1. Chapter 21: Mitochondrial DNA Profiling

2.  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 2

3.  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

4.  Mitochondria = Sub-cellular organelles which are generated ATP from breakdown of food 4

5.  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 5

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7.  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 7

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9.  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) 9

10.  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 10

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13.  Current method developed by Sanger  Uses modified nucleotides called ddNTPS ▪ Dideoxynucleotide triphosphates (ddATP, ddGTP, ddTTP, ddCTP) 13

14.  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 14

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16.  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 nucleotide 16

17.  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) 17

18.  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

19.  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 19

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21.  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 21