Location And Structure Of mtDNA

The vast majority of the human genome is located within the nucleus of each cell (see Figure 2.3, Table 10.1). However, there is a small, circular genome

Tabe 10.1

Comparison of human nuclear DNA and mitochondrial DNA markers.

Characteristics

Nuclear DNA (nucDNA) Mitochondrial DNA (mtDNA)

Size of genome Copies per cell

Percent of total DNA content per cell

Structure

Inherited from Chromosomal pairing Generational recombination Replication repair Unique

Mutation rate Reference sequence

~3.2 billion bp

2 (1 allele from each parent)

Linear; packaged in chromosomes

Father and Mother

Diploid

Unique to individual (except identical twins)

Described in 2001 by the Human Genome Project

Circular

Mother Haploid No No

Not unique to individual (same as maternal relatives)

At least 5-10 times nucDNA

Described in 1981 by Anderson and co-workers found within the mitochondria, the energy-producing cellular organelle residing in the cytoplasm. The number of mitochondrial DNA (mtDNA) molecules within a cell can vary tremendously. On average there are 4-5 copies of mtDNA molecules per mitochondrion with a measured range of 1-15 (Satoh and Kuroiwa 1991). Because each cell can contain hundreds of mitochondria (Robin and Wong 1988), mathematically there can be up to several thousand mtDNA molecules in each cell as in the case of ovum or egg cells. However, the average has been estimated at about 500 in most cells (Satoh and Kuroiwa 1991). It is this amplified number of mtDNA molecules in each cell that enables greater success (relative to nuclear DNA markers) with biological samples that may have been damaged with heat or humidity. Consider though that mtDNA makes up less than one percent (about 0.25%) of the total DNA content of a cell if we assume that there are 1000 copies of mtDNA (16 569 bp) in a cell and two copies of nuclear DNA (3.2 billion bp).

Mitochondrial DNA has approximately 16569 base pairs and possesses 37 'genes' that code for products used in the oxidative phosphorylation process or cellular energy production. There is also a 1122bp 'control' region that contains the origin of replication for one of the mtDNA strands but does not code for any gene products and is therefore referred to sometimes as the

16024

16365 1 73

16365 1 73

HV1

HV2

Control region (D-loop) O

16024'

Control region (D-loop) O

Heavy (H) strand

Heavy (H) strand

Forensic Amp Reapting Nucletides

COIII

1/16569.

F 12S " rRNA

16S rRNA

'16569' bp

COIII

ATP6 r COII ATP8U COII D

F 12S " rRNA

16S rRNA

1/16569.

'16569' bp

M ND2

9 bp deletion

22 tRNAs 2 rRNAs 13 genes

M ND2

9 bp deletion

'non-coding' region. The total number of nucleotides in a mitochondrial genome (mtGenome) can vary due to small mutations that are either insertions or deletions. For example, there is a dinucleotide repeat at positions 514—524, which in most individuals is ACACACACAC or (AC)5 but has been observed to vary from (AC)3 to (AC)7 (Bodenteich et al. 1992, Szibor et al. 1997). The 37 transcribed 'genes' of mtDNA found in the 'coding region' include 13 proteins, two ribosomal RNAs (rRNA), and 22 transfer RNAs (tRNA) (Figure 10.1). The nucleotide positions for each coding and non-coding segment of the mtGenome are indicated in Table 10.2. Note that the genes are very tightly

Figure 10.1

Schematic showing the circular mitochondrial DNA genome (mtGenome). The heavy (H) strand is represented by the outside line and contains a higher number of C-G residues than the light (L) strand. The 37 RNA and protein coding gene regions are abbreviated around the mtGenome next to the strand from which they are synthesized (see Table 10.2). Most forensic mtDNA analyses presently examine only HV1 and HV2 in the non-coding control region or displacement loop shown at the top of the figure. Due to insertions and deletions that exist around the mtGenome in different individuals, it is not always 16569bp.

Table 10.2 Mitochondrial DNA information and genes.

Nucleotide Position

Strand Transcribed

Abbreviation Description

Non-coding

16024-16569, 1-576 16104-16569, 1-191 16158-16172 531-568

D-loop control region 1122

OH replication origin (H-strand) 658

D-loop termination signal 15

H-strand transcription promoter 38

1122

Nucleotide Position

Strand Transcribed

Abbreviation

Description

Size (bp)

Non-coding

577-647

H

F

tRNA phenylalanine

71

648-1601

H

12S

12S rRNA

954

1602-1670

H

V

tRNA valine

69

1671-3229

H

16S

16S rRNA

1559

3230-3304

H

L1

tRNA leucine 1

75

3305-4263

H

ND1

NADH dehydrogenase 1

959

4263-4331

H

tRNA isoleucine

69

4329-4400

L

Q

tRNA glutamine

72

4401

non-coding

1

1

4402-4469

H

M

tRNA methionine

68

4470-5511

H

ND2

NADH dehydrogenase 2

1042

5512-5579

H

W

tRNA tryptophan

68

5580-5586

non-coding

7

7

5587-5655

L

A

tRNA alanine

69

5656

non-coding

1

1

5657-5729

L

N

tRNA asparagine

73

5730-5760

OL

L-strand origin

31

31

5761-5826

L

C

tRNA cysteine

66

5826-5891

L

Y

tRNA tyrosine

66

5892-5900

non-coding

9

9

5901-7445

H

COI

Cytochrome c oxidase I

1545

7445-7516

L

S1

tRNA serine 1

72

7517

non-coding

1

1

7518-7585

H

D

tRNA aspartic acid

68

7586-8294

H

COII

Cytochrome c oxidase II

709

8295-8364

H

K

tRNA lysine

70

8365-8572

H

ATP8

ATP synthase 8

208

8527-9207

H

ATP6

ATP synthase 6

681

Nucleotide Position Strand Abbreviation Description

Size

Non-coding

Transcribed

(bp)

9991-10058

10059-10404

10405-10469

10470-10766

10760-12137

12138-12206

12207-12265

12266-12336

12337-14148

14149-14673

14674-14742

14743-14746

14747-15887

15888-15953

15954

15955-16023

COIII G

ND3 R

ND4L

ND5 ND6 E

Cyt b T

Cytochrome c oxidase III 784

tRNA glycine 68

NADH dehydrogenase 3 346

tRNA arginine 65

NADH dehydrogenase 4L 297

NADH dehydrogenase 4 1378

tRNA histidine 69

tRNA serine 2 59

tRNA leucine 2 71

NADH dehydrogenase 5 1812

NADH dehydrogenase 6 525

tRNA glutamic acid 69

non-coding 4

Cytochrome b 1141

tRNA threonine 66

non-coding 1

tRNA proline 69

packed with only 55 nucleotides in the 15 447 bp of the coding region not being Table 10.2 used to transcribe a protein, rRNA, or tRNA molecule. Thus, the genes within (Continued) mtDNA are economically packaged with no introns and none or only a few non-coding nucleotides between the coding regions.

An asymmetric distribution of nucleotides gives rise to 'light' and 'heavy' strands when mtDNA molecules are separated in alkaline CsCl gradients (Scheffler 1999). The 'heavy' or H-strand contains a greater number of guanine nucleotides, which have the largest molecular weight of the four possible nucleotides, than the 'light' or L-strand. Replication of mtDNA begins with the H-strand in the non-coding 'control region', also known as the displacement loop or D-loop (Figure 10.1). A total of 28 gene products are encoded from the H-strand while the L-strand transcribes eight transfer RNAs (tRNAs) and an enzyme called ND6 (Table 10.2).

Since the D-loop does not code for gene products, the constraints are less for nucleotide variability and polymorphisms between individuals are more abundant than in similar sized portions of the coding region. More simply, there can be differences in the D-loop region because the sequences do not code for any substances necessary for the cell's function. Most of the focus in forensic DNA studies to date has involved two hypervariable regions within the control region commonly referred to as HV1 and HV2, which will be described in more detail below.

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