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Strange Evidence

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Anomaly Physical Evidence Investigation Group & DNA Investigations

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Strange Evidence

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IUR F SPRING 1999 4 Peter Khoury pt 11

FORENSIC INVESTIGATION: In early 1998, Bill Chalker provided this hair sample to the Anomaly Physical Evidence Group, whose members as professional scientists choose to remain anonymous at this time.

A standard forensic investigation was then carried out on the hair shaft, using well established protocols (see M. R. Wilson, et al., “Extraction, PCR Amplification and Sequencing of Mitochondrial DNA from Human Hair Shafts,” Biotechniques 18 (1995): 662–669), and also on control hair samples from the young man and his wife (married in 1990). The goal of such analysis is to establish a precise DNA base sequence of Mitochondrial Hypervariable Region I, spanning nucleotides 16,000 to 16,400 of the circular mitochondrial DNA.

Such DNA is present in hundreds of copies within each human cell, and thereby acts as an easily amplified genetic marker for the polymerase chain reaction (PCR), even in moderately degraded samples. (See the Web based Mitomap for more information: infinity.gen. emory.edu/mitomap.html for “Mitomap: A Human Mito-chondrial Genome Database,” Center for Molecular Medicine, Emory University, Atlanta, 1999.)

Any sequence variation within that hypervariable region 16,000 to 16,400 then provides a “microscopic genetic fingerprint” of the individual involved, which is used commonly as forensic evidence in many criminal investigations. In order to extract fragments of mitochondrial DNA from the hair of the tall, blonde female, a two cm piece of hair shaft (located just above the root) was excised and transferred to a sterile Eppendorf tube. Similar pieces were excised also from both control hair samples, as obtained from the young man and his wife, in the presence of Bill Chalker.

These three hairs could be easily distinguished by their appearance under a microscope, since the hair from the tall blonde female was extremely thin and almost clear Mitochondrial DNA sequence from the blonde woman’s hair strand. (very little melanin), whereas the hair from the young man was of normal thickness and black; hair from his wife was of normal thickness and brown. In fact, that blonde hair was almost invisible to the naked eye on a glass surface, due to its unusual optical clarity, and hence it could only be handled under reflected light.

Further investigation of this thin, almost clear hair by high resolution darkfield microscopy showed it to lie at the lower end of normal human hair thickness, and also to show a pronounced “mosaic” structure, perhaps due to the near absence of melanin.

PROCEDURE The three hair samples to be tested:

(a) tall blonde,

(b) the young man, and

(c) his wife, were washed twice with PCR quality water, twice with 70% ethanol, then once with extraction buffer (7 M guanidinium hydrochloride, 100 mM Tris, pH 7.0, 1% Triton X100 and 5 mM EDTA) for one hour at 20° C. to remove any contaminating DNA from the outside of the shaft. Next, each washed hair was extracted in the same guanidinium Triton buffer plus 10 mM dithiothreitol (DTT) for two days at 50° C.

Finally, each hair sample was fragmented using a sterile disposable pestle for 1.5 ml Eppendorf tubes, then subjected to several cycles of boiling and freezing on dry ice, in order to release the DNA from its protein matrix. Each fully treated hair solution was then extracted twice using phenol/chloroform/isoamyl alcohol (25/24/1) to remove organic impurities, especially melanin.

The purified DNA was then supplemented with 2 micrograms of UV sterilised glycogen as a carrier, and precipitated with ethanol. Each DNA pellet was next resuspended in 10 mM Tris, 0.1 mM EDTA and used in small aliquots for PCR amplification. No washes of any hair, prior to extraction with guanidinium Triton and DTT, produced any detectable mitochondrial DNA in subsequent PCR steps.

Other attempts to extract DNA from pieces of that hair far from the root were unsuccessful. In order to amplify Mitochondrial Hypervariable Region I from a broad mixture of DNA fragments, two oligonucleotide DNA primers were synthesized first over mitochondrial locations 15,993–16,022 (upper strand) or 16,401–16,430 (lower strand), because the nucleotides in those locations are highly conserved among all primates, including chimpanzee, gorilla, orangutan and man.

Next, two more primers were synthesized within the hypervariable region itself, so that one might amplify the whole DNA of 16,023–16,400 in two overlapping fragments (mitochon-drial DNA from a hair shaft is often degraded, so that chains longer than 300 bases become difficult to find).