DNA Analysis
Paternity Tests
Genomic Pictures
Final Considerations
In 1892, fingerprints began to be used in personal identification. The discovery that an individual's fingerprint is a unique characteristic (i.e., no other person would have the same pattern in the population) stimulated their use for identification purposes. Fingerprints are so unique that not even identical twins possess the same fingerprints.
The judicial system has been able to convict a great number of suspects from fingerprints left on surfaces such as furniture, telephones, and glass. On the other hand, a comparable number of individuals have also been cleared of criminal charges because of this identification tool.
Similarly, molecular individual identification, or DNA fingerprinting (see Chapter 9, “Molecular Markers”), has become widely used to solve forensic cases. The first significant case of the use of DNA as criminal evidence occurred in 1986 in England, when a homicide suspect was released after DNA analysis of evidence collected at the crime scene was compared with the suspect's DNA fingerprint. Beginning in 1987, the U.S. Federal Bureau of Investigation (FBI) and several other criminal laboratories in different countries began to use DNA as biological evidence in criminal cases. Samples of blood, saliva, hair, semen, and other human cells found at the crime scene became strong evidence for the imprisonment or release of suspects.
As seen in Chapter 9, molecular markers can be used to characterize an individual's DNA in a pattern or profile of fragments that is unique to him or her. As opposed to normal fingerprints that can be altered by surgery, a person's DNA cannot be deliberately altered. Consequently, the DNA profile has been considered an important and reliable method of individual identification.
The genetic information contained in DNA is determined by the sequence of the letters of the genetic alphabet (A, C, G, and T). In humans, about 3 billion of these letters are arranged in the same order in the chromosomes of each cell in the human body. It is the order in which the letters are arranged in the chromosomes that makes each individual unique from all others. Obviously, the more dissimilar the individuals are, the more distinct is the order of the nucleotides (letters) in the genome. Similarly, individuals who are genetically related (e.g., siblings, parents, and children) have proportionally larger similarity in their gene sequences. Ultimately, only identical twins have the same DNA sequence. Therefore, a DNA profile is a simple and fast way to compare DNA sequences of two or more individuals.
DNA profiles have many varied applications. They can be employed in criminal and civil cases, used for paternity tests, help in determination of succession of properties by inheritance, used for identification of bodies, or used to determine property rights of crop varieties, among other things.
In December 1954, Sam Sheppard's trial in Cleveland, Ohio, occupied the media across the United States. Convicted to life in prison for the murder of his pregnant wife, Sheppard repeatedly maintained his innocence. In 1966, the Supreme Court threw out the trial because of trial errors. Sheppard was freed, but he died four years later. In 1992, a book was published accusing Richard Eberling, a neighbor of Sheppard, of committing the crime. Eberling had been convicted of murdering another person and he died in jail. After his death, other inmates advised authorities that Eberling had admitted to the murder of Sheppard's wife. In 1997, Sheppard's son requested authorization to exhume his father's body with the objective of obtaining a DNA analysis to substantiate his father's claims of innocence. The analysis indicated that his father's DNA did not correspond to the evidence collected at the crime scene, refuting the possibility that Sheppard had committed the crime.
Between 1989, when the FBI began to use DNA analysis in rape cases, and 1996, the agency investigated about 10,000 cases of sexual abuse. DNA tests excluded about 25 percent of the primary suspects in those cases. In many criminal cases, eyewitnesses, or more frequently the victims themselves, are needed to positively identify the suspect. DNA has shown those eyewitnesses are not always reliable. When properly collected, manipulated, stored, and analyzed, DNA has the potential to eliminate numerous errors in the criminal justice system. DNA is valuable in all situations, and even more important when eyewitnesses cannot be found. It is almost impossible to commit a crime without leaving DNA evidence such as hair, skin cells, and body fluids at the crime scene.
The Innocence Project at the Benjamin Cardozo Law School in New York is a clinical law program for students supervised by law professors and administrators. The Innocence Project provides pro bono legal assistance to inmates challenging their convictions based on DNA testing of evidence, although clients must obtain funding for the testing. Founded in 1992 by Barry Scheck, Professor of Law, and Peter Neufeld, the Project has represented or assisted in many cases in which convictions have been reversed or overturned in the United States. To date, this project has been responsible for the exoneration of more than 100 people.
DNA analysis is transforming biological evidence as an irrefutable instrument for incrimination or absolution of suspects. It eliminates some of the ambiguities of the justice system. Even so, in the controversial case in which O. J. Simpson was charged with murdering his ex-wife Nicole Brown, the evidence failed to provide conclusive results. In a highly disputed outcome, Simpson was acquitted of the charges, in part because his lawyers challenged all biological evidence. As in the Simpson case, skilled lawyers are always challenging the chain of custody, hoping to convince juries that DNA evidence has been contaminated. As with all evidence collected, an important requirement for the validity of DNA tests in criminal trials is the integrity of those who collect, process, and store the evidence.
The DNA digestion with restriction enzymes produces fragments of different lengths, according to the individual's genome sequence. For instance, an individual that has the sequence AAGCTT, the cut site for Hind III, will have his or her DNA cut by this enzyme in as many fragments as that sequence occurs. Therefore, if the DNA of a suspect S1 has 50 restriction or cutting sites, whereas suspect S2 has 55 restriction sites, the fragmentation of the DNA of the two individuals will produce different patterns. The size of each fragment depends on the distance between sites, and the number of fragments depends on the number of cut sites. This variation of fragment number and size is usually referred to as polymorphism, due to the multiple forms in which the DNA is cleaved.
The DNA profile of an animal, plant, or microorganism can be obtained by analyzing its DNA.
A simplified protocol might include seven steps:
Harvesting the biological sample: Blood, saliva, semen, hair, tooth, bones, or any other cellular tissue or fluid from the individual (Figure 10-1).
DNA extraction: DNA should be isolated from the sample. Depending on the preferred method, a tiny amount of sample might be sufficient, such as the defoliated cells from the skin of an individual's forehead found in a hat. Alternatively, a saliva droplet left on a telephone handset or postage stamp might also contain enough DNA for the analyses.
DNA digestion: The following step is the DNA cleavage with a restriction enzyme. The enzymes Hind III and EcoR I, among others, have frequently been used for this purpose. After the DNA is treated with the restriction enzyme, it is then made up of a number of fragments of different sizes.
Fragment separation: The DNA fragments are separated by size using electrophoresis. This procedure consists of submitting the DNA fragments, inside of a gel, to an electric field. The gel is usually made of agarose, a substance extracted from sea algae that is similar to gelatin in composition. The DNA is placed in a small well on the gel close to the negative electrode. An electric field causes the DNA fragments to migrate toward the positive electrode. Small DNA fragments move quicker than large fragments, allowing separation by size.
DNA transfer: After the separation of the fragments, they are transferred to a nylon membrane by capillarity. Once they are attached to the membrane, the fragments can be manipulated for viewing.
Hybridization with probes: The addition of fluorescent labeled or radioactive probes to the nylon membrane allows the visualization of the fragments that are complementary to the probes. Each probe typically highlights some of the fragments in the membrane.
DNA profile: The final DNA profile is obtained after the hybridization of various probes to the membrane. The result is a pattern of bands (dark spots) of different sizes (Figure 10-2).
DNA as criminal evidence or as a means for individual identification is revolutionizing law enforcement. Consider the following example, in which two individuals, S1 and S2, are rape suspects. Analysis of the semen or any other tissue collected at the crime scene, here designated E, can be used to identify the individual responsible for the crime without submitting the victim to the additional stress of testifying at the trial. This is especially important when the victim is unable to identify his or her aggressor.
The DNA analysis of the two suspects and of the biological evidence can provide unmistakable evidence for the true perpetrator of the crime. After DNA analysis, one of the possible profiles is presented in Figure 10-3. Examining the profiles produced with the four probes in this figure, suspect S1 can be excluded as the instigator of the crime because his DNA profile is different from that of E (probes 1, 2, and 4). Technically, it would be more accurate to say that suspect S1 could be cleared of having left the biological evidence at the crime scene. Good criminologists understand that the relationship between the evidence, the crime, and its authorship should receive equal attention. However, it is clear from the DNA analysis that E does not have the same profile as suspect S1.
Figure 10-3. DNA profile of two suspects, S1 and S2, and of the evidence E collected at a crime scene.
The DNA of suspect S2 corresponds perfectly with that of E in the four regions of the DNA analyzed with the four probes. That doesn't necessarily mean that the suspect S2 is the author of the crime. Obviously, to know with what certainty the evidence should be considered, it is necessary to know with what frequency that same DNA profile is found in the human population, and that is a matter of probabilities. After the DNA analysis with a certain number of probes, the laboratory produces a report indicating the probability that the DNA of suspect S2 and E would be the same by coincidence. Usually, the level of statistical stringency is extremely high to prevent costly errors.
Besides RFLPs and RAPDs, other types of molecular markers have been used in DNA analyses for criminal purposes. For instance, several forensic laboratories, including the FBI, are using STR in their analyses. Those markers are highly discriminatory, and the analysis of 13 regions of the human genome, with markers now available, allows the generation of reports stating the probability of up to 1 in 82 billion individuals.
DNA profiling is a powerful identification tool. Most countries have fingerprint databases that are used in criminal cases. In crimes against properties, fingerprints can often be found. However, in violent crimes, fingerprints are not typically left at the crime scene. Rapists usually touch the victim's body, leaving no fingerprints. However, semen on the victim's clothes or body can be used as evidence for solving such cases.
Currently, many countries are establishing DNA databases from individuals with a history of violent crimes. Those databases would allow the identification of suspects by simply cross-checking the DNA profile of the evidence with those stored in the database. However, several ethical issues have been raised about those databases. More details on DNA and genetic privacy are discussed in Chapter 14, “Bioethics.”
Although a DNA profile is considered irrefutable proof of identification, it is necessary to establish standards of analysis and accuracy levels in the statistical calculations. Additionally, the laboratories that provide these services should be submitted to double-blind tests, in which neither the lab nor the technicians know what the samples are, to ensure that they are working with acceptable quality control.
A different approach has been given to the analysis of DNA in mitochondria, an organelle inside the cells. In cases where the amount of DNA is extremely small or in cases where the victim has been carbonized, partially destroying the nuclear DNA, the analysis of the mitochondrial DNA is an alternative. However, it should be acknowledged that mitochondrial DNA tends to be much less variable, because the mitochondria is maternally inherited; that is, an individual's mitochondrial DNA is exactly identical to that of his or her mother and siblings, and to all uncles from his or her mother's side of the family, and so forth. The use of mitochondrial DNA for individual identification was extremely important in Argentina in cases in which children were separated from their families during a military dictatorship. The movement known as “The Grandparents of May” in Argentina was able to reunite many children with their blood families on the basis of mitochondrial DNA analysis. The same analysis was also used in the identification of some bodies from the World Trade Center terrorist attack on September 11, 2001 in New York City.
DNA tests are the most accurate and reliable technology used for paternity identification today. Usually, samples from mother, child, and alleged father are tested to determine if the alleged father is the biological father of the child.
The paternity test report should clearly indicate one of these two alternatives:
The tested individual is excluded and, therefore, he or she cannot be a biological parent of the child.
The tested individual is not excluded as the biological parent of the child. The statistics in this case should indicate the probability that the alleged individual can be a biological parent.
Paternity tests can prove with complete certainty that an individual is not a child's biological parent. However, there is no available test that can prove with 100 percent certainty that an individual is the child's biological parent. Paternity tests can guarantee at most 99 percent probability of paternity.
The following situations can result in reduced precision of the tests:
Interracial marriage or nonstandardized populations. This basically means it is more difficult to use highly specific DNA fingerprinting methods because of the large sample population. In those cases, the laboratories are forced to use generic tables in the analysis.
A biological parent is related to the alleged parent. Such cases are more serious in close relationships as in brothers, or father and son. The paternity test cannot exclude an individual when the alleged parents are identical twins.
A paternity test report should provide the following information:
Identification of the tested individuals and their pictures
Details of the procedures used for collecting of the biological samples
Description of the procedures of confidentiality and processing of samples
Description of the molecular techniques used in the test
Identification of the locus used in the test and of the alleles found in each tested individual
Number of analyzed loci
Reference to the genetic database used in the analysis
The digitized pictures of the genetic analyses that prove the presented results
In cases of exclusion, the paternal alleles not expected to be found in the alleged parent that indicate that he or she could not be the child's biological parent
In cases of inclusion, the three statistics of paternity used
The same considerations for individual identification presented in this chapter apply for the identification of both animals and plants. The same methods used in forensic DNA testing are also used in patent litigation to determine property rights for some crop varieties. The DNA profile of hybrid corn varieties was used in a long and controversial lawsuit between two seed companies in the United States that argued over the improper acquisition of parental inbred lines. The DNA analysis of the inbred lines was used to settle the case. Today, many seed companies use molecular identification for its elite germplasm.
Considering that all phenotypic characteristics, such as color of eyes, hair, skin, face format, and so on, are defined by the genome, many forensic scientists believe that in the future it will be possible to substitute for the composite picture drawn based on information reported by an eyewitness with a composite drawn on the basis of the analysis of biological samples left as evidence at the scene of a crime (see Figure 10-4). Some still speculate that in the near future computer software will compose the individual's picture automatically, considering the individual propensity for obesity, baldness, and other characteristics. However, an individual's physical appearance is the result not only of his or her genes, but also of environmental factors. Many characteristics are affected for a great number of genes. This type of technology is a long way off, but the unveiling of the human genome is allowing scientists to gain a better picture of how DNA influences each one of us, and is facilitating the recognition of each unique individual.
Considering that DNA analysis is a powerful identification technique, it should be used carefully. The sensitivity level of many DNA tests is so high that cells from a technician's hands or from a sneeze could contaminate the sample.
Therefore, care in the collection, custody, and manipulation of the biological sample is of great importance for the validity of these analyses. Finally, human beings can make mistakes. Technicians can mislabel a flask, change codes, change names, and so on. Due to the many possible errors, many laboratories use double reading in each step of the analysis. They also save part of the sample for possible reanalysis. Even so, mistakes will continue to happen, and in many cases it is left to skilled lawyers to question and criticize unexpected results.
Some scientists believe that in the future the DNA profile will be part of the personal identification used in identity cards (Figure 10-5).
