The era of forensic DNA typing began in the 1980s when ABO and isoenzymes were the forensic tools for biological identification. As was the case with ABO blood grouping, DNA profiling was adapted from research in molecular biology. However, migration from the research laboratory to the forensic laboratory involves far more than buying new equipment. Forensic methods and techniques must satisfy two diverse communities – the scientific and the judicial. There is a common misconception that science and justice both seek “truth” and are natural partners. This assessment is oversimplified. At best, the disciplines manage to work together in a strained relationship. Before we move on to the science of DNA profiling, we need to explore how DNA found acceptance in the courts.

When a new scientific method is employed in a case, the courts must decide whether the data will be admitted into evidence that will be seen by those who will pass judgment, such as a judge or jury.

DNA profiling of STR loci is a mature technology. Improvements continue in sensitivity and additional STR loci, but the process, kits, and instrumentation are established. Courts, police, and the legal system accept and rely on DNA evidence, and databases continue to grow. However, this does not mean that the field has become static. Research continues, and newer concepts are being evaluated and adopted by the forensic community. Some of these are evolutionary, while others could be revolutionary.

We have come a long way in a short time. From the first use of DNA typing for a criminal investigation in 1986 to now, over 35 years have passed. Those years have brought a revolutionary change in human identification, from ABO blood typing to analysis of complex mixtures, probabilistic genotyping software, and investigative genetic genealogy. Forensic DNA typing now applies to STRs (still the primary method), Y-STRs, mitochondrial DNA, and SNPs. We have law enforcement databases and consumer databases that are used in current and cold cases. We have seen how portable DNA instruments can be used in mass fatalities and police booking stations.

There are several key takeaways from this journey, including the need to correct several common misunderstandings, as summarized in the next section.

So far, we have focused on DNA types in which one allele is from the father and one from the mother. However, three other sources of DNA come from only one parent, and all can be employed in forensic testing. One is mitochondrial DNA (from the mother in all her children), and the other two are STR sites on the Y chromosome (from the father in his sons) and STR sites on the X chromosome (from the mother in her sons). These DNA sources are lineage markers, since they can be traced back generations through our family trees. Lineage markers are valuable in missing person cases where DNA from the person of interest is not available. Mitochondrial DNA (mtDNA) has been used in historical cases, such as identifying soldiers killed in past conflicts. We will explore these and other examples in this chapter.

Forensic samples are among the most complex encountered. Blood is best known, but other biological matrices also carry genetic information. Cheek swabs (buccal swabs) collect cells from the inside of the mouth and have the advantage of being a non-invasive sample collection compared to a blood draw. Hair, depending on the presence of the root, is amenable to DNA typing. Semen, vaginal fluids, and vaginal swabs are collected in sexual assault cases. Any surface on which biological fluids (blood, oral fluid, vaginal fluid, etc.) are deposited becomes a potential DNA source.

The initial deposition (called the primary transfer) occurs from a person to a surface. It is the deposition of blood, saliva, semen, or other biological substance directly from the body onto a surface. This process could be a victim’s blood dripping onto an assailant’s clothing, saliva on a cigarette, or seminal fluid on a bedsheet.

Forensic DNA typing was developed to improve our ability to conclusively identify an individual and distinguish that person from all others. Current DNA profiling techniques yield incredibly rare types, but definitive identification of one and only one individual using a DNA profile remains impossible. This fact may surprise you, as there is a popular misconception that a DNA profile is unique to an individual, with the exception of identical twins. You may be the only person in the world with your DNA profile, but we cannot know this short of typing everyone. What we can do is calculate probabilities. The result of a DNA profile translates into the probability that a person selected at random will have that same profile. In most cases, this probability is astonishingly tiny. Unfortunately, this probability is easily misinterpreted, a situation we will see and discuss many times in the coming chapters.

The last chapter outlined the basic concepts of mixture analysis. Now we move on to the much more challenging situations arising from low-level DNA samples and complex mixtures. These topics go together. Early DNA methods such as RFLP and initial PCR methods were less sensitive (which means they were unable to detect very small quantities of DNA) than today’s techniques. As a result, DNA present in tiny quantities was not seen. Now the technologies afford much better detection, which is a mixed blessing. Rather than simply detecting the DNA from the major contributor(s), now trace levels of DNA can be recovered and typed, and not all of it is pertinent to the crime under investigation. Very small amounts of DNA, much less than in typical samples, are referred to as low copy number (LCN) DNA.

Low copy number and complex mixtures are related.

The relentless advance of DNA typing capabilities leads to complications and concerns. It is one thing for someone to be able to obtain your ABO blood type from a tiny spot of blood and another thing to know your eye color and ancestry. Some of the concerns are due to misconceptions, but others can pit personal privacy against perceived security. Practices and policies have not caught up with capability. We highlight a few of these current dilemmas in this chapter.

The last chapter discussed how peaks in an instrument output are converted into a DNA profile and how the random match probability is calculated using the product rule. Now we delve into how these profiles are analyzed and interpreted. Once a DNA profile has been developed from crime-scene evidence, it is compared to the profile(s) from known reference samples. These include elimination samples and samples from a person or persons of interest. If these comparisons do not provide helpful information, the profile can be submitted to a DNA database to search for investigative leads. Our focus is on DNA samples from a single person or simple mixtures such as a well-separated sample from a sexual assault case. Complex and low-level mixtures are much more challenging. We tackle those in the next chapter using the foundation we will build in this one.

In the last chapter, we discussed the first method (RFLP) used in DNA typing. This procedure targeted relatively long DNA fragments (VNTRs) containing many repeated units of a base pair sequence. We ended by noting that they were not amenable to automation and therefore not destined for widespread forensic applications. However, by the early 1990s, many factors coalesced to set the stage for a leap in DNA typing capabilities. For example, the forensic and legal community had adjusted to DNA evidence, and analysts had moved from serological techniques such as ABO to genetic typing utilizing multiple DNA markers. Additionally, researchers in molecular biology, including in genomic sequencing, had identified many shorter repeat sequences that exhibited variation among individuals in a population.