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In cells of human beings Deoxyribonucleic acid (DNA) is found in the nucleus as well as in mitochondria. The mitochondrial organelle is referred to "power house of cell" because molecular products produced by it, provides the energy to the cell and responsible for oxidative phosporylation. Each nucleus in the cell has 2 copies of DNA i.e. one from each parent. But it's not the case with mitochondria. Human cells have several mitochondria and the DNA in each mitochondrion is copied number of times (Goodwin.W, 2007). Other variable features between these are: -
a) Nuclear DNA is different for each individual(except identical twins) and provides a higher discrimination power than mt-DNA (Butler,2005). Mt-DNA is the same for all maternal relatives, so any maternal sample can provide the same information.
b) Codon for tryptophan in mt-transcription is UGA while in nuclear DNA UGA is referred as a stop Codon (Butler &Coble 2006).
c) High mutation rate occurs in mt-DNA because of the less efficient repair mechanism. Continuous proof reading mechanism by polymerase enzyme reduces the mutation rate in nuclear-DNA
Mt-genome makes approximately 16569 base pairs and has 37 genes which codes for the different products in the mitochondrial cell.
This above stated fact makes it enormously useful for solving many of the violent crime cases where DNA was highly degraded (like by the environmental factors). Apart from that the mt-DNA analysis is useful in the cases where nuclei DNA is absent like hair shafts, teeth and bones (Butler, 2005). STR (short tandem repeats) analysis do not work well in degraded sample however the power of discrimination is very low in mt-DNA analysis. The STR product length is very small (upto 350 base pairs) which provides more detailed study (Budowle & Daal, 2008). The detection limit in some of the samples is up to 33-330 femtograms of mt-DNA genome (Allen et al. 1998).
Mitochondrion DNA and Y chromosomes is specific lineage marker. Mt-DNA is inherited from the mother. The reason behind this is when the sperm head fused or fertilizes an egg, the other portion of the sperm like midsection and tail are left behind (usually have 50-70 number of mitochondria).
This left over portion is rich in mt-DNA which never reaches inside of egg (Goodwin, 2007). In consequence, all the mt-DNA comes from egg (1000's of mitochondria) i.e. mother which ultimately makes it highly useful for providing maternal relationship in many of the important cases, missing person and mass disaster cases. In one of the study in England shows that complete absence of parental mt-DNA inheritance is impossible. In some of the cases they do inherent the parental mt-DNA (Schwartz M, 2002). This is purely based upon changes, as millions of mt-DNA samples showed maternal inheritance.
Study of mt-DNA mutation proves effective in medical field and helps scientist to link with various diseases. Comparison between the different species is made by mt-DNA in some of the cases (Butler, 2005).
2. Mitochondrial DNA Genome:
Mt-DNA is circular in structure and has 16569 base pairs which make it less prone to exo-nucleases and ultimately help in forensic DNA profiling. But the total amount of nucleotide in mt-Genome varies. The main reason of variation is mutation that either causes deletion or insertion in the actual genome (Butler & Coble 2006). This can change the total number of mt-Genome. The common example for this is that many of the individual have 5 times repetition of (AC) base pairs but it varies from 3 to 7 repetitions of this base pair (Butler, 2005) and age independent.
Figure 1. mt-DNA genome showing 37 RNA and protein coding region. HVI and HVII are the regions for forensic interest. These hyper variable regions are located in displacement loop; Photo Courtesy: (Holland & Huffine, 2001)
Control region: Its 1122 bp and responsible for the replication of mt-DNA strands. It does not code for the gene products and also known as non coding region. Transcribed genes code for 13 different proteins, 22 for t-RNA and 2 for r-RNA (Butler, 2005). The coding genes are closely packaged to each other which lead to exclusion of introns and reduce the variability.
When these mt-DNA molecules separated in the CsCl gradients then this invariable nucleotide leads to heavy (guanine content is high as compared to light) and light chains (Holland & Huffine, 2001).
H Strand (Displacement loop)
Replication of mt-DNA (in control region)
28 genes products
D-loop is variable in nature because it does not code for any products.
Transcribes 8t-RNA, ND6 enzyme
This in total makes 37 genes product.
Table1: Information of some important mt-DNA genome and genes. Table courtesy (Butler, 2005)
Replication origin (H-strand)
D-loop termination signal
H-stand transcription promoter
According to Dr.Amarjit chahal (2010) another feature of mt-DNA is heteroplasmy. It's the presence of more than one mt-DNA in an individual. Various factors give rise to heteroplasmy and it can be more than one mt-DNA type in a single tissue or the two tissue samples of the suspect have different heteroplasmy or sample may contain homoplasmy.
It's usually of two types and increases the power of discrimination.
1. Point heteroplasmy: It is observed when the 2 nucleotide is present at single site which in the analysis result shows 2 overlapping peaks.
2. Length heteroplasmy: It occur in the c-stretches or polycytosine stretches (homopolymeric) of hyper variable region 1 (16184-16193) and 2 (303-310) (Lee.H et al. 2004). In Anderson reference sequence tyrosine is found in both HVI (at 16189) and HV II (at 310). In some samples this tyrosine is replaced by the cytosine which results in c stretches.
Due to sequencing method limitations it's hard to interpret ate length heteroplasmy. These various length variants cause various common diseases like diabetes mellitus (Lee.H et al. 2004).
Triplasmy was recorded in some of the cases but in very less frequencies as compare to single site heteroplasmy (Tully et al.2000). The main positions of the heteroplasmy in hyper variable region I are eight and five in hyper variable region II. The region why heteroplasmy is important for the individual identification is that it remain stable over generation and can be inherited (Butler,2005).
Another variation in mt-DNA analysis is caused by 'nuclear Pseudo genes'. Sometimes what happened is that parts of mt-Genome are migrated to nuclear genome. Most commonly this mt-DNA control region appears in 11th chromosome number and account for 7.5 percent sequence variation (Butler & Coble, 2004). It creates variation in forensic studies when genomic mt-DNA copy amplifies up instead of target mt-DNA but careful design of the primers with less PCR cycles can reduce this problem (Butler, 2005).
3. Mitochondrial-DNA analysis
The basic principle in mt-DNA analysis is to target the variation that occurs between the individual Displacement loops which is found in the control region of circular mt-DNA (Butler.JM, 2004). PCR amplify the hyper variable region I (342bp evaluated)& II (268 bp evaluated) and the results are compared with the reference sequence. When HV I and HV II failed to produce the result then the HV III region (nucleotide position 438-574) sometimes provides the valuable base for analysing (Butler,2005).
3.1 Method: -The first basic key issue in the analysis is contamination. The test sample which is to be analysed by this techniques is usually degraded so extra effort is needed to avoid contamination by proper wearing of protective clothes and keep the lab clean by continuously washing the working slab.
Extraction of the mt-DNA requires special care because the source of it like teeth, hair and bones provides very small amount of DNA and many of the samples like bones and teeth is needed for further investigation and its uses in anthropological studies (Butler, 2005).
Mt-DNA extraction from hair fibres is little bit difficult and costly therefore if Scene of Crime Officers found such kind of evidence (hair fibers) then they go for microscopic examination rather than mt-DNA extraction.
However mt-DNA in hair shaft is available by grinding tissue and extracting mt-DNA from keratin of hair. In one of the studies it shows that adding of Bovine serum albumin helps to carry out the successful PCR process by inhibiting the effects of melanin (Butler, 2005).
Quantification of extracted sample is necessary to see the availability of mt-DNA to carry out the rest of the process. Instead of comparison of quantity and fixing the ratio with nuclear DNA real time PCR provides the direct quantification of mt-DNA molecules (Butler.JM, 2004).
Amplification of the mt-DNA by PCR usually requires 35 cycles on an average which in turn increases the sensitivity and more chances of contamination. It amplifies the whole control region or specific region. The sequencing is done by Sanger method where fluorescent ddNTPs was processed with DNA. A primer binds to the specific site anneals and extension occurred by polymerase enzyme.
The concept behind including ddNTPs is that while extension process is carried out by dNTPs, ddNTPs stops some of the products to extend and marked that product because ddNTPs is labelled with different colour brighter dye. Ultimately it provides the best resolution with clear indication of the bases. Various filtration processes like removal of primers and dNTPs is done by spin filtration and enzymatic action. Fluorescent dye is removed by spin column filtration.
The key difference between STR analysis and mt-DNA analysis is that in mt-DNA sequencing, separation medium is required in multiple colour detection instruments to resolve single base and products are analysed by capillary electrophoresis.
Results are then compared with the reference sequence and comparison was made with databases to identify and estimate haplotype frequency. In NCBI database with genbank accession: M63933 the mt-DNA sequence was noted which is used to compare the new sequence. This sequence is referred as 'Cambridge Reference Sequence.
Earlier in 1981 these sequence was reported by the Anderson from the single individual and uses Bovine sequence to fill the gaps in between these reference sequence but later in 1999 placenta material was used to re-sequence.
Various results are compared by the L-strand reference sequence for example if Thymine is present at 16126 position in the CRS and the result displayed Cytosine then it would be considered as 16126Cytosine. This is the common variant which results into c-stretches in both hyper variable regions.
In many of the forensic cases the hyper variable region '1' and '2' of control region is considered and the result is matched with these reference sequences.
4. Technologies associated to mitochondrial-DNA analysis
4.1: Continuous improvement in measuring mt-DNA variation makes the technique more reliable. Earlier low resolution use of 5-6 restriction enzymes in RFLP (restriction fragment lengths polymorphism) analysis improved to high resolution analysis by PCR amplification (Butler, 2005). PCR amplifies the 9 overlapping fragment and then 12-14 restriction enzymes act on it to produce good profile of target sequence and ultimately use of fluorescent dye to sequence the mt-DNA (Holland & Huffine, 2001).
4.2: Various approaches are made in quantification of mt-DNA in the mixture of human and non-human DNA source. In forensic investigation it's difficult to identify the source of DNA so one of the approaches was taken to quantify sample mixture down to 1.7fg mt-DNA (Tobe et al. 2008). It uses cytochrome b and 12S ribosomal gene on the mt-Genome.
The specific fragments are quantifed using SYBR Green and amplifed. They take 24 human and 27 non human sample into account for quantification analysis.
4.3: Whole mt-Genome Research- Sequencing the whole mt-Genome helps in reconstruction of phylogentics of human and helps in understanding genome evolution and is useful in forensic identification (Naraa.A et al. 2009) The specific arrangement of genes in mt-Genome makes it more useful than shorter sequence of individual genes (Jeffrey L. Boore et al. 2005).
In this 48 primers are used in PCR amplification process and target the sizes of 765bp to 1162bp which results in 200 bp of overlap (Butler, 2005) between different product of PCR.
Results of the polymorphic nucleotide sequence in the genome can be seen and compared in Mito Analyser database. It is bioinformatics platform for mt-Genome analysis and information (Lee & Levin 2010).
4.4: Rapid Screening of mt-DNA types: Expensive and laborious effort in data analysing and chances of error in sequencing, screening assay helps to overcome from these entire problem. Screening methods provides quick result by excluding non-matching sample and helps the scientist to get involved in mt-DNA analysis.
Dr. John Butler and his team give two effective methods of screening. First method is 'pattern matching' in which 3 different mt-DNA sequences can be distinguished from each other. Second one is 'sequencing at specific position' (Butler et al. 1998).
This can help in study of ancestry pattern with mt-DNA The unique mt-DNA inheritance from mother makes it more powerful tool to study the human migration pattern. The haploid pattern that is autosomal DNA does not provide so much ancestry pattern because of shuffling effects of recombination (Butler, 2005).
Mitochondrial DNA analysis and detection of DNA from the degraded material makes it useful to study the ancient bones and helps in finding its origin. This can be done by analysing HV I and HV II region with the use of same type of DNA markers (Butler.JM, 2004).
Other important techniques are Snapshot in which primer specific to target allele extended with 11 coding region.
Single nucleotide polymorphism in addition with single multiplex amplification and detection assay makes this technique more useful and fast. In luminex 100 liquid beads assay 30 single nucleotide polymorphism was studied and used in HV I and HV II.
The allele specific hybridization technique sequence specific oligonucleotide probe (SSO) was used and labelled with different coloured beads and later on with the help of flow cytometry beads are separated (Origene Technologies,2011).
5. Mitochondrial-DNA analysis in Forensic Cases
5.1: Case Study 1: In one of the investigation of 4 missing person mt-DNA analysis proves to be effectual and linked 3 unidentified skeletal remain to the missing person. In one of the case they fail to link it properly but further analysis confirm successfully that the bone was not matched to the missing person (Bender et al. 2000). According to Klaus Bender (2000) and his team the results can be obtained from the degraded bones up to 75 years.
Mt-DNA can be obtained from a variety of sources like epithelial cells (5000 mt-DNA) for further analysis. In this research work DNA was extracted and amplified by the following process:
Bones was cleaned and removed by high speed cutter
Clean part of the bones was cut in specific size and grinded to fine powder by frozing the bone in liqid nitrogen.
Decalcified in EDTA solution and extracted by Phenol chloroform protocol (Sullivan et al. 1994).
Amplification was done on HV I & HV II with the use of platinum Taq polymerase, and 4 sequence specific primers in forward and reverse reaction with 35-40 PCR cylces
For sepration and purification use of 1.5 agrose gel and QIA quick purification provides the good result (Bender et al. 2000).
In 2 of the cases police obtained the mouth swab from their relatives, scientist amplified it and matched with the target sample. They find the identical differences with the Anderson and D loop databases for reference. Interpretation of the results in 1st case showed the insertion of cytosine in both region but insertion in HV I was uncommon and set as unique identification (Butler, 2005).
In 2nd case of missing person heteroplasmy in HV II region was seen which was also present in his brother (Bender et al. 2000). In 3rd case of missing child (according to anthropologist data) result was different from the sample obtained from his/her mother by comparing 7 sequence position. 2 insertions, few exchanges and heteroplasmy in HV II region exclude the results.
In 4th case both mt-DNA analysis and STR typing was done because of the complexity. Clear mismatch of the HV I and HV II region bases were seen with the sample of mother and father DNA (Blender et al. 2000). These all results indicate that mt-DNA provides the clue and confirmation to the police of missing persons and can successfully reveal the identity of the person.
5.2 Case study 2: In many of the crime scene searches the Scene of Crime officer came across the hair fibres and blood of dogs which shows their involvement but it's hard to identify them and separate them with other fibres (Saferstein.R, 2011). In one of the thesis carried out by Sanne Tambeur and his team sucessfully showed the mt-DNA anaylsis helps to identify and characterise domestic dogs hairs and blood (Tambeur et al. 2010).
SNPs and polymorphisims in mt-DNA provide the information which helps in forensic case work. They divide the breeds in four haplogroups and ultimately create the mt-DNA database for the breeds of dogs in Belgium. In many of the samples heteroplasmy and 74 polymorphic positions was observed which can increase the sequence identification. The uniqueness of these polymorphic positions was that 46 positions in total showed nucleotide position chances.
It will be easy for the forensic analyst to observe the particular haplotype groups and helps them to identify the origin from the data set and data point (Tambeur et al. 2010).
5.3 Case Study 3: The problem with mt-DNA analysis is that it provides the low discrimination power because of the inefficiency of recombination and did not provide the much greater help in forensic investigation. One research paper report assay technique for the mt-DNA analysis which can give power of discrimination (Nilsson et al. 2007). By reviewing the data from Caucasian mt-DNA sequences they found coding region assays gives 46% more discrimination power.
Pairwise aligment gives insight of Caucasian mt-DNA sequences. Evaluating HV I & II region and non HV regions provides some of the unique sequences (Nilsson et al. 2007). But its hard to distinguish the population from the same ethnic origin. HV III region can be used to discriminate.
Apart from its expensive and laborious task various forensic laboratories are now using mt-DNA analysis and its screening method for solving crimes in UK and other part of countries. The techniques is widely accpeted by the courts but Youth Justice and Criminal Evidence Act 1999 and Criminal evidence and Justice Act 2003 have many exclusionary powers and statutes that can exclude the mt-DNA evidence. So expert should be very careful while preparing witness statement. They should include probability and statistics of particular findings.
FBI had created one of the database called 'National Missing Person DNA Database' (National Institute of Justice,2010) to provide the details of lost person and widely uses mt-DNA analysis to resolve the things. Array has some problem in relation with deletion, insertion and polymorphic sequence which are closed to each other
A SNPs sites can be assayed now and provide the answer to the above stated problem and give more discrimination power and avoid further sequencing (Coble, 2006). According to Micheal Coble (2006) various approches are undertaken by NIST which can provide with the cheaper option for mt-DNA analysis.