Amplification of Ancient DNA. Applications in Forensic Analysis

The application of PCR technology to amplify DNA from ancient sources has made PCR an invaluable tool in the fields of anthropology, archaeology, and evolutionary studies. Many important samples exist in museums, zoological collections, and various institutions that could be of great use if the DNA in these samples could be analyzed. Some examples of these are preserved ancient microorganisms, Egyptian mummy tissue, and animal skins from rare or extinct animal species.

The direct analysis of these samples is typically hampered by an insufficient amount of tissue and the danger of destroying the samples. PCR can be used to amplify regions of DNA from these samples with a minimal amount of starting material required. Although PCR can generate sufficient quantities of material for extensive qualitative analysis, there are two problems with the amplification of ancient DNA.

One is the problem of the quality of the DNA. Old DNA is heavily modified and typically degraded to a large degree so that only small fragments of several hundred base pairs are present in preserved samples. Therefore, the size of DNA that can be amplified and examined is limited. The second problem is the design of primers used in the PCR reaction.

Because ancient samples are divergent in their DNA from modern organisms, guesses must be made for the sequence of the primers. Typically, primers are designed against highly conserved regions, such as that of mitochondrial DNA segments, to give the greatest probability of hybridization to target sequences. Despite these drawbacks, PCR has been used to examine the sequences of DNAs from rare sources that were previously inaccessible by routine methods.

Applications in Forensic Analysis. Because PCR enables the amplification of minute amounts of sample tissue, the technique has become an invaluable tool in forensic analysis. Prior to PCR, DNA was utilized as a fingerprint in criminal investigaitons and in paternity determination, but a significant amount of starting material was required. PCR allows the analysis of a single hair, a small number of cells, or even a single sperm to identify a suspect or confirm identity.

In order to be of use, the DNA region of analysis must be sufficiently polymorphic in the population as to allow the differentiation of individuals. The common DNA region examined is that of the class II major histocompatibility complex or HLA-D genes. [See Major Histocompatibility Complex (MHC).]

The HLA-D genes are organized into three regions, HLA-DR, -DQ, and -DP, each of which encode an a and ß gene. These genes encode proteins that are expressed on a number of cell types, including В lymphocytes, macrophages, and activated T lymphocytes. Their diversity arises from the fact that they are part of the immune response and are responsible for the recognition and binding of foreign antigens in the human body.

The HLA gene routinely used in analysis is DQα. To date, eight unique alleles of DQα have been identified in the human population. To analyze evidence, forensic samples are subjected to PCR using primers that recognize conserved regions outside the highly polymorphic region of the DQα gene to generate a 242-bp DNA fragment. The DNA is then fixed to a substrate and hybridized to a series of radiolabeled or colorimetrically labeled allele-specific oligonucleotides (ASOs). These ASOs can differentiate among the variants of the DQα gene and reveal which two of the alleles are present in a forensic sample.

These can then be compared to allelic samples obtained from a suspect. Allele frequency data have been accumulated for Caucasian, Black, and Asian populations and it therefore can be determined with what frequency the alleles will appear at random in a specific individual. The average frequency or occurrence of two specific DQα alleles in an individual in the population ranges from 0.5 to 15%. Although this is not a conclusive fingerprint, it may aid in the elimination of potential suspects in a criminal investigation.

In addition, using the DQα as a fingerprint can be used in combination with other HLA-D regions. The DPβ gene has 21 allelic variants that can be characterized by similar methods used for the DQα gene. The use of this loci in combination with DQα and other loci can effectively multiply the discriminatory power of the genotyping and function essentially as a conclusive fingerprint. Presently, a variety of other loci are being examined for use in DNA fingerprinting, and population statistical data are being generated for these loci. Because of its ability to generate an individual identity from a minimal amount of evidence, PCR has become an invaluable tool in forensic analysis.