Chapter 21: Mitochondrial DNA Profiling

DNA found in mitochondria Bacteria-like

Circular No recombination Short and “no-nonsense”

Main advantages in forensics: More copies in cell; less subject to

degradation▪ Hair shaft▪ Bones▪ Decomposed samples

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Main disadvantages in forensics Low power of discrimination▪ Maternally inherited▪ No recombination

Less polymorphic than nuclear DNA▪ Most common Caucasian type found in 7.1%

of all Caucasians More subject to contamination during

analysis than nuclear DNA▪ More copies per cell – including cells from

analyst or other sources 3

Mitochondria = Sub-cellular organelles which are generated ATP from breakdown of food

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16,569 nucleotides (more or less) and 37 genes 13 genes coding for proteins in ETC 24 genes coding for tRNA and rRNA

A person’s mtDNA sequence is called a mitotype

10x higher mutation rate than nuclear genome

Revised Cambridge Reference Sequence First human mtDNA genome sequenced Later revised to correct mistakes: rCRS Used as reference for mitotype nomenclature

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Located in control region called D-Loop No genes in D-loop

Hypervariable regions: HV1 (16,024-16,365; 342 bp) HV2 (73-340; 248 bp) HV3 (438-574; 137 bp) Most commonly region in forensics:▪ HV1▪ HV2

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Mitotype reporting Reported against rCRS Sequence polymorphisms▪ E.g. 16233T; 73A

Length polymorphisms▪ Insertions or deletions▪ Insertions: E.g. 524.1A, 524.2C▪ Deletions: E.g. 16296d

Heteroplasmy More than one detectable mitotype in a

person or some tissues in a person (esp. hair)

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Two methods of detection: Allele-specific oligonucleotide (ASO) assay▪ Rapid▪ Can be used to screen and eliminate suspects

prior to DNA sequencing▪ Targets 18 most common sequence

polymorphisms DNA Sequencing▪ More labor intensive▪ Better methods are helping reduce time and labor involved

▪ All polymorphisms can be detected so more informative than ASO

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Current method developed by Sanger Uses modified nucleotides called ddNTPS▪ Dideoxynucleotide triphosphates (ddATP,

ddGTP, ddTTP, ddCTP)

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Reaction contains: Denatured “template” DNA Short, synthetic single-stranded DNA primer Large concentration of dNTPs (normal

nucleotide triphosphates) Small concentration of ddNTPs▪ Labeled with different fluorescent dyes (e.g. G

black, A green, T red, C purple) DNA polymerase Salts and buffers

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Scientific working group on DNA analysis methods (SWGDAM) and International Society of Forensic Genetics (ISFG) Cannot exclude▪ Questioned sample has same sequence as reference

sample Exclusion▪ Two or more nucleotides differences between

questioned sample and reference sample Inconclusive▪ Questioned and reference sample differ by one

nucleotide16

Russian Tsar Nicholas II and family removed from power and murdered during Bolshevik Revolution in 1918 Shot by firing squad, doused with sulfuric

acid, buried in a shallow pit under a road Remains went undiscovered until 1991▪ Nine skeletons discovered (4 male adults, 2

female adults, and 3 female children)▪ Unrecognizable by any method other than DNA▪ Too decomposed for nuclear DNA typing (RFLP)

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mtDNA extracted from femur of each skeleton

Blood samples obtained from maternally-related descendants Tsarina Alexandra▪ Prince Phillip (England) is grand nephew of unbroken

maternal descent▪ His sequence matched that of one adult female

skeleton (the Tsarina) and all 3 female children’s skeletons

Tsar▪ Sequence of adult male skeletons compared to two

relatives of unbroken maternal descent to Tsar 18

Single nucleotide difference found at 16,169▪ Tsar had heteroplasmy at this site (T and C

detected in sequencing reaction)▪ Putative relatives had only T▪ Inconclusive

Body of Tsar’s brother exhumed and tested▪ Tests showed same heteroplasmy as Tsar▪ Tsar’s identity confirmed▪ Eye-witness reports indicate that the footman, the

family cook, and the family doctor were the other 3males in the grave; the Tsarina’s assistant was the second adult female

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Tsar had four daughters and one son▪ One daughter and the son not accounted for

In 2007, two additional bodies found in same area▪ mtDNA testing confirmed that they belonged to the

missing daughter and son

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