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sizes of the fragments and their quantities, and these are represented as peaks on a graph (Figure ST 5.4). Typically, automated systems analyze dozens of samples at a time, and the analysis takes less than an hour. After DNA profiling, the profile can be directly compared to a profile from another person, from crime scene evidence, or from other profiles stored in DNA profile databases (Figure ST 5.5). The STR profile genotype of an individual is expressed as the number of times the STR sequence is repeated. For example, the profiles shown in Figure ST 5.5 would be expressed as shown in Table ST 5.1. An electropherogram showing the STR profiles of four samples from a rape case. Three STR loci were examined from samples taken from a suspect (male), the per- son who was sexually assaulted (female), and two fractions from a vaginal swab taken from the female. The x-axis shows the DNA size ladder, and the y-axis indicates relative fluorescence intensity. The number below each allele indicates the number of repeats in each allele, as measured against the DNA size ladder. Notice that the STR profile of the sperm sample taken from the female matches that of the suspect. Scientists interpret STR profiles using statistics, probability, and population genetics, and these methods will be discussed in the section Interpreting DNA Profiles. Y-Chromosome STR Profiling In many forensic applications, it is important to differentiate the DNA profiles of two or more people in a mixed sample. For example, vaginal swabs from rape cases usually contain a mixture of female somatic cells and male sperm cells. In addition, some crime samples may contain evidence material from a number of male suspects. In these types of cases, STR profiling of Y-chromosome DNA is useful. There are more than 200 STR loci on the Y chromosome that are useful for DNA profiling; however, fewer than 20 of these are used routinely for forensic analysis. PCR amplification of Y-chromosome STRs uses specific primers that do not amplify DNA on the X chromosome. One limitation of Y-chromosome DNA profiling is that it cannot differentiate between the DNA from fathers and sons or from male siblings. This is because the Y chromosome is directly inherited from the father to his sons, as a single unit. The Y chromosome does not undergo recombination, meaning that less genetic variability exists on the Y chromosome than on autosomal chromosomes. Therefore, all patrilineal relatives share the same Y-chromosome profile. Even two apparently unrelated males may share the same Y profile, if they also share a distant male ancestor. Although these features of Y-chromosome profiles present limitations for some forensic applications, they are useful for identifying missing persons when a male relative’s DNA is available for comparison. They also allow researchers to trace paternal lineages in genetic genealogy studies. Mitochondrial DNA Profiling Another important addition to DNA profiling methods is mitochondrial DNA (mtDNA) analysis. Between 200 and 1700 mitochondria are present in each human somatic cell. Each mitochondrion contains one or more 16-kb circular DNA chromosomes. Mitochrondria are passed from the human egg cell to the zygote during fertilization; however, as sperm cells contribute few if any mitochondria to the zygote, they do not contribute these organelles to the next generation. Therefore, all cells in an individual contain multiple copies of specific mitochondrial variants derived from the mother. Like Y-chromosome DNA, mtDNA undergoes little if any recombination and is inherited as a single unit. Scientists create mtDNA profiles by amplifying regions of mtDNA that show variability between unrelated individuals and populations. After PCR amplification, the DNA sequence within these regions is determined by automated DNA sequencing. Scientists then compare the sequence with sequences from other individuals or crime samples, to determine whether or not they match. The fact that mtDNA is present in high copy numbers in cells makes its analysis useful in cases where samples are small, old, or degraded. mtDNA profiling is particularly useful for identifying victims of mass murders or disasters, such as the Srebrenica massacre of 1995 and the World Trade Center attacks of 2001, where reference samples from relatives are available. The main disadvantage of mtDNA profiling is that it is not possible to differentiate between the mtDNA from maternal relatives or from siblings. Like Y-chromosome profiles, mtDNA profiles may be shared by two apparently unrelated individuals who also share a distant ancestor—in this case a maternal ancestor. Researchers use mtDNA profiles in scientific studies of genealogy, evolution, and human population migrations. Single-Nucleotide Polymorphism Profiling Single-nucleotide polymorphisms (SNPs) are single-nucleotide differences between two DNA molecules. They may be base-pair changes or small insertions or deletions. SNPs occur randomly throughout the genome, approximately every 500 to 1000 nucleotides. This means that there are potentially millions of loci in the human genome that can be used for profiling. However, as SNPs usually have only two alleles, many SNPs (50 or more) must be used to create a DNA profile that can distinguish between two individuals as efficiently as STRs. Scientists analyze SNPs by using specific primers to amplify the regions of interest. The amplified DNA regions are then analyzed by a number of different methods such as automated DNA sequencing or hybridization to immobilized probes on DNA microarrays that distinguish between DNA molecules with single-nucleotide differences. Forensic SNP profiling has one major advantage over STR profiling. Because a SNP involves only one nucleotide of a DNA molecule, the theoretical size of DNA required for a PCR reaction is the size of the two primers and one more nucleotide (i.e., about 50 nucleotides). This feature makes SNP analysis suitable for analyzing DNA samples that are severely degraded. Despite this advantage, SNP profiling has not yet become routine in forensic applications. More frequently, researchers use SNP profiling of Y-chromosome and mtDNA loci for lineage and evolution studies. DNA Phenotyping An emerging and controversial method, known as DNA phenotyping, is gaining popularity as a new DNA forensics tool. Unlike DNA profiling, which is used to confirm or exclude sample identities, DNA phenotyping uses DNA sequence information to reveal a person’s physical features and ancestral origins. Currently, DNA phenotyping methods can predict a person’s eye, hair, and skin colors based on their DNA SNP patterns. For example, scientists have found six SNPs in six genes that are related to blue and brown eye color, Using statistical models based on these six SNPs, it is possible to predict with 95 percent accuracy whether a person has brown or blue eyes. Using 22 SNPs associated with 11 genes, it is possible to predict with 90 percent accuracy whether a person has black hair and 80 percent accuracy whether a person has red or brown hair. Skin color predictions involve 36 SNPs associated with 15 genes, with prediction accuracies similar to those for hair colors. Both biological sex and geographic ancestry can also be accurately determined from a person’s DNA sequence. Some researchers and private companies have taken DNA phenotyping well beyond prediction of these features. Their algorithms claim to predict three-dimensional facial structures which allow them to compile full-color photo- graphic representations of a person’s face, based only on their DNA sample. At the present time, DNA phenotyping has not been validated sufficiently to be presented in court. However, police are using the method to help identify unknown missing persons and to provide leads in cold cases, ST 5.2, Interpreting DNA Profiles After a DNA profile is generated, its significance must be determined. In a typical forensic investigation, a profile derived from a suspect is compared to a profile from an evidence sample or to profiles already present in DNA data- bases. If the suspect’s profile does not match that of the evidence profile or database entries, investigators can conclude that the suspect is not the source of the sample(s) that generated the other profile(s). However, if the suspect’s profile matches the evidence profile or a database entry, the interpretation becomes more complicated. In this case, one could conclude that the two profiles either came from the same person—or they came from two different people who share the same DNA profile by chance. To determine the significance of any DNA profile match, it is necessary to estimate the probability that the two profiles are a random match. The profile probability or random match probability method gives a numerical probability that a person chosen at random from a population would share the same DNA profile as the evidence or suspect profiles. The following example demonstrates how to arrive at a profile probability The first locus examined in this DNA profile (D5S818) has two alleles: 11 and 13, Population studies show that the 11 allele of this locus appears at a frequency of 0.361 in this population and the 13 allele appears at a frequency of 0.141. In population genetics, the frequencies of two different alleles at a locus are given the designation p and q, following the Hardy—Weinberg law described earlier in the text (see Chapter 21).