DNA and Mitochondrial DNA

1. Outline one advantage and one limitation of analysing mitochondrial DNA when compared to genomic DNA [20 marks]

Advantage:

• Greater sensitivity in degraded or small samples: Mitochondrial DNA (mtDNA) is present in thousands of copies per cell, as opposed to genomic DNA, which is only present in two copies. This means that mtDNA is more abundant, making it particularly useful in forensic cases where the available DNA sample is degraded or in low amounts. For instance, in cases where nuclear DNA is too damaged to be analyzed (e.g., old or compromised samples), mtDNA can still provide valuable genetic information.

Limitation:

• Lower discriminatory power: Unlike genomic DNA, mtDNA is inherited only from the mother, so it does not provide the same level of genetic variation. In family members, mtDNA will be identical because it is maternally inherited, making it less useful for distinguishing between individuals in cases where family members are involved. This lack of variability limits its ability to definitively identify individuals, especially in cases where multiple people share the same maternal lineage.

2. Write an account of the role of mitochondrial DNA in forensic science with reference to the features of the mtDNA genome, inheritance pattern, analysis of mtDNA sequence and applications of mtDNA analysis. [60 marks]

Role of Mitochondrial DNA in Forensic Science:

Features of mtDNA:

• Mitochondrial DNA (mtDNA) is distinct from genomic DNA and exists in the mitochondria of cells, which are responsible for energy production. Each mitochondrion contains several loops of circular DNA. Unlike nuclear DNA, which is linear and contains 23 pairs of chromosomes, mtDNA is a small circular genome with approximately 16,500 base pairs. It encodes 37 genes, which are involved in cellular energy generation (e.g., encoding proteins for the electron transport chain).

• mtDNA is inherited only through the maternal lineage, which means it is passed from mother to offspring, regardless of the offspring’s sex. This inheritance pattern is useful in tracing maternal ancestry.

Inheritance Pattern:

• Unlike genomic DNA, which is inherited from both parents, mtDNA follows a strictly maternal inheritance pattern. This means that children inherit their mtDNA solely from their mother, making it identical for all individuals on the maternal side of the family.

• The inheritance of mtDNA allows forensic scientists to trace maternal lineage over several generations and is valuable in cases where nuclear DNA is not available or has degraded.

Analysis of mtDNA Sequence:

• To analyze mtDNA, forensic scientists typically focus on the hypervariable regions of the mitochondrial genome. These regions show more sequence variability between individuals, which makes them useful for identification. One such region is the D-loop, which is highly polymorphic and can show significant differences in the mtDNA sequence between individuals.

• The mtDNA is extracted from the biological sample and amplified using PCR (Polymerase Chain Reaction), specifically targeting the hypervariable regions. Sequencing of the amplified regions can reveal unique patterns, which can then be compared with databases containing known sequences.

Applications of mtDNA Analysis:

• When Nuclear DNA Is Not Available: mtDNA is particularly useful when nuclear DNA is degraded, limited, or unavailable. For instance, it is often used in cases involving ancient or highly degraded biological samples, such as bones, hair, or teeth.

• Identification of Remains: mtDNA is used in the identification of human remains in cases such as disaster victim identification, missing persons, and historical investigations. Because mtDNA is inherited maternally, it can also be helpful when reference samples from the victim's nuclear DNA are unavailable, but maternal relatives (e.g., mother, maternal siblings) are available for comparison.

• Maternal Lineage Tracking: In forensic cases where an individual is unknown, mtDNA can be used to trace their maternal lineage, which may provide insights into their identity, especially when other DNA profiling methods fail.

Limitations of mtDNA in Forensic Science:

• The primary limitation of mtDNA is its lack of genetic diversity. Since mtDNA is inherited only from the mother, individuals in the same maternal lineage will share identical or very similar mtDNA. This makes it difficult to distinguish between individuals who share the same maternal ancestry.

• Additionally, mtDNA analysis is slower and less discriminative than nuclear DNA-based methods such as STR (Short Tandem Repeat) profiling. It is typically used as a last resort or supplementary method when nuclear DNA is unavailable.

3. Describe the process of variable number tandem repeat (VNTR) DNA profiling. In your answer, state the limitations of VNTR analysis when compared to short tandem repeat (STR) profiling [50 marks]

Process of VNTR DNA Profiling:

1. DNA Isolation: The process begins with the isolation of DNA from the sample (e.g., blood, hair, or tissue). This is done by breaking down the cell membranes and separating the DNA from other cellular components.

2. Restriction Enzyme Digestion: Once the DNA is isolated, it is treated with restriction enzymes that cut the DNA at specific sequences. These enzymes are chosen so they cut at sites adjacent to the VNTR regions, which are areas where short sequences of DNA are repeated multiple times.

3. Gel Electrophoresis: The resulting DNA fragments are then separated by size using gel electrophoresis. The gel is subjected to an electric field, causing the DNA fragments to move. Smaller fragments move faster, while larger fragments move more slowly.

4. Southern Blotting: After electrophoresis, the DNA fragments are transferred from the gel to a nylon membrane in a process called Southern blotting. This membrane is then incubated with a probe – a short piece of DNA that binds to the VNTR regions.

5. Detection: The membrane is treated with a radioactive or chemiluminescent probe to allow visualization of the fragments containing the VNTRs. When the membrane is exposed to X-ray film or a chemiluminescent detector, the locations of the VNTR fragments can be seen as bands, which are used to create a DNA profile.

Limitations of VNTR Analysis Compared to STR Profiling:

• Larger DNA Samples Required: VNTRs are much larger than STRs (typically 15-100 base pairs vs. 3-5 base pairs), meaning that VNTR profiling requires a larger amount of DNA for analysis. This is a limitation when working with degraded or low-quantity samples.

• Time-Consuming and More Complex: VNTR analysis is more time-consuming than STR analysis because it involves multiple steps such as restriction enzyme digestion, gel electrophoresis, and Southern blotting. In contrast, STR analysis is faster and more efficient, particularly due to the use of PCR.

• Less Precision: VNTR analysis is less precise compared to STR profiling. STR regions are much shorter and more numerous, allowing for higher-resolution profiling, especially when DNA samples are degraded or mixed.

• Difficulty with Multiple Alleles: VNTR analysis can be more difficult to interpret when there are multiple alleles, as the large size of the VNTRs can cause overlapping bands or complicated patterns that are harder to distinguish.

4. Describe the process by which DNA is profiled by STR analysis. Include reference to the type of DNA regions analysed, PCR, analysis of PCR products and an electropherogram in your answer. [50 marks]

Process of STR DNA Profiling:

1. DNA Extraction: DNA is first extracted from the biological sample, which could be blood, saliva, hair, or tissue. The process involves breaking down the cell membrane and isolating the DNA from other cellular components.

2. Amplification of STR Regions: Short Tandem Repeats (STRs) are regions of the genome where a short sequence (typically 3-5 nucleotides) is repeated multiple times. In STR profiling, the focus is on specific loci where these short repeats are found. The number of repeats can vary between individuals, providing a basis for differentiation.

   • PCR (Polymerase Chain Reaction) is used to amplify the STR regions of interest. PCR uses primers that specifically bind to sequences flanking the STR loci, allowing for the amplification of these regions.

3. Analysis of PCR Products: Once the STR regions are amplified, the PCR products are analyzed. The PCR products vary in size due to differences in the number of repeats at each STR locus. These products are run through a gel electrophoresis system or an automated capillary electrophoresis system, where they are separated based on size.

4. Electropherogram Generation: The separation of STR fragments by size results in the creation of an electropherogram. This is a graphical representation of the PCR products, where the x-axis represents the size of the fragments, and the y-axis represents the fluorescence intensity. Each peak in the electropherogram corresponds to a specific STR allele, with the size of the peak representing the length of the STR fragment. The number of peaks (representing alleles) at each locus can be used to create a DNA profile.

5. Interpretation: STR profiles typically show two peaks at each locus (one from each parent). If an individual has identical alleles at a locus (homozygous), the profile will show one peak. If the individual has different alleles (heterozygous), two distinct peaks will be visible. The combination of alleles across multiple loci creates a unique profile that can be used for identification.

Applications:

• Forensic Identification: STR profiling is widely used in forensic science for criminal investigations, paternity testing, and identification of human remains. By comparing STR profiles from a suspect, victim, and crime scene, forensic scientists can determine potential matches.

• Database Comparison: STR profiles can be compared to DNA databases (such as CODIS) to find potential matches for unidentified individuals or to link crimes based on shared genetic markers.

Advantages of STR Profiling:

• High Discriminatory Power: STRs are highly variable between individuals, which makes STR profiling an effective method for identifying individuals.

• Speed and Sensitivity: STR profiling can be performed quickly and is sensitive enough to work with small or degraded samples, making it suitable for forensic applications.