Forensic Biology by Richard Li

1. Lecture 7: Mitochondrial DNA Profiling Forensic Biologyby Richard Li

2. Outline • Mitochondrial DNA and forensics • Human mitochondrial genome • Polymorphic regions • DNA sequencing • Interpretation of Results • Identifying Tsar Nicholas and family • The mystery of Anna Anderson

3. mt DNA and Forensics • 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

4. mt DNA and Forensics • 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

5. Human Mitochondrial Genome • Mitochondria = Sub-cellular organelles which are generated ATP from breakdown of food

6. Human Mitochondrial Genome • 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

8. Polymorphic Regions • 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

10. Polymorphic Regions • Mitotype reporting • Reported against rCRS • Sequence polymorphisms; requires DNA sequencing • 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)

11. DNA Sequencing • Current method developed by Sanger • Uses modified nucleotides called ddNTPS • Dideoxynucleotide triphosphates (ddATP, ddGTP, ddTTP, ddCTP)

12. DNA Sequencing • 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. Chromas Lite CODISmt

15. Interpretation of Results • 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. Identifying Tsar Nicholas & Family • 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. Identifying Tsar Nicholas & Family • 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. Identifying Tsar Nicholas & Family • 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

20. Identifying Tsar Nicholas & Family • 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. Mystery of Anna Anderson