Mt DNA

Mitochondrial
DNA

Jitendra Kumar
MFK-1109
Department of Fisheries Resource Management)
jitenderanduat@gmail.com
College of Fisheries, Mangalore

DNA
99%
100%
80%
60%
40%

1%

20%
0%
Mitochondrial
DNA


Nuclear DNA

Molecular Tools
Allozyme markers;

Amplified fragment length polymorphism
(AFLP);

Nuclear DNA markers (nDNA);
Tandemly repeated DNA (mini and
Mitochondrial DNA markers (mtDNA);

microsatellites);

Restriction fragment length polymorphism

Single nucleotide polymorphism (SNP);

(RFLP);
Expressed sequence tags (ESTs);
Random amplified polymorphic DNA
(RAPD);


Mitochondrial DNA
mtDNA is small genome and double stranded
circular DNA molecule
Haploid in nature
mt DNA contain 37 genes all of essential for normal
mitochondrial function
13 genes making enzymes involved
phosphorylation (ATP production )

in

oxidative

process involved use of oxygen and simple sugars to form
ATP

Nuclear DNA vs. Mitochondrial DNA
• Nuclear DNA

• Mitochondrial DNA

– found in nucleus of the cell
– 2 sets of 23 chromosomes

– found in mitochondria of the cell
– each mitochondria may have
several copies of the single
mtDNA molecule

– maternal and paternal
– can "discriminate between
individuals of the same
maternal lineage“
– double helix
– bounded by a nuclear
envelope
– DNA packed into chromatin

– maternal only
– cannot "discriminate between
individuals of the same maternal
lineage“
– Circular
– free of a nuclear envelope
– DNA is not packed into chromatin


Nuclear DNA vs. Mitochondrial DNA


Maternal Inheritance of mtDNA
During fertilization, the sperm
only contributes its nucleus
(23 chromosomes)

Mitochondria of the sperm cell are located at the
mitochondrial sheath which is destroyed upon
fertilization
Only available mitochondria (mtDNA) is that of the
mother's; this is why mtDNA is of maternal origin

Maternal Inheritance of mtDNA


The Mitochondrial Genome
• 16,569 base pairs (bp) in length (16-18 kbp)

• encodes 37 genes, 13 proteins, 22 tRNAs, and 2 rRNAs
two general regions:
– coding region: responsible for the production of various biological
molecules involved in "cellular respiration"

– control region: responsible for the regulation of the mtDNA
molecule

• It evolves faster than nuclear DNA
(Brown et al. 1982),
• Probably due to inefficient replication repair
(Clayton 1984)
• Mitochondrial DNA is maternally inherited in most
species.
(Gyllesten et al. 1991)

• Generally mitochondrial DNA does not recombine
(Hayashi et al. 1985)
Though some evidence of recombination events has recently
been reported
(Eyre-Walker et al. 1999, Hagelberg et al. 1999).

Molecular markers also show significant
promise for aquaculture applications
(i) In comparison of hatchery and wild stocks;

(ii) Genetic identification and discrimination of hatchery stocks;
(iii)Monitoring inbreeding or other changes in the genetic variation;
(iv) Assignment of progeny to parents through genetic tags;

(v) Identification of quantitative trait loci (QTL) and use of these
markers in selection programmes;
(vi)Assessment of successful implementation of
manipulations such as polyploidy and gynogenesis.

genetic

Source: (Magoulas, 1998 ; Davis and jitenderanduat@gmail.com
Hetzel, 2000; Fjalestad et al., 2003; Subasinghe et al.,
2003)

Uses for mtDNA in Forensics
•mtDNA will be used when "biological evidence
may be degraded [i.e. charred remains] or in small
quantity“
•Cases in which evidence consists only of:
–hairs
–bones
–Teeth
•Missing Persons Cases (use of skeletal remains)
•Establishing Individuals as suspects (hair evidence)

Methods for mtDNA analysis
DNA Extraction
• cellular homogenate is
“exposed to a mixture
of organic chemicals
that separate the DNA
from other biological
molecules, such as
proteins”
• mixture is spun in a
centrifuge

• DNA settles
• top layer is filtered and concentrated
• DNA sample is now purified

Polymerase Chain Reaction (PCR) Amplification
• PCR is a “procedure that
makes many copies of a small
amount of DNA.”
• DNA is heated at 94 C to
separate the two strands of the
DNA double helix in the
sample
• new DNA strands are then
made from the template
(initially separated strands) of
DNA
by
using
DNA
polymerase, primers, and free
nucleotides
• the process is repeated
multiple times, doubling the
amount of DNA after each
cycle

(PCR) Amplification


Post amplification Purification and Quantification
• Purification is performed
“using filtration devices
that remove the excess
reagents used in the PCR
from the sample.”

• Quantification is performed “using
capillary electrophoresis (CE),”
which compares the amount of DNA
in the PCR product to “a known DNA
standard
to
determine
the
concentration of the DNA in the
PCR-amplified sample.”


DNA Sequencing
• Gel Electrophoresis:
– DNA products are separated by length of bp
– pore size of the gel influence how far the
DNA fragments will travel when placed in
an electric field
– smaller fragments will travel faster and
appear further from the wells in the gel
– larger fragments will travel slower and
appear closer to the wells in the gel
– fluorescent detector “records the emitted
wavelength of the fluorescent dyes on each
base as the fragments travel past the
detection area of the instrument”
– a chromatogram is generated, showing
the colors of the labeled fragments
– “the sequence of the mtDNA is determined
from a series of cycle sequencing
reactions”

Data Analysis
mtDNA sequences are generated by a computer and
edited by a DNA examiner to obtain the final sequence
Difference(s) is/are recorded by comparing the finalized
sequence to the Anderson reference sequence

If sequence concordance (“the presence of the same base
or a common base at every position analyzed”) is
observed,
then both mtDNA samples could be considered as
originating from the same source

Functions of mtDNA in Ichthyotaxonomy
Individual identification

Mixed Stock Fishery Analysis(MSFA)
To identify the phylogenetic relationship b/w the different
species of fishes
To identify and arrangement of the species and stock on the
basis of their genetic affinity

To identify the genetic diversity with in the stock & cultured
species

References
•
•
•

•

•

•

Ibrahim Okumus and Y. Çiftci / Turk. Turkish Journal of Fisheries and Aquatic Sciences 3: 5179 (2003)
ARIAGNA LARA,* JOSE LUIS PONCE DE, Molecular Ecology Resources (2010)
10, 421–430
Vallone, P.M., Just, R.S., Coble, M.D., Butler, J.M., Parsons, T.J. (2004) A multiplex allelespecific primer extension assay for forensically informative SNPs distributed throughout the
mitochondrial genome. Int. J. Legal Med., 118: 147-157. [Protocol for 11plex SNP assay
developed at NIST] [Genotyper macro for mtSNP 11plex]
Coble, M.D., Just, R.S., O'Callaghan, J.E., Letmanyi, I.H., Peterson, C.T., Irwin, J.A.,
Parsons, T.J. (2004) Single nucleotide polymorphisms over the entire mtDNA genome that
increase the power of forensic testing in Caucasians. Int. J. Legal Med., 118: 137-146.
Coble, M.D. (2004) The identification of single nucleotide polymorphisms in the entire
mitochondrial genome to increase the forensic discrimination of common HV1/HV2 types in
the Caucasian population. PhD dissertation, George Washington University, 206 pp.
www.google.co.in/mtdna/wikipedia/in


Mt DNA

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