DNA Forensics

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DNA Forensics

• Physical Evidence

2
Physical Evidence

• Perspective
• Recognition of Evidence
• Collection of Physical Evidence
• Preservation of Physical Evidence
• Preparation of the Physical Evidence
• Evaluation and Quantification of the Evidence

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Forensic science makes determinations about the
state of physical evidence at the time of
collection.

• Forensic science does not establish innocence or
  guilt.
• Forensic science contributes information about
  who, what, where why and how.
• Forensic science establishes associations.

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Paradigm of the Principles of Evidence Formation

• Physical Evidence is generated when matter from
  persons and objects at the crime scene is divided
  and transferred.

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Locard Transfer Theory

• When two objects come in contact, traces from
  one will be transferred to the other, in both
  directions. These traces may not always be
  detectablebut they are always present.

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Paradigm of the Principles of the Process of
Evidence Analysis

• Evidence Recognition Collection
• Evidence Identification (according to appearance
  and composition)
• Evidence Classification
• Evidence Individualization (in an attempt to
  determine a source)
• Association of the evidence to link a person with
  the crime scene.
• Reconstruction of events

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Identification

• Answers the question What is it?

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Classification

• Preliminary step that leads to individualization
• Sort evidence according to a class of
  characteristics.
• Firearmscaliber
• Shoestread, size

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Classification Fiber or
hair animal or human
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Individualization

• Evidence that exhibit traits that are are so
  unique that when considered alone or in
  combination with other traits can reduce the
  evidence source from a class to one individual.
• Evidence that can indicate that two samples share
  a common unique source or origin.

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Association

• Description of the relationship between two
  objects items, or people.
• Concept used in a crime scene analysis for
  reconstruction.
• Involves the evaluation of evidence to infer a
  common source.
• Does not prove a crime.

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Reconstruction

• the ordering of associations in space and time.
• Attempts to answer Where? How? When?

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Traits that Indicate Individuality

• Fingerprintsare a result of several genes and
  other nongenetic events. Has been accepted as
  unique for each individual (even identical twins)
  
• DNAearly results suggested individuality except
  in identical twins but in reality more like a
  partial print.

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Conventional Blood Typing as a Model of Analyzing
Results of DNA Typing

• A blood sample tested and found to be Type A and
  PGM type 1
• 35 of the Caucasian population is Type A
• 19 of the Caucasian population is PGM type 1
• 0.35 X 0.19 0.067
• 6.7 of the population included as possible donors

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Comparison of DNA Typing Methods and Power of
Discrimination
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RFLP Methods

• Have a high power of discrimination 20-80
  different alleles may be possible at one
  location analyzed in combination can be used to
  determine an individualized type.
• RFLP procedures are labor intensive multi-locus
  probes are difficult to automate single-locus
  probes can be used in serial fashion.
• Require ample supply of high grade DNA.

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Mitochondrial DNA (mtDNA)

• Lowest power of discrimination
• Longest sample processing time
• Can be very helpful in forensic cases involving
  severely degraded DNA samples

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ABO Blood Typing Groups or D1S80 Short Tandem
Repeat (STR)

• Rapid Results
• Useful for excluding an individual not very
  useful for inclusion
• Single STRD1S80 is done using PCR fast but not
  discriminating.

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STRs

• Short Tandem Repeats Repeating units of an
  identical DNA sequence, length is often between 2
  5 bp in length. The repeat units are arranged
  in direct succession of each other, and the
  number of repeat units varies between individuals
  (subgroup of VNTRs)

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Multiplex STRs

• High power of Discrimination
• Rapid Analysis
• Analysis can be automated and 3 or more locations
  can be analyzed at a time.

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Validation of STR Techniques

• 1991Fluorescent STR markers first described
• 1993First STR kit available
• 1996First multiplex STR kits available
• 199713 core STR loci defined Y-chromosome STR
  described
• 1999Multiplex STR kits validated
• 2000FBI and other labs stop running RFLP and
  convert to multiplex STRs.

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Example of STR Multiplex
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A Typical DNA Profile
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Example of STR Multiplex
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Sources of DNA for Testing

• Blood
• Semen
• Tissue
• Bone (Marrow)
• Hair Root
• Saliva
• Urine
• Tooth (Pulp)

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Sources of DNA for Testing BLOOD

• Extracting DNA from dried blood on glass, metal,
  hard plastics or lightweight cloth is
  straightforward
• Extracting DNA from dried blood on vinyl, carpet,
  car seats or dense or heavily colored fibers
  require additional steps of purification

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Sources of DNA for Testing BLOOD

• Extracting DNA from concrete is difficult because
  of separation issues.
• Extracting DNA from soil is almost impossible

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Sources of DNA for Testing BLOOD

• Blood stains may be mixtures of blood from more
  than one person and can produce complex DNA
  profiles
• The most straightforward samples are liquid whole
  blood.
• Examplars specimens drawn from suspects or
  victims usually liquid whole blood.

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Sources of DNA for Testing BLOOD

• Best storage method for blood is frozen
• Most common handling method for forensic blood
  samples following collection is a series of
  dime-sized stains on washed cotton sheeting

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Examplars

• Whole blood
• Buccal swabs from inside the cheek
  non-invasive
• Military recruits give both blood and buccal cell
  samples.
• Guthrie Cards collected at birth for newborn
  screening of genetic diseases.

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Sources of DNA for Testing SEMEN

• Sperm cells as dried stains are identifiable for
  years.
• Must consider all possible partners within 72
  hours of event.
• Mixtures of cells are a confounding factor
• Differential lysis is used to separate the sperm
  cells from the non-sperm cells (the female
  fraction)

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Sources of DNA for Testing TISSUE

• Isolation of DNA from tissues is a simple and
  straightforward.
• DNA survival in soft tissues is low (liver,
  kidney)
• Remnants of tissues such as brain can be
  scattered by gunshot.
• DNA can be successfully tested from tissues
  chemically treated with formaldehyde or embalming.

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Sources of DNA for Testing TISSUE

• DNA survival in dense bone and teeth is the
  longest of all types of tissues.

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Sources of DNA for Testing Hair Roots

• One to five hair roots can contain enough tissue
  for RFLP analysis
• One is sufficient for PCR methods
• Mitochondirial sequencing was used to identify
  one of Napoleons hairs and members of the
  Romanoff family

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Sources of DNA for Testing Saliva

• DNA can be typed from saliva deposited on
  envelope flaps or stamps or cigarette butts or
  cups or telephone mouthpieces.

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Sources of DNA for Testing Urine

• Not common because healthy persons do not shed
  nucleated cells into their urine.

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Sources of DNA for Testing Products of
Conception

• Non-living products of conception may be analyzed
  in cases of allegations of criminal paternity

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Preservation of Evidence

• Air dry (no heat) all stains and swabs
• Store frozen in paper bags, not plastic
• Store tissue samples frozen preferably without
  preservatives (formalin or embalming fluid)
• Liquid blood can be refrigerated up to a few
  weeks (longer term, freeze for DNA testing)

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Sample Collection

• Avoid contamination of the area where DNA might
  be present.
• Use clean latex gloves for collection.
• Change gloves between handling of different items
• Package each item separately

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Sample Collection

• Bloodstains, semen stains, and other types of
  stains need to be air-dried prior to packaging.
• Samples should be packaged in paper envelopes or
  paper bags. Avoid plastic because water
  condenses in them.
• Packages should be clearly marked
• Stains on unmovable surfaces may be transferred
  with sterile cotton swabs distilled water.
  Allow the swab to dry

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Factors Leading to DNA Degradation

• Time
• Temperature
• Humidity
• Light
• Exposure to chemicals

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Important Considerations in Sample Collection

• Preserve the biological integrity of the sample.
• Avoid contamination
• Document the chain of custody of the evidence
  throughout the system

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Preservation of Physical Evidence

• Most biological evidence is best preserved when
  stored dry and cold.
• DNA samples are stored either at 4oC or 20oC.
• Extracted DNA samples may be stored at 70oC
• Most labs have freezers dedicated to DNA storage

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DNA Extraction Methods

• Organic or phenol chloroform
• Chelex
• FTA Paper

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Organic Extraction Method

• Has been used the longest period of time
• Can be used for either RFLP or PCR typing.
• High molecular weight DNA needed for RFLP methods
  may be obtained most effectively with organic
  extraction.

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Organic Extraction Method

• SDS, DTT and proteinase K are added to break open
  the cell walls and break down DNA binding
  proteins.
• Phenol/choloroform mixture is added to separate
  proteins from DNA
• Concentrate or ethanol precipitate

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Organic Extraction Method

• Time consuming
• Involves the use of hazardous chemicals
• Requires the transfer of the sample to multiple
  tubes during the process

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Chelex Extraction Method

• More rapid than organic extraction method
• Involves few steps and fewer opportunities for
  contamination
• Produces single-stranded DNA
• Only useful for PCR procedures

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Chelex Extraction Method

• Chelex is a chelating resin suspension that can
  be added directly to the sample
• Biological samples are added to a 5 Chelex
  suspension and boiled for several minuets to
  break open the cells
• Centrifuge to separate the resin from the DNA
  solution
• Sample is ready for PCR reaction

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Chelex Extraction Method

• Not effective for RFLP typing
• It removes inhibitors of PCR
• Uses only a single tube
• The addition of too much whole blood can inhibit
  PCR.

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FTATM Paper Extraction Method

• Absorbent cellulose paper
• DNA is immobilized within the matrix of the paper
• A small punch of the bloodstain is place in a
  tube for washing.
• The punch is washed with solvent
• The punch is added directly to the PCR reaction
  tube

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FTATM Paper Extraction Method

• Consistent results without quantification
• Procedure may be automated
• Punch may be reused for sequential DNA
  amplifications and typing.
• Can be used with buccal cells

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Other Extraction Methods

• Differential ExtractionModified version of
  organic extraction used in FBI Labs to isolate
  female and male fractions.
• Microwave Extraction
• QIAamp spin columns

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Next Lecture February 23

• Methods for evaluation of the integrity and
  quantification of the DNA samples
• Forensic DNA Typing Systems (Chapter 5)

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Vocabulary

• Physical Evidence
• Classification
• Identification
• Association
• Individualization
• Inclusion
• Excluded

• Examplars
• Buccal
• Contamination
• Chelate
• Nuclease
• Denature

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Lecture Sources

• TextChapters 1,2 5
• DNA in the Courtroom Coleman Swenson, GeneLex
  Press 1994
• Forensic DNA Typing John M. Butler, Academic
  Press 2001
• More Chemistry and Crime Samuel M. Gerber
  Richard Saferstein, American Chemical Society,
  1997