Discovery of Three Newly Described Single Nucleotide Polymorphisms in Mitochondrial DNA Hypervariable Region I (HVI) and Estimation of Variants and Haplotypes Encompassing Nucleotide Positions 16024-16365

The mitochondrial DNA (mtDNA) is a small circular genome located within the mitochondria in the cytoplasm of the cell. The mitochondrial genome can be divided into two sections: a large coding region, which is responsible for the production of various biological molecules involved in the process of energy production in the cell, and a smaller 1.2 kilobase pair fragment, called the control region. It is found to be highly polymorphic and harbors three hypervariable regions HVI, HVII and HVIII [1]. Mitochondrial DNA comprising of about 37 genes coding for 22 tRNAs, two rRNAs and 13 mRNAs are a small circle of DNA [2]. Mitochondrial DNA does not recombine and thus there is no change between parent and child, unlike nuclear DNA [3,4]. There is more sequence divergence in mitochondrial than in nuclear DNA [5,6]. This may be caused by a faster mutation rate in mtDNA that may result from a lack of repair mechanisms. Sequencing of highly polymorphic segments of the control region of mitochondrial DNA (mtDNA) is today a routine method of analysis of biological traces which are not suitable for STR analysis due to insufficient concentration of nuclear DNA or heavy degradation processes [7,8]. A promising approach in this context seems to be analysis of selected single nucleotide polymorphisms (SNPs) that are useful for identification purposes. The aim of this study was to sequence the portion of the noncoding region of mtDNA in order to ascertain the degree of variation present in this fragment and to find those particular polymorphic positions that fulfill the conditions necessary for their future application in the identification process.


Introduction
The mitochondrial DNA (mtDNA) is a small circular genome located within the mitochondria in the cytoplasm of the cell. The mitochondrial genome can be divided into two sections: a large coding region, which is responsible for the production of various biological molecules involved in the process of energy production in the cell, and a smaller 1.2 kilobase pair fragment, called the control region. It is found to be highly polymorphic and harbors three hypervariable regions HVI, HVII and HVIII [1]. Mitochondrial DNA comprising of about 37 genes coding for 22 tRNAs, two rRNAs and 13 mRNAs are a small circle of DNA [2]. Mitochondrial DNA does not recombine and thus there is no change between parent and child, unlike nuclear DNA [3,4]. There is more sequence divergence in mitochondrial than in nuclear DNA [5,6]. This may be caused by a faster mutation rate in mtDNA that may result from a lack of repair mechanisms. Sequencing of highly polymorphic segments of the control region of mitochondrial DNA (mtDNA) is today a routine method of analysis of biological traces which are not suitable for STR analysis due to insufficient concentration of nuclear DNA or heavy degradation processes [7,8]. A promising approach in this context seems to be analysis of selected single nucleotide polymorphisms (SNPs) that are useful for identification purposes. The aim of this study was to sequence the portion of the noncoding region of mtDNA in order to ascertain the degree of variation present in this fragment and to find those particular polymorphic positions that fulfill the conditions necessary for their future application in the identification process.

Population data
Unrelated 324 healthy blood samples from the middle and south of Iraq provinces. The age of the donors was between 20 to 30 years old, due to the mtDNA will get more mutation after 30 years old in human (Figure1).

Extraction of DNA
Blood samples were taken by FTA (FTA TM paper DNA extraction) cards and sent it to the genetics laboratory.

Amplification of mitochondrial DNA
A portion of noncoding region encompassing positions from 16024 to 16365 amplified in accordance with the Anderson reference sequence [9] GenBank: J01415. This portion of DNA was amplified in two primers: the first one is HVI-F (16024-16045) 5'-TTCTTTCATGGGGAAGCAGATT-3' and the second HVI-R has a position (16365-16345) 5'-AGTCAAATCCCTTCTCGTCCC-3'. Add in 20 μL of Master Mix into PCR tube. Change the pipette tip and add 20 μL of Primer Mix into PCR tube. Add 10 μL of extracting DNA into the PCR tube after changing the pipette tip again. Allow all the liquid settles at the bottom of the tube, and not elsewhere. Check the volume in the PCR tube using the PCR tube with 50 μL in it. 95°C hold for 10 minutes, 30 cycles of: 94°C for 30 seconds, 52.5°C for 30 seconds, 65°C for 1 minute. 72°C hold for 10 minutes. 4°C hold, ∞ infinity is the cycling protocol for amplification of mtDNA PCR. scrutinize the products of the amplification. Gel electrophoresis is used to do this. Here the electricity is used to force the movement of DNA fragments through a special gel. Since the DNA is negatively charged, it will move to the positive electrode in the electric field. The electric force causes the shorter portions of the DNA to move faster than the longer ones.

Purification of mitochondrial DNA
Using a special binding buffer, EZ-10 spin column purification kits using a silica gel membrane selectively absorb up to 10 μg of DNA fragments. Since Nucleotides, oligos (<40-Meir), enzymes, mineral oil and other impurities do not EZ-10 Spin Column bind to the membrane, they are just removed. Here the DNA fragments can be separated in small amounts and can be used in further applications without any further treatment.

Cycle sequencing and sequence analysis
The DNA Sequencing of the PCR products was done using the BigDye TM Terminator. Utilizing POP-7 polymer (Applied Biosystems) polymer lot number 1206453. The separation of the cycle sequencing products was carried out. Detection was by using the ABI 3730×L DNA Analyzer, cap array size 96, cap array length 50. The reference sequence described by Anderson [9] was compared to the data observed.

Cycle sequencing interpretation guidelines
Within the noncoding region Mitochondrial DNA, sequencing results are studied from a consensus sequence derived from multiple sequence results. Data were analyzed by Sequencher TM (SEQUENCHER TM 4.7 User Manual for Windows © 1991-2007) [10] and aligned with the Anderson sequence using the sequence Navigator software. They are accepted by stating the nucleotide position followed by the code for the polymorphic base (for example, 263G).

Statistical analysis
Genetic diversity for the analyzed DNA fragment was calculated according to the formula: D=1-∑p².
Where p: frequencies of the observed haplotypes [11].

Results
The basic aim of this work was to assess the degree of variation characterizing a selected segment of the noncoding region of mtDNA. The study enabled identification of 103 different haplotypes and 28      16030 16032 16038 16041 16042 16051 16052 16064 16084 16105 16117 16119 16120 16124 16129 16141 16154 16156 16197 16221 16227 16253 16266 16303 16312 16334 16346 16349 Anderson C  T  A  A  G  A  C  T  G  T  T  A  A  T  G  A  T  G  C  C  A  A  C  G  A  T  G  Comparative analysis of our results with previously published Iraqi data revealed significant differences in varying patterns [14,15]. This observation supports the thesis that different SNP-type polymorphisms can be strongly associated with a given population. Table 3 presents a summary of the Iraqi data in comparison with other global populations [16][17][18]. Significant assistance for the research was provided by Mitomap computer database, which contains information concerning human mtDNA [19]. This database includes data about currently known variable positions, their possible association with genetic diseases, and references to the literature. There is also a simple program called Mito Analyzer attached to the database which enables convenient access to information concerning polymorphic positions.

Discussion
The presence of more than one mitochondrial DNA (mtDNA) variant within a cell, tissue, or individual is emerging as an important component of eukaryotic genetic diversity. Yet the variations may vary from person to person. Therefore, to understand the polymorphisms at different sites, it is of critical importance to investigate the sequencing of mtDNA coding region's transmission. The first entire human mtDNA sequence was explained by Anderson et al. [9]. Cambridge Reference Sequence CRS is the name given to the published sequence used as a reference standard. For the coding function of the analyzed DNA fragment. Sequencing of the mitochondrial DNA coding region in the 300 unrelated donors showing a new polymorphic position 16046, 16105 and 16141 are described may in future be suitable sources for identification purpose. Sequence the portion of the noncoding region of mtDNA is in order to verify the degree of variation present in the fragment. It is also to identify those particular polymorphic positions that meets the conditions necessary for their future use in the identification process.
Earlier writings for a detailed description of molecular biology, genetics, sequence determination procedures, interpretation practices, and utility of mtDNA sequence analysis in forensic casework and human identification [20,21]. In cases where there is an abundance in the sample, for example mass graves in mass disasters, there are newly discovered forensically validated methods such as ESI-MS [22] Certainly, all such applications should have a strong grasp of the mtDNA variation that is present in the populations concerned. As an example, describing and frequency estimates of common mtDNA types and any population sub-structuring must be at hand [23]. Consequently, this may also increase the pool of samples with degraded and insufficient nuclear DNA for mitochondrial DNA analysis.

Conclusion
It will become easier to handle minute amounts of DNA or DNA