Medical Genetics - V. M. Zaporozhan 2005

Diagnostic Methods for Hereditary Diseases
Molecular Genetic Methods (DNA Diagnostic Methods)
Application of Molecular Genetic Methods in Forensic Medicine for Personal Identification and Paternity/Kinship Testing

In recent years, Methods based on the detection of individual genetic DNA differences have been increasingly applied for paternity testing and forensic medical examinations.

Such variations can be identified by examining highly polymorphic (highly variable or hypervariable) DNA regions, such as regions with a variable number of tandem repeats (VNTR) — minisatellite DNA, or short tandem repeats (STR) — microsatellite DNA.

Regions with a variable number of repeats can be located within a single locus on one chromosome or across multiple loci on different Chromosomes.

When analyzing VNTRs or STRs localized across multiple loci, the entire Human Genome is examined. This technique is known as genomic fingerprinting. Genomic fingerprinting was first performed by the English geneticist Alec Jeffreys in 1985. For personal identification, he used minisatellite DNA whose repeating unit was complementary to an intron of the Myoglobin Gene. The scientist analyzed restriction fragment length polymorphism (RFLP) using Southern blot Hybridization with radioactively labeled probes targeted to the myoglobin intron.

Today, variable regions are repeatedly replicated using the Polymerase Chain Reaction (PCR) method and analyzed after gel Electrophoresis for the length polymorphism of amplified DNA fragments (amplified fragment length polymorphism, AFLP). This yields a unique pattern of DNA fragment lengths for each individual, known as a "genomic fingerprint." A drawback of this method is the simultaneous generation of A large number of fragments, which complicates the analysis of results.

Modern techniques are primarily based on the analysis of VNTRs localized within a single locus. An example is the D1S80 locus, located on the first chromosome. This locus contains minisatellite DNA. The repeat length is 16 bp, but the number of repeats can vary. Thus, in the same individual, the locus has one length on one chromosome and a different length on the other. For example, Fig. 10.13 illustrates a hypothetical chromosome number 1 containing 3 repeats, and a second homologous chromosome with 7 repeats. These chromosomal regions are inherited according to Mendel's Laws as codominant traits. A child inherits one chromosome from the mother (with a specific number of repeats and corresponding locus length) and the second from the father (also with a specific Number and Length). Paternity testing is based on comparing the DNA fragment lengths of the child, the mother, and the putative father. Personal identification is possible due to the uniqueness of the DNA fragment length pattern in each individual.

Polymerase chain reaction is also used to analyze variable DNA regions. Primers specific to the analyzed locus (e.g., the D1S80 locus) are added to the PCR reaction mixture. Following electrophoresis in agarose or polyacrylamide gel, the length polymorphism of the amplified fragments (AFLP) is analyzed. During the Amplification of the D1S80 locus, the size of the amplified fragments ranges from 375 to 850 bp.

Fig. 10.14 presents an example of paternity establishment and exclusion using the AFLP method (based on the D1S80 locus). In the case of confirmed paternity, it can be seen that the mother has alleles 6 and 9 (differing in repeat number and length), and the father has alleles 3 and 9. The child exhibits the same alleles as the mother (6 and 9). Since the child could inherit alleles 6 and 9 from their mother, one of these alleles must come from the biological father. Indeed, allele 6 is absent in the putative father, meaning he cannot be excluded as the child's father. In the mixture of the child's and father's DNA, it is evident that allele 9 is shared between them.

In the case of unconfirmed paternity, the mother is homozygous for allele 11, the child has alleles 11 and 3, and the putative father has alleles 13 and 9. The child received allele 11 from the mother, meaning they should have received allele 3 from the biological father. However, this allele is absent in the putative father, and therefore paternity must be excluded.

Class="center">

Fig. 10.13. Hypothetical chromosome 1 containing 7 repeats (top) and 3 repeats (bottom)

In most cases, to obtain reliable results in paternity testing using the PCR-AFLP method, four or five independently inherited loci are analyzed.

Currently, microsatellite DNA is more frequently used for personal identification and paternity testing, meaning that STR length polymorphism is determined. An individual's genotype is analyzed simultaneously across multiple STR sites located on different chromosomes. Multiplex PCR variants are used for this purpose.

Mitochondrial DNA can also be used for personal identification. It is characterized by maternal inheritance. The Study of mitochondrial DNA was instrumental in confirming that the famous Yekaterinburg remains belonged to the Romanov family.

Over time, it will become possible to create a database of genomic fingerprints for any individual (similar to a fingerprint database).

The advantages of the method include:

1. DNA uniqueness — every individual is genetically distinct (except for identical twins, who are essentially clones).

2. Genetic constancy of the Organism — Genetic information remains unchanged throughout life and does not depend on the type of Cells from which the DNA was extracted.

3. Sensitivity of the method — for modern DNA methods, DNA extracted from just a few cells (theoretically, even a single Cell) is sufficient.

4. Relative stability of DNA — DNA molecules exhibit high resistance to environmental influences. This property of DNA makes identification possible even after very long periods of time, or when human remains cannot be recognized by any other methods.

Fig. 10.14. Example of paternity establishment and exclusion using the AFLP method at the D1S80 locus (MS — marker allele ladder; M — mother; C — child; PF — putative father; C+PF — mixture of child and putative father DNA)