Forensic DNA Issues

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Forensic DNA Issues

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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Summary

• Degraded DNA
• PCR Inhibition
• Contamination
• Mixtures

• Amelogenin
• Y-Chromosome
• DAB Guidelines

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Low Copy Number DNA

• Low quantity or quality of DNA referred to as Low
  Copy Number (LCN) DNA.

• Can result from small sample
• Can result from degraded samples
• May requires a different approach that is less
  cost efficient than normal analysis.

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LCN DNA
1 cm of DNA 3 x 106 nucleotides http//www.mediz
in.uni-tuebingen.de/med_virologie/exp_viro/Exp_Vir
o_de/Forschung/Sonstiges/Useful_remarks.html  

• 1 ng good quality DNA for good PCR results
• 6 pg per cell, 3 pg per copy or .003 ng
• 333 intact copies needed for DNA analysis

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LCN DNA
Only 50 copies good DNA will not amplify
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LCN DNA
350 copies (1ng) will amplify
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LCN DNA

• Degraded DNA

• Nuclease activity Nucleases found all over the
  place in nature
• Moisture allows bacterial growth (increases
  amount of nucleases)
• Light can cause degradation

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LCN DNA
350 copies (1ng) will amplify
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LCN DNA
350 copies (1 ng) degraded DNA could be only 50
copies intact DNA Will not amplify
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LCN DNA

• Amplifying Degraded DNA

• Use more extract (risk adding PCR inhibitors)
• Amplify different area smaller PCR area less
  risk of degradation

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LCN DNA

• For normal STR amplification,

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LCN DNA

• For normal STR amplification, degradation may
  knock out 60 of copies

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LCN DNA

• For shorter STR amplification, degradation may
  only knock out 40 of copies

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PCR Inhibition

• Usually due to inhibition of DNA polymerase.

• Dyes or heme have been shown to inhibit
• Found in sample gets in extract

Solutions

• Dilute extract
• More polymerase
• Separation (concentrators)

• BSA (bovine serum albumin)
• NaOH

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Contamination

• Contamination DNA enters sample from source
  other than donor(s).

• DNA from environment (during collection,
  extraction, or PCR set-up)
• Cross contamination between samples
• Amplified DNA into reaction.

Mixtures arent necessarily contamination
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Contamination

• Consequences of contamination

• Usually leads to false exclusion.
• False inclusions are prevented by
• Running known and unknown samples separately
  (prevents cross contamination)
• Running known samples after sample (prevents
  amped product from getting into sample)

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Mixtures

• Mixtures are when 2 or more genotypes result from
  one sample

• May be due to contamination.
• May be that sample is a mixture of 2 individuals
  biological fluids.

Examples swab from rape kit, mixed blood, blood
transfusion, etc.
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Mixtures

• Indicators of a mixture

• More than 2 peaks
• Peak imbalance
• Stutter product high

These should occur at several loci if the
combination is a mixture.
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Mixtures

• Mixtures can be in equal ratios or one
  contributor may dominate.

• All possible combinations of alleles should be
  considered. Calculations can become quite
  complex.

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Amelogenin

• Gene that codes for protein in tooth enamel.

• Amelogenin gene is found on each sex chromosome,
  but is longer on Y.

• 6 bp deletion is within intron 1 of the X
  chromosome

• Male gives 2 peaks, female gives one peak.

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Sex Identification

• Y chromosome-specific loci

• Lineage Marker (similar to mtDNA) passed down
  unchanged.
• Also helps to identify males in ?? mixture
• Like mtDNA, haplotype is determined not very
  discrimatory

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Sex Identification

• Sample B and Swab match
• For CODIS, STRs would multiple frequencies.
• 720, 1118, 834, 148 0.1
• For Y-STR, haplotype is reported.
• 8 matches in 1500 0.5

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SNPs

• Single Nucleotide Polymorphisms (SNPs)
• Occur throughout genome

Example At a specific location on chromosome 5,
25 have a C, 75 have a T.

• Amplicons can be small.
• Non-sequencing detection.
• More needed (25-45 as specific as 13 STRs)

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Alu Repeats

• Are short, interspersed nuclear elements (SINES)
  repeated DNA inserted at different locations in
  genome

• Named Alu because sequence contains an AluI
  restriction site.
• Can be inserted, but are not deleted

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Alu Repeats
Alu Repeat
2 alleles
Alu Repeat
Repeat inserted 400 bp long
Repeat absent 100 bp long
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Alu Repeats

• Use
• Low variation and arduous detection methods dont
  make it a good identification technique.
• More useful for tracing ancestry, since Alu
  repeats arent lost.

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DAB Guidelines

• 5.3 Examiner/analyst shall have
• 5.3.1 at a minimum a BA/BS degree or its
  equivalent degree in biology-, chemistry- or
  forensic science- related area and must have
  successfully completed college course work
  (graduate or undergraduate level) covering the
  subject areas of biochemistry, genetics and
  molecular biology (molecular genetics,
  recombinant DNA technology) or other subjects
  which provide a basic understanding of the
  foundation of forensic DNA analysis, as well as
  course work and/or training in statistics and
  population genetics as it applies to forensic DNA
  analysis.

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DAB Guidelines

• 5.3.2 a minimum of six (6) months of forensic
  DNA laboratory experience, including the
  successful analysis of a range of samples
  typically encountered in forensic case work prior
  to independent case work analysis using DNA
  technology.
• 5.3.3 successfully completed a qualifying test
  before beginning independent casework
  responsibilities.

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DAB Guidelines

• 5.2 The technical manager or leader shall have
  the following
• 5.2.1 Degree requirements The technical manager
  or leader of a laboratory shall have at a minimum
  a Masters degree in biology-, chemistry- or
  forensic science- related area and successfully
  completed a minimum of 12 semester or equivalent
  credit hours of a combination of undergraduate
  and graduate course work covering the subject
  areas of biochemistry, genetics and molecular
  biology (molecular genetics, recombinant DNA
  technology), or other subjects which provide a
  basic understanding of the foundation of forensic
  DNA analysis as well as statistics and/or
  population genetics as it applies to forensic DNA
  analysis.

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DAB Guidelines

• 5.2.2 Experience requirements A technical
  manager or leader of a laboratory must have a
  minimum of three years of forensic DNA laboratory
  experience.