DNA as Evidence — The Technology of Identification
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《新英格兰医药杂志》
In 1983, in a village near Leicester, England, a local girl named Lynda Mann was found raped and murdered. Three years later, a second girl, Dawn Ashworth, was found dead under similar circumstances. The similarities between the two cases led the police to believe that the same person had committed both crimes. After extensive inquiries, an arrest was made. The suspect confessed to the murder of Lynda Mann but denied having killed Dawn Ashworth. Convinced that they had the right man, the police approached Sir Alec Jeffreys, a professor of genetics at the University of Leicester, with a request to conduct tests using a new method that he called "DNA fingerprinting," which had not yet been used in a real case.
The results were surprising: the suspect was exonerated, and the DNA profiles in the two murder cases were the same, indicating that a single, unknown person had committed both crimes. This finding led to the screening of all 5000 men in the area, using both conventional blood-group methods and DNA testing. The screening failed to identify a suspect — because, as it turned out, the perpetrator, Colin Pitchfork, had paid a colleague to give a DNA sample in his place. When the colleague was overheard bragging to a friend about the incident, Pitchfork was quickly apprehended, analysis of a DNA sample confirmed his guilt in both murders, and he was duly convicted in 1988.1
Thus, the first criminal case in which DNA was used provided a vivid demonstration of the method's potential — not only for convicting the guilty but also for exonerating the innocent. It also demonstrated for the first time that a DNA fingerprint could be used to find a perpetrator from within a population.
In 1985, a year after the development of DNA fingerprinting, the polymerase chain reaction (PCR) was discovered.2 The discovery would revolutionize the field of molecular biology, though the method would not come into routine use in forensic cases until the early 1990s, since new platforms and biochemical tools were needed in order to take full advantage of the potential of PCR. In particular, new automation technology was key, and the advent of the automated fluorescent DNA sequencer in the early 1990s was a major step forward. More generally, forensic DNA analysis has benefited substantially from the Human Genome Project, for the genome could be sequenced only with automated equipment that permitted high-throughput processing. Because forensic science could use the same equipment and biochemical tools that gene sequencing used, new methods were rapidly developed in the early 1990s that would have been considered impossible just a few years earlier.
Perhaps the best example of this adjunct benefit of genomics was the development of national DNA databases. Since its inception in 1995, the National DNA Database for England and Wales has expanded to include more than 2.75 million reference DNA profiles, against which all specimens obtained from the scene of a crime ("crime stains") are routinely compared.3 The likelihood that a match will be found is approximately 30 percent. Many other countries have since followed suit, and the benefits of such databases are considerable, since persons who commit serious crimes such as murder usually have a previous criminal record. The United Kingdom's policy permits the collection of DNA profiles from all convicted criminals, as well as from anyone suspected of committing a crime that could lead to a prison sentence — and the law allows authorities to retain the DNA profile even if the suspect is found innocent. Consequently, persons who later commit more crimes can be identified and apprehended quickly.
DNA-profiling technology takes several forms. Short tandem repeats (STRs) are universally used as the workhorses of national DNA databases. The length of these stretches of DNA varies from person to person, making them useful as genetic identifiers similar to the bar codes that identify items for sale (see diagram). In the United States, 13 STRs are amplified together in a multiplex PCR. When a complete DNA profile has been obtained, the probability of a match with a randomly chosen person is less than 1 in 1 trillion. Profiling efforts in European countries apply the same principles but generally use fewer STRs; for example, in the United Kingdom, 10 STRs are used, resulting in a match probability of less than 1 in 1 billion.
Schematic Diagram of a Short-Tandem-Repeat Marker.
Interspersed throughout the human genome are stretches of DNA comprising short blocks of repeated sequence known as short tandem repeats (STRs). Typically, "forensic" STRs include four bases, arranged in a specific order. Each STR site that is used for forensic analysis has an internationally recognized identifying name. Here, the STR locus is called HUMTH01, and each block consists of the DNA bases thymine, cytosine, adenine, and guanine. In this example, the person carries 8 repeats on one chromosome and 11 repeats on the other (hence, HUMTH01 8.11). The number of repeats carried by each allele is determined by amplifying the DNA with the polymerase chain reaction and then measuring the size of the amplification products. A total of 3 to 14 repeats may be observed at this particular site. Ten separate STR sites, each denoted by two digits, are used in analyses performed in the United Kingdom. Consequently, any person may be defined by a code comprising 20 digits, which can easily be stored in a searchable computer database. The rarity of each genetic combination is estimated on the basis of population surveys and is used to calculate the strength of the evidence, estimated as the likelihood that a random (unrelated) person has the same DNA profile as a suspect.
With the use of conventional STR-based methods, it is possible to analyze small samples that consist of approximately 60 cells; sensitivity can be improved simply by increasing the number of PCR cycles. Theoretically, just a single cell is required to obtain a result, although more are usually analyzed; analysis of such small samples is known as low-copy-number DNA profiling.4 Caution is required when using this technique, however, because when sensitivity is increased, the potential for analyzing contaminant DNA from a person unrelated to the crime (especially, with contemporary DNA, from a crime-scene investigator or a scientist working on the case) increases as well. Moreover, DNA profiling does not tell us when the sample was deposited, so considerable attention must be paid to the use of ultraclean laboratories and handling with DNA-free materials at all stages of the analysis. The use of low-copy-number DNA profiling has expanded the range of evidentiary samples that can be analyzed, permitting the recovery of a DNA profile even from the skin cells found in a fingerprint.
Low-copy-number analysis does not work in every instance, however. Certain types of evidence, such as hair shafts, have little or no nuclear DNA, and STR analysis often fails with highly degraded materials such as bone.
Fortunately, mitochondrial DNA (mtDNA) may also be used. This type of DNA is found in the cytoplasm of the cell, where there are high copy numbers. However, mtDNA is inherited in the vast majority of cases from the mother, is much smaller than nuclear DNA, and lacks the discriminatory power of STRs; moreover, data derived from mtDNA are incompatible with those in national DNA databases. Hence, mtDNA offers both advantages and disadvantages to the investigator. The main advantage is that it can give results when all other techniques fail — which is why mtDNA analysis is the preferred method for ancient samples. Because of its nearly exclusive inheritance through the female line, mtDNA can help to solve historical mysteries revolving around maternal lineage. Perhaps the best example is the 1994 analysis of remains thought to be those of the Russian royal family, the Romanovs, which revealed mtDNA that matched samples taken from living descendants of the maternal line, such as Prince Philip of Britain.
The male counterpart of mtDNA is the Y chromosome. Unlike mtDNA, it resides in the nucleus and does not have a high cellular copy number. Both Y-specific STRs and single-nucleotide polymorphisms (SNPs) — single-base variants that occur at specific positions in the genome — can be used to characterize the Y-chromosome haplotype. Sometimes in specimens combining material from persons of both sexes, the male component can be completely masked by the female DNA, rendering identification impossible. Under these circumstances, analysis of the Y chromosome is warranted. However, the discriminating power of such analysis is low (as in the case of mtDNA), and the results are incompatible with STR databases. But Y-chromosome DNA is useful in the investigation of historical mysteries involving paternal lineages. The use of mtDNA and Y-chromosome DNA can yield population-specific genetic signatures that offer investigative leads in unsolved cases in which there are no firm suspects. The Y chromosome might also be used as a marker of family surname; people with unusual surnames often have a common genealogy.
The World Trade Center disaster has illustrated the importance of having the entire gamut of techniques available in order to maximize the chances of identifying victims: STRs, mtDNA, and SNPs were all used. More than 19,000 samples of human remains were recovered from more than 2700 victims, and many of these samples were highly degraded, having been subjected to considerable pressure and fire.5 To date, more than 1500 victims have been positively identified — an undertaking that would have been impossible without DNA technology. DNA profiling is also playing a major role in the identification of victims of the recent Asian tsunami.
The efforts to identify bodies after these disasters underscore the importance of having national DNA databases that can accommodate new types of data, without which there is a danger of becoming locked into old technology. The best way to facilitate change (given that the overriding concern is the maintenance of compatibility) is an important and pressing issue.
Although it is difficult to make predictions, I believe that the use of STRs will probably remain the system of choice for the foreseeable future, because of its advantages over the use of SNPs, including a relative ease of interpretation. SNPs may find a special niche in the analysis of highly degraded material and in the context of massive disasters. Undoubtedly, the usefulness of both methods will benefit from new biochemical tools and new platforms such as DNA microarrays. Automation, miniaturization, and expert systems will all have critical roles in advancing forensic analysis in the coming years, just as they did during the sequencing of the human genome.(Peter Gill, Ph.D.)
The results were surprising: the suspect was exonerated, and the DNA profiles in the two murder cases were the same, indicating that a single, unknown person had committed both crimes. This finding led to the screening of all 5000 men in the area, using both conventional blood-group methods and DNA testing. The screening failed to identify a suspect — because, as it turned out, the perpetrator, Colin Pitchfork, had paid a colleague to give a DNA sample in his place. When the colleague was overheard bragging to a friend about the incident, Pitchfork was quickly apprehended, analysis of a DNA sample confirmed his guilt in both murders, and he was duly convicted in 1988.1
Thus, the first criminal case in which DNA was used provided a vivid demonstration of the method's potential — not only for convicting the guilty but also for exonerating the innocent. It also demonstrated for the first time that a DNA fingerprint could be used to find a perpetrator from within a population.
In 1985, a year after the development of DNA fingerprinting, the polymerase chain reaction (PCR) was discovered.2 The discovery would revolutionize the field of molecular biology, though the method would not come into routine use in forensic cases until the early 1990s, since new platforms and biochemical tools were needed in order to take full advantage of the potential of PCR. In particular, new automation technology was key, and the advent of the automated fluorescent DNA sequencer in the early 1990s was a major step forward. More generally, forensic DNA analysis has benefited substantially from the Human Genome Project, for the genome could be sequenced only with automated equipment that permitted high-throughput processing. Because forensic science could use the same equipment and biochemical tools that gene sequencing used, new methods were rapidly developed in the early 1990s that would have been considered impossible just a few years earlier.
Perhaps the best example of this adjunct benefit of genomics was the development of national DNA databases. Since its inception in 1995, the National DNA Database for England and Wales has expanded to include more than 2.75 million reference DNA profiles, against which all specimens obtained from the scene of a crime ("crime stains") are routinely compared.3 The likelihood that a match will be found is approximately 30 percent. Many other countries have since followed suit, and the benefits of such databases are considerable, since persons who commit serious crimes such as murder usually have a previous criminal record. The United Kingdom's policy permits the collection of DNA profiles from all convicted criminals, as well as from anyone suspected of committing a crime that could lead to a prison sentence — and the law allows authorities to retain the DNA profile even if the suspect is found innocent. Consequently, persons who later commit more crimes can be identified and apprehended quickly.
DNA-profiling technology takes several forms. Short tandem repeats (STRs) are universally used as the workhorses of national DNA databases. The length of these stretches of DNA varies from person to person, making them useful as genetic identifiers similar to the bar codes that identify items for sale (see diagram). In the United States, 13 STRs are amplified together in a multiplex PCR. When a complete DNA profile has been obtained, the probability of a match with a randomly chosen person is less than 1 in 1 trillion. Profiling efforts in European countries apply the same principles but generally use fewer STRs; for example, in the United Kingdom, 10 STRs are used, resulting in a match probability of less than 1 in 1 billion.
Schematic Diagram of a Short-Tandem-Repeat Marker.
Interspersed throughout the human genome are stretches of DNA comprising short blocks of repeated sequence known as short tandem repeats (STRs). Typically, "forensic" STRs include four bases, arranged in a specific order. Each STR site that is used for forensic analysis has an internationally recognized identifying name. Here, the STR locus is called HUMTH01, and each block consists of the DNA bases thymine, cytosine, adenine, and guanine. In this example, the person carries 8 repeats on one chromosome and 11 repeats on the other (hence, HUMTH01 8.11). The number of repeats carried by each allele is determined by amplifying the DNA with the polymerase chain reaction and then measuring the size of the amplification products. A total of 3 to 14 repeats may be observed at this particular site. Ten separate STR sites, each denoted by two digits, are used in analyses performed in the United Kingdom. Consequently, any person may be defined by a code comprising 20 digits, which can easily be stored in a searchable computer database. The rarity of each genetic combination is estimated on the basis of population surveys and is used to calculate the strength of the evidence, estimated as the likelihood that a random (unrelated) person has the same DNA profile as a suspect.
With the use of conventional STR-based methods, it is possible to analyze small samples that consist of approximately 60 cells; sensitivity can be improved simply by increasing the number of PCR cycles. Theoretically, just a single cell is required to obtain a result, although more are usually analyzed; analysis of such small samples is known as low-copy-number DNA profiling.4 Caution is required when using this technique, however, because when sensitivity is increased, the potential for analyzing contaminant DNA from a person unrelated to the crime (especially, with contemporary DNA, from a crime-scene investigator or a scientist working on the case) increases as well. Moreover, DNA profiling does not tell us when the sample was deposited, so considerable attention must be paid to the use of ultraclean laboratories and handling with DNA-free materials at all stages of the analysis. The use of low-copy-number DNA profiling has expanded the range of evidentiary samples that can be analyzed, permitting the recovery of a DNA profile even from the skin cells found in a fingerprint.
Low-copy-number analysis does not work in every instance, however. Certain types of evidence, such as hair shafts, have little or no nuclear DNA, and STR analysis often fails with highly degraded materials such as bone.
Fortunately, mitochondrial DNA (mtDNA) may also be used. This type of DNA is found in the cytoplasm of the cell, where there are high copy numbers. However, mtDNA is inherited in the vast majority of cases from the mother, is much smaller than nuclear DNA, and lacks the discriminatory power of STRs; moreover, data derived from mtDNA are incompatible with those in national DNA databases. Hence, mtDNA offers both advantages and disadvantages to the investigator. The main advantage is that it can give results when all other techniques fail — which is why mtDNA analysis is the preferred method for ancient samples. Because of its nearly exclusive inheritance through the female line, mtDNA can help to solve historical mysteries revolving around maternal lineage. Perhaps the best example is the 1994 analysis of remains thought to be those of the Russian royal family, the Romanovs, which revealed mtDNA that matched samples taken from living descendants of the maternal line, such as Prince Philip of Britain.
The male counterpart of mtDNA is the Y chromosome. Unlike mtDNA, it resides in the nucleus and does not have a high cellular copy number. Both Y-specific STRs and single-nucleotide polymorphisms (SNPs) — single-base variants that occur at specific positions in the genome — can be used to characterize the Y-chromosome haplotype. Sometimes in specimens combining material from persons of both sexes, the male component can be completely masked by the female DNA, rendering identification impossible. Under these circumstances, analysis of the Y chromosome is warranted. However, the discriminating power of such analysis is low (as in the case of mtDNA), and the results are incompatible with STR databases. But Y-chromosome DNA is useful in the investigation of historical mysteries involving paternal lineages. The use of mtDNA and Y-chromosome DNA can yield population-specific genetic signatures that offer investigative leads in unsolved cases in which there are no firm suspects. The Y chromosome might also be used as a marker of family surname; people with unusual surnames often have a common genealogy.
The World Trade Center disaster has illustrated the importance of having the entire gamut of techniques available in order to maximize the chances of identifying victims: STRs, mtDNA, and SNPs were all used. More than 19,000 samples of human remains were recovered from more than 2700 victims, and many of these samples were highly degraded, having been subjected to considerable pressure and fire.5 To date, more than 1500 victims have been positively identified — an undertaking that would have been impossible without DNA technology. DNA profiling is also playing a major role in the identification of victims of the recent Asian tsunami.
The efforts to identify bodies after these disasters underscore the importance of having national DNA databases that can accommodate new types of data, without which there is a danger of becoming locked into old technology. The best way to facilitate change (given that the overriding concern is the maintenance of compatibility) is an important and pressing issue.
Although it is difficult to make predictions, I believe that the use of STRs will probably remain the system of choice for the foreseeable future, because of its advantages over the use of SNPs, including a relative ease of interpretation. SNPs may find a special niche in the analysis of highly degraded material and in the context of massive disasters. Undoubtedly, the usefulness of both methods will benefit from new biochemical tools and new platforms such as DNA microarrays. Automation, miniaturization, and expert systems will all have critical roles in advancing forensic analysis in the coming years, just as they did during the sequencing of the human genome.(Peter Gill, Ph.D.)