mt dna

Mitochondrial DNA

Prepared byJitendra Kumar

MFK-1109Department of Fisheries Resource Management)

College of Fisheries, Mangalorejitenderanduat@gmail.com

Mitochondrial DNA Nuclear DNA

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Molecular Tools

Allozyme markers;

Nuclear DNA markers (nDNA);

Mitochondrial DNA markers (mtDNA);

Restriction fragment length polymorphism

(RFLP);

Random amplified polymorphic DNA

(RAPD);

Amplified fragment length polymorphism

(AFLP);

Tandemly repeated DNA (mini and

microsatellites);

Single nucleotide polymorphism (SNP);

Expressed sequence tags (ESTs);

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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 in oxidative phosphorylation (ATP production )

process involved use of oxygen and simple sugars to form ATP

Mitochondrial DNA

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jitenderanduat@gmail.com

• Nuclear DNA

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

– maternal and paternal– can "discriminate between

individuals of the same maternal lineage“

– double helix– bounded by a nuclear

envelope– DNA packed into chromatin

• Mitochondrial DNA

– found in mitochondria of the cell– each mitochondria may have

several copies of the single mtDNA molecule

– 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

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Nuclear DNA vs. Mitochondrial DNA

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jitenderanduat@gmail.com

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

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Maternal Inheritance of mtDNA

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• 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

The Mitochondrial Genome

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• 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). jitenderanduat@gmail.com

(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 genetic manipulations such as polyploidy and gynogenesis.

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

Molecular markers also show significantpromise for aquaculture applications

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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)

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jitenderanduat@gmail.com

• 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

DNA Extraction

Methods for mtDNA analysis

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• 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

Polymerase Chain Reaction (PCR) Amplification

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(PCR) Amplification

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• 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.”

Post amplification Purification and Quantification

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• 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”

DNA Sequencing

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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

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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

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References

• Ibrahim Okumus and Y. Çiftci / Turk. Turkish Journal of Fisheries and Aquatic Sciences 3: 51-79 (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 allele-specific 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

jitenderanduat@gmail.com

jitenderanduat@gmail.com