chapter 21: mitochondrial dna profiling. dna found in mitochondria bacteria-like circular no...
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Chapter 21: Mitochondrial DNA Profiling
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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
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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
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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
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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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