repeating units of an identical (or similar) DNA
  sequence, where the repeat sequence is 2-5 base
  pairs in length. The repeat units are arranged
  in direct succession of each other, and the
  number of repeat units varies between
  individuals. (Textbook)

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The value of STR markers in forensics

• STR markers are easily amplified.
• The number of repeats can be highly variable
  among individuals.
• Thousands of polymorphic STR loci have been
  identified in human genome.
• STR markers are scattered throughout the genome.

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Advantages for using STRs over VNTRs for forensic
DNA typing

• Smaller size advantageous where degraded DNA is
  common
• Preferential amplification of the smaller allele
  not significant problem
• Single base resolution easier for fragments lt 500
  bp

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Isolation of STR Markers
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Methods for Identification of STR Markers

• Search DNA sequence databases such as GenBank for
  regions with more than six or so contiguous
  repeat units (Weber May, 1989)
• Perform molecular biology isolation methods
  (Edwards et al, 1991)

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Types of STR Repeat Sequences

• Length of Repeat Unit
• Number of Repeats
• The Level of Conformity of the Repeated Units

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Length of Repeat Unit

• Mononucleotide repeats
• Dinucleotide repeats
• Trinucleotide repeats
• Tetranucleotide repeatshave become the most
  popular markers for human identification (AGAT or
  GATA)
• Pentanucleotide repeats
• Hexanucleotide repeats

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The Conformity of the Repeated Motif

• Simple Repeats contain units identical in length
  and sequence.
• Compound Repeats contain two or more adjacent
  simple repeats.
• Complex Repeats may contain several repeat blocks
  that vary in length or may have variable
  intervening sequences.
• Complex Hypervariable Repeats may exist as
  non-consensus alleles that differ in size
  sequence.

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Desirable Characteristics of STRs Used in
Forensic DNA Typing

• High variation among individuals
• Separate Chromosomal locations
• Reproducible results when multiplexed
• Low yield of stutter products
• Low mutation rate
• Allele length range of 90-500 bp.

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Stutter Products Amplicons that are typically
one or more repeat units less in size than the
true allele
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Minisatellite VNTR alleles (400-1000 bp) can
exhibit preferential amplification.
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Advantages of using tetranucleotide STR loci in
forensic DNA typing

• Conducive to multiplexing
• Reduced allelic dropout
• Capable of generating PCR product from degraded
  DNA samples
• Reduced stutter product formation
• Heterozygotes are easier to differentiate
• More common than pent- or hexa-nucleotide
  repeats.

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Male-specific Y Chromosome STRs

• Useful for analyzing male-female mixtures from
  sexual crimes.

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Nomenclature for STR Alleles

• International Society of Forensic Haemogenetics
  (ISFH) issued guidelines in 1994 1997
• Aid in reproducibility and comparison of data

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Nomenclature for STR AllelesChoice of Strand

• If within a coding region, coding strand should
  be used
• If not within a coding region, use the sequence
  first described in public database
• If nomenclature established prior to the
  guidelines, the original nomenclature is
  maintained.

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TH01 Note that using the top strand versus the
bottom strand results in different repeat motifs.

• 1 2 3 4
  5 6
• 5-TTCCC TCAT TCAT TCAT TCATTCAT TCAT
  CACCATGGA-3
• 3-AAGGG AGTA AGTA AGTA AGTA AGTA AGTA
  AGTGGTAACCT-5
• 6 5 4
  3 2 1

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Nomenclature for STR AllelesChoice of Repeat
Sequence Motif

• The first 5-nucleotide repeat defines the motif.
  
• 5-GG TCA TCA TCA TGG-3
• (3 X TCA repeats) or (3 X CAT repeats)
• According to ISFH guidelines (3 X TCA)

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Nomenclature for STR AllelesChoice of Allele
Designation

• Number of repeats.Number of bp in incomplete
  repeat
• EX A common microvariant of TH01 is allele 9.3
• 9 tetranucleotide repeats and one incomplete
  repeat of three nucleotides.

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Allelic Ladders

• A mixture of common alleles present in the human
  population for a particular marker
• Generated with the same primers as the DNA
  samples and the reference DNA
• Can be created within the lab or can be purchased
  by commerical manufacturers.

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Allelic Ladder for D1S80Manual Gel
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Allelic Ladder on Automated Gel
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Commonly used STR markers

• Developed by Dr. Thomas Caskey at the Baylor
  College of Medicine and Forensic Science Service
  in England.
• Commercialized by Promega and PE Applied
  BioSystems

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Availability of STR Marker Kits have
revolutionized forensic DNA.

• Cover a common set of markers.
• Permit amplification of gt 8 markers in a
  multiplex reaction.
• Generate matching probabilities that exceed 1 in
  a billion.
• Utilize 1 ng of DNA template.
• Can produce results in a few hours.l

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13 Core STR loci
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Commercial STR Multiplex Kits
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PowerPlex 16 Sample Analysis
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Molecular Dynamics FluorImager
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Schematic of Hitachi FMBIO 100 Fluorescent Scanner
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Processing Steps
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Analysis of STRs and Amelogenin Using a Static
Detection System
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ABI PRISM 377 DNA Sequence
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ABI PRISM 310 Genetic Analyzer
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Process
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STR Data Collection Using ABI Prisim
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STRBase

• http//www.cstl.nist.gov/biotech/strbase

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STR Product Artifacts

• Stutter Products
• Non-template Nucleotide Addition
• Microvariants
• Allele Dropout
• Mutations

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Stutter Product Peaks

• Stutter peaks are typically a few bases smaller
  than the allele peak.
• Proposed that a portion of the primer-template
  complex becomes mismatched causing slippage.
• Sometimes difficult to determine if the small
  peak is a stutter peak or a real allele.

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Non-Template Addition

• Frequently Taq Polymerase will add an extra
  nucleotide to the 3 end of the PCR product.
• The addition is usually an A.
• The addition to only some of the PCR products
  results in peak broadening and poor resolution
  that can impact analysis

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Schematic of Non-Template Addition
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Microvariants

• Rare alleles that differ from common alleles by
  one or more base pairs
• Often called off-ladder alleles
• Most common in the more polymorphic STR loci

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Example of a Microvariant Allele
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Alleles of the Same Length but Different Sequences

• Complex repeat sequences can contain variable
  repeat blocks that are the same length but differ
  in nucleotide sequence.
• D21S11 has four alleles all of 210 bp when
  amplified.
• Only detectable by complete sequence analysis

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Three-Banded Patterns

• Sometimes observed at a single location in a
  multiplex STR profile.
• The extra peak is not from a mixture but is a
  reproducible artifact.
• The extra peak may or may not be smaller in size.

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Three-banded Pattern
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Allele Dropout

• A base pair change can occur in the DNA template
  that disrupts primer hybridization.

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Mutations

• Mutations can occur at STR loci
• Most STR mutations involve the gain or loss of a
  single repeat unit.
• Can be difficult to determine from which parent
  contributed the mutant allele.
• Low mutation rates are important for paternity
  testing.

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Vocabulary

• Short Tandem Repeats (STR)
• Microvariants
• Amplicon
• Polymorphic
• Stutter Product

• Allelic Dropout
• International Society of Forensic Haemogenetics
  (ISFH)
• Adenylation

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References

• Textbook
• Forensic DNA Typing John M. Butler Academic
  Press, 2001.
• Biotechnology ExplorerChromosome 8 Gene
  Amplification Alu-TPA PCR Kit by BioRad.