Mitochondrial AnalysisFrom Hair

Examinations to DNA Sequencing

State of Connecticut Forensic Laboratory

Trace EvidenceTrace evidence: items or debris found in the process of investigating a crime Generally small in size, may be

transferred when physical contact occurs between two individuals or an individual and an object.

Hairs: a commonly encountered example of trace evidence.

HairA biological outgrowth from the skin of mammals.Three areas of a hair: Root, Shaft, TipThree cross-sectional regions of a hair: Cuticle, Cortex, Medulla

Hair EvidenceFound in many crimes, submitted to the laboratory. Can include homicides, hit and runs, sexual

assaults, kidnappings, burglaries, etc. Can associate a suspect to a victim, weapon and/or a crime scene. Suspect

Object

Scene

Victim

Why Is Hair Good Evidence?

Abundant; the average person has >100,000 hair follicles on their head aloneEasily transferable; the average person loses 100 hairs per dayConsiderable variability; comparing body area alone, human hair can be from the head, pubic, anal, eye, nose, ear, underarm, leg, arm, chest, trunk, or beard area, not including the fine hair covering the entire body except on a person’s palms and soles.Durable; hairs have been recovered from mummies dating back 2,000 years

Steps of AnalysisHairs are collected from evidence submittedHairs are identified and preserved onto glass microscope slidesHairs are examined both macroscopically and microscopically If a known standard exists, hairs are comparedA report containing all the results of the comparison is writtenExaminer testifies in a court of law to the results written in the report.

Processing

Hairs are identified from the collected debris and mounted

onto glass microscope slides

Type of evidence submitted is anything on which a hair could possibly be found: includes clothing, letters, guns, knives, bomb devices, bedding, car parts, furniture, carpets, flooring, tape, etc.Hairs are collected by either scraping, picking, taping or vacuuming.

ExaminationMacroscopic characteristics of hair: length, coloration, shaft shape, and texture. Microscopic characteristics of hair include:

The hair shaft (optical cross section, diameter variation, tip appearance, buckling, artifacts, other)

The cuticle (thickness, appearance, color, damage) The cortex [color, pigment granule size, pigment

granule shape, pigment granule distribution, pigment granule density, cortical inclusions, cortical fusi (size, distribution, and density), ovoid bodies (size, distribution, and density), cortical texture]

The medulla (color, thickness, texture, continuity pattern, abnormalities)

The hair root (condition, color, tissue, cortical fusi)

ResultsA qualified hair examiner can provide the following informationfrom a hair examination and comparison:

1. Whether there is hair(s) recovered from the item(s) submitted. 2. What the racial origin of the hair is (Caucasian-type, Negroid-type,

Mongoloid-type)3. The area of the body that the hair came from (head, pubic, body)4. The nature of the hair’s removal from the body5. Whether the hair has been artificially treated6. Whether there has been damage done to the hair 7. Whether there are similarities and/or differences between the

questioned hair and known sample.

*How much information an examiner can provide depends upon the suitability of the hair.

Hair Examination Conclusions

A hair examiner can conclude the following:1. The questioned hair exhibits similar characteristics (no significant differences are present) to the known sample. Thus, the source of the known sample cannot be excluded as being the source of the questioned hair.2. The questioned hair exhibits dissimilar characteristics to the known sample. Thus, the source of the known sample can be excluded as being the source of the questioned hair. 3. The questioned hair exhibits both similarities and differences to the known sample. Thus, no conclusion can be reached as to whether the questioned hair can or cannot be excluded as coming from the same source as the known sample.4. The known hairs are insufficient or unsuitable for comparison purposes.5. The questioned hair(s) are insufficient or unsuitable for comparison purposes.

Further Testing: A Complement To Forensic Hair Examinations

Hair examinations can not identify a specific individual Results can be supplemented by performing other

independent analyses Further testing will not change conclusions stated by

the hair examiner Can provide alternative and additional information about the

hair Further testing is deemed suitable and/or probative based upon:

If the hair root has tissue attached, Nuclear DNA analysis If there is no tissue and/or root, Mitochondrial DNA analysisNuclear DNA

Mitochondrial DNA

Mitochondrial DNA Analysis:

Mitochondria are considered the “powerhouse” of the cell Where the energy to run cellular

functions (ATP) is madeHas its own DNA (mtDNA)

Nuclear DNA vs. Mitochondrial DNA

NUCLEAR DNA Linear 2 Copies per cell Very large (3

billion base pairs) Inherited from

both parents Unique to the

individual (except identical twins)

MITOCHONDRIAL DNA Circular Thousands of copies per

cell Small (Just over 16,000

base pairs) Inherited only from the

mother Not unique

When Would We Use Mitochondrial DNA?

Hairs missing the RootOld Bones, Teeth Missing persons casesMass Disasters

Since mitochondrial DNA has a high copy number, it is more resistant to degradation

Why Isn’t Mitochondrial DNA Unique?

All Yellow =Same mtDNAtype as Mother

Mother

FemaleMale

How Is Mitochondrial Testing Done

Extraction of DNA from sampleAmplification of extracted DNA, to make many exact copies of the DNA we extractedQuantitation of amplified DNA, to determine how much DNA is thereDetermine the sequence of two specific regions of the mitochondrial genome

Mitochondrial DNA Analysis

Evidence tested:

purified DNA

PCR Amplification& sequencing

Computer analysis and mtDNA profile comparisons.

mtDNA gel image

Regions of mtDNA

Control region

mtDNA

HV1(hypervariable region 1)

HV2(hypervariable region 2)

16,569 bases

Case #1 Case #2Questioned GCATTATTGGCGCCCTA GCATATTGCGCCTAKnown GCACCATTAACGTTCTA GCATATTGCGCCTA

*EXCLUSION *CANNOT EXCLUDE

Now What?Compare sequences of questioned items to known sequencesAre they different?

*This is a simplification, the regions scrutinized are larger, andanalysis is far more complicated. This is meant to assist in avery basic understanding.

And Last…Compare the attained sequence with the Mitochondrial CODIS DatabaseHow many times does the sequence we’ve attained appear in the databaseFrom that number, we can estimate the frequency of that sequence in the general population

Interesting Forensic Casework Involving Mitochondrial DNA

Tsar Nicholas II Romanov Did we find Anastasia? NO

Anna Alexander claimed to be the missing child of Nicholas Romanov

Her mitochondrial DNA did not match those of unearthed bones, or of maternal relative Philip of Edinburg

Interesting Forensic Casework Involving Mitochondrial DNA

Tennessee Vs. Ware First case in US where mitochondrial

testing was introduced. September, 1996 Murder case in which the only evidence

found was red hairs in throat of victim and on the victim’s bed

Paul Ware could not be excluded as the source of the hairs.

Interesting Forensic Casework Involving Mitochondrial DNA

Connecticut Vs. Pappas Robbery, first CT case in which mitochondrial DNA

analysis was allowed in the courts Mitochondrial DNA analysis on two head hairs

from sweatshirt found on train tracks adjacent to where some of the robbed money was located

The defendant could not be excluded as the source of the questioned hair sample

Case appealed up to CT Supreme Court, who ruled that this type of testing is acceptable

Interesting Forensic Casework Involving Mitochondrial DNA

Connecticut Vs. Torres Murder/Sexual Assault, verdict March, 2002 Amount of DNA recovered not sufficient for

nuclear DNA testing Mitochondrial profile from semen matched

that of the suspect

*For more detail on this case, click here to see description from the laboratory’s trace section, who had a major contribution to the outcome.