Mitochondrial DNA

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Mitochondrial DNA

• Jason Linville, Ph.D.
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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Mitochondrial DNA

• MtDNA background
• PCR of mtDNA
• Sequencing mtDNA
• Human identification
• Species identification

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Mitochondrial DNA

• Mitochondria - the powerhouse of the cell.

Mitochondria have their own DNA
Mitochondria
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Mitochondrial DNA
Ring of DNA YES
Double Helix YES
Chromosomes NO
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Mitochondrial DNA
Mitochondrial DNA is 16,569 letters long.
(compared to 3 billion in nuclear DNA)
D Loop
There is a 900 base pair region where the
sequence is different among individuals.
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Mitochondrial DNA

• Nuclear DNA vs. Mitochondrial DNA

Double Helix
Double Helix
One Ring
46 Chromosomes
Multiple copies in each mitochondria
One copy per cell
Multiple mitochondria in each cell
MtDNA used for old or degraded samples
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Mitochondrial DNA

• Advantage of mtDNA

• Can be amplified from old or degraded samples
  when STR analysis is not possible.

Disadvantage of mtDNA (sort of)

• Not specific to an individual.
• Maternal relatives will have same sequence.
• Random individuals may have same sequence.

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Mitochondrial DNA

• mtDNA is maternally inherited

male
female
2
1
6
4
5
3
10
9
8
7
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Mitochondrial DNA

• mtDNA is maternally inherited

male
female
2
1
6
4
5
3
10
9
8
7
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Mitochondrial DNA

• mtDNA is maternally inherited

male
female
2
1
4
6
8
3
5
7
14
11
12
10
9
13
15
7
6
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Mitochondrial DNA

• mtDNA is maternally inherited

male
female
2
1
4
6
8
3
5
7
14
11
12
10
9
13
15
7
6
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Mitochondrial DNA

• Uses of mtDNA

• Can be used to help identify the source of
  degraded evidence if STRs cannot be amplified.
• Can be used to help identify human remains by
  comparing DNA to maternal relative.
• Can be used to identify the species of an unknown
  sample.

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Mitochondrial DNA gt Sequencing

• Analysis of mtDNA

• PCR amplification
• Yield Gel
• Clean PCR Product
• Sequencing Reaction
• Clean Sequencing Product
• Load and run on 310

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Forensic DNA Analysis
One Segment of DNA 10 million copies
Sample Buccal swab Blood stain Semen stain
Agarose Gel
Cycle Sequencing
PCR
One Segment of Sequenced DNA Millions of copies
Extraction
All DNA Hundreds of copies
Run on Genetic Analyzer
PCR
16 Segments of DNA 10 million copies
Run on Genetic Analyzer
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Mitochondrial DNA gt Sequencing

• PCR Amplification

• PCR reaction similar to STR amplifications.
• In mtDNA, typically one region amplified (one
  primer set)

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Mitochondrial DNA gt Sequencing

• PCR Amplification

• Components can be added individually.
• May be premixed w/ Taq (Promega PCR MasterMix)
  only add primers and DNA
• Commonly used primers are published and can be
  made by anyone.

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Mitochondrial DNA gt Sequencing

• Yield Gel

Band Sample worked
No band Sample failed
Samples run on ethidium bromide stained gel. This
tells you if the amplification was successful.
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Mitochondrial DNA gt Sequencing

• Yield Gel

• Ethidium bromide is intercalating dye.
• Fluoresces when hit with UV light.
• Indicates success of reaction.

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Mitochondrial DNA gt Sequencing

• Cleaning PCR Product

• Amplified samples cleaned using silica column
  (similar to Qiagen extraction).
• Removes all primers, left over Taq, etc.
• Small amount of clean PCR product used for
  sequencing reaction.

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Sequencing

• Sequencing DNA

F15
PCR product (millions of copies)

• Primer anneals to ss DNA. Often, it is one of the
  primers used for PCR.
• Extension by DNA polymerase
• Thermal cycling similar to PCR (see sheet)

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Sequencing gt Cycle Sequencing

• dNTPs and dideoxyNTPs (ddNTP) added.
• Each ddNTP labeled with a different color
• (A green, T red, G yellow, C blue)

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Sequencing gt Cycle Sequencing

• Results in termination of product when ddNTP is
  added.
• Ending position varies because normal dNTPs are
  also in the mixture.

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Sequencing gt Cycle Sequencing
PCR Product GCTAGCGATGTACGATTAGCTAGCGCT
CS Product GCTAGCGATG
F15
CS Product GCTAGCGATGT
F15
CS Product GCTAGCGATGTA
F15
CS Product GCTAGCGATGTAC
F15
Only the last base will be a ddNTP. Only the
last base will be labeled.
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Sequencing gt Cycle Sequencing
Since millions of copies are there at the start
of the reaction, million of copies of random
lengths will occur with each cycle.
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Sequencing gt Cycle Sequencing
GGTACTTT
GGTACTT

• Separated by length
• 1 bp difference

GGTACT
GGTAC
GGTA
GGT
GG
G
F15
F15
F15
F15
F15
F15
F15
F15
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Sequencing gt Cycle Sequencing
For mtDNA Sequence is examined
Different colored peaks correspond to a different
base.
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Sequencing gt Cycle Sequencing

• Cleaning Sequencing Product

• Sample run through sephadex column
• Impurities cling to sephadex, cleaned product
  washes through.
• Dried down to eliminate water

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Sequencing gt Cycle Sequencing

• 310 Separation and Analysis

• Set up same way as STR analysis
• Cleaned cycle sequncing product
• Formamide
• Size Standard is not needed.
• Allelic Ladder is not needed

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Mitochondrial DNA
Human Identification

• Within the control region, there are two highly
  variable regions (1 - 3).
• Human hypervariable region (HVI, HVII)

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Mitochondrial DNA
Haplotype often reported as differences from
reference sequence.

• Also called Anderson sequence or Cambridge
  reference sequence
• Human mtDNA first sequenced in 1981 by Anderson,
  et al. in Cambridge.

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Mitochondrial DNA gt Anderson Sequence
141
151
161
Anderson CTGCCTCATT CTATTATTTA TCGCACCTAC
Sample 1 CTGCTTCATT CTATTATTTG TCGCACCTAC
Sample 2 CTGCCTCATT CTATTATCTA TCGCACCGAC
Sample 3 CTGCCTCGTT CTATCATTTA TCCCACCTAC
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Mitochondrial DNA gt Anderson Sequence
141
151
161
Anderson CTGCCTCATT CTATTATTTA TCGCACCTAC
Sample 1 CTGCTTCATT CTATTATTTG TCGCACCTAC
Sample 2 CTGCCTCATT CTATTATCTA TCGCACCGAC
Sample 3 CTGCCTCGTT CTATCATTTA TCCCACCTAC
Sample 1 145 T, 160 G
Sample 2 158 C, 168 G
Sample 3 148 G, 155 C, 163 C
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Mitochondrial DNA
Species Identification

• Cytochrome b used
• Being a protein coding gene, there is not much
  variation within a species (lt1)
• Enough change that even closely related species
  will have more significant sequence variation
  (gt3)

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Mitochondrial DNA
Mitochondrial DNA is 16,569 letters long.
(compared to 3 billion in nuclear DNA)
D loop Control region
There is a 900 base pair region where the
sequence is different among individuals.
cyt b gene
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Sequencing gt Cycle Sequencing
For mtDNA Sequence is examined
Different colored peaks correspond to a different
base.
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(No Transcript)
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Mitochondrial DNA
Heteroplasmy

• Different haplotypes in the same individual will
  result in two peaks at one location.
• Can be difficult to distinguish from background.
• May also be due to mixture