DNA Testing: Introduction


	DNA Testing: Introduction and Index http://arbl.cvmbs.colostate.edu/hbooks/genetics/medgen/dnatesting/ -------------------------------------------------------------------------------- The focus of most criminal investigations is on linking evidence from the crime scene to suspects, and for more than a century, science has played an increasingly important role in this process. Fingerprinting was applied to criminal investigations beginning in the 1880's. Shortly after the principle of ABO blood typing was reported in 1900, its relevance to forensic investigations became apparent. In the 1960's human leukocyte antigen (HLA) typing became the premier serologic tool for personal identification, although in practice, it was useful for only a small percentage of samples. Finally, the 1980's ushered in the age of DNA testing, which permits investigators to perform almost unbelieveable feats of identification. With current techniques, it is possible for a single person to be differentiated from all the people that have ever lived using DNA from a single hair root. The principles and techniques used for forensic DNA typing are also quite useful for other purposes. DNA profiles are widely used in resolving issues of parentage in man and animals, and are rapidly replacing serologic analysis (i.e. blood typing) for that purpose. Additionally, DNA testing is an indispensible tool for positional cloning, a technique by which a previously unknown gene is identified by finding associations or links between DNA markers and the inheritance of a disease. Strengths, Limitations and Controversies of DNA Testing -------------------------------------------------------------------------------- DNA testing has a number of real advantages over serological methods such as blood typing and HLA analysis for use in forensic investigations: Unsurpassed discriminatory potential: Complete blood group testing allows discrimiation of one person in several thousand and HLA typing one in several million. DNA typing can routinely provide exclusion probabilities on the order of one in billions. Exquisite sensitivity: Standard DNA typing can be conducted with DNA extracted from the roots of a few hairs. In contrast to proteins, DNA can be amplified, and by using polymerase chain reaction methods, even smaller sample sizes are adequate. One important consequence of this great sensitivity is that it allows rather small samples to be split and submitted for testing to more than one laboratory, which can identify laboratory errors more commonly serves to nullify objections that laboratory erros were committed. Application to any body tissue: Complete serologic testing requires blood, but because DNA testing can be conducted with any sample having nucleated cells, it is applicable to such samples as hairs, semen, urine and saliva. DNA is stable in comparison to proteins: In comparision to protein, DNA in quite resistant to degradation by common environmental insults. DNA testing can therefore often be performed on samples that have been exposed to detergents, acids and bases, gasoline, salt, and bacterial contamination. Importantly, DNA is also long-lived in comparison to protein. It does degrade over time, but reliable information can be obtained from samples that are years old. Overall, DNA is remarkably robust as a sample for foresic testing, which, for example, has allowed it to be used on skeletenized remains for identification of soldiers missing in action. DNA typing has often been portrayed in the media and the courtroom as a controversial technology, largely because it has been so characterized by many defense attorneys. When DNA evidence demonstrates that the odds that someone other than your client committed the crime are one in a billion, there is really nowhere else to go but to attack the basic technology of DNA testing. With increased experience and standardization of testing methods, these assaults are heard less frequently. There have been some scientifically legitimate criticisms of DNA testing, based on concerns about allele frequencies in certain populations. These frequencies are used in calculation of calculating probability of identification. The worry was that the chances of a random match may be higher than stated because the database used was inappropriate for the subpopulation of people containing the suspect. For example, the frequency of a specific allele under test may be 4% in Asians instead of 1% as it is in Northern Europeans. However, most experts concluded that such differences in allele frequency have rather little impact on the final diagnosis - it makes little difference whether the probability of innocence or guilt is one in 10 million or one in 100 million. Nevertheless, the basic premise of the argument is valid and has been incorporated into recommendations about how forensic DNA testing be conducted and interpreted. DNA Polymorphisms: The Basis of DNA Typing -------------------------------------------------------------------------------- All DNA testing is based on the observation that the genome of each person or animal is unique (except of course identical twins). The myriad of small and large differences in nucleotide sequence among individuals are known as DNA polymorphisms. Two fundamentally different types of polymorphisms have been widely exploited for DNA typing: tandem repeats and retriction fragment length polymorphisms. Tandemly Repeated DNA The eukaryotic genome is densely populated with islands of short sequences that are repeated over and over in small to large arrays called minisatellites and microsatellites. Another term commonly used to describe these sequences is variable number tandem repeats or VNTRs. For a given repetitive locus, the number of repeats is highly variable among individuals and heterozygosity is high (i.e. the number of repeats at the locus is usally different on the two pairs of chromosomes of one individual). Analyzing the number of repeats at one or more such loci provides a highly sensitive measure of individual identity and is the technique most often used for forensic DNA typing.


	The figure above depicts a VNTR locus with 8 versus 3 repeats. Digestion with the restriction enzyme Hinf1 will yield fragments of two lengths that will hybridize to the (red) probe. Variability in Restriction Sites Single base changes in DNA often introduce or obliterate a restriction enzyme site. For example, a mutation that changes the sequence AGATCC to GGATCC will introduce a BamH1 site into that segment of DNA. Such sequence variability is exceedingly common, particularly in non-coding regions of DNA, and determining whether or not a particular group of restriction sites exists in DNA is a very sensitive means of differentiation one individual from many others. Because polymorphisms in a restriction sites translates into variability in the length of fragments after digestion of DNA with that restriction enzyme, these DNA markers are called restriction fragment length polymorphisms or RFLPs.


	The figure depicts a BamH1 RFLP in which the top strand of DNA has only two GGATCC sites while the lower has three. Digestion followed by hybridization with the (red) probe will reveal two fragments of differing length. Techniques for DNA Testing -------------------------------------------------------------------------------- DNA typing is performed by demonstrating differences in length of specific DNA sequences. This can be done by digestion of DNA with restriction enzyme(s), followed by Southern blot hybridization using a probe specific for the polymorphic site. Polymerase chain reaction (PCR) techniques are becoming widely applied to the same task, and have several advantages over Southern blotting - for example, much less DNA is required and in many cases, typing can be done using partially degraded DNA. For PCR analysis, the primers are designed to flank the VNTR locus and the size of the PCR product is dependent on the number of repeats. The general term "DNA fingerprinting" is used to describe all these procedures for characterizing VNTRs, RFLPs and other sequence polymorphisms. Two conceptually different types of fingerprinting are commonly performed for either VNTR or RFLP analyses: Single locus DNA fingerprinting: Polymorphism at a single locus is characterized, usually through use of a specific probe or specific PCR primers. Because the single loci detected by this method are characterized, one obtains a DNA genotype from single locus methods. Multilocus DNA fingerprinting: Polymorphism at multiple loci is simultaneously identified. This can be performed by application of a mixture of single locus probes or application of a single probe that identifies multiple similar sequence polymorphisms. In the latter case, one is detecting unidentified fragments of DNA and the result is therefore a DNA phenotype rather than a genotype. Each of these methods has advantages over the other in specific situations. For example, single locus but not multilocus methods are useful when the DNA is degraded and for mixed (i.e. victim and pertetrator) samples. On the other hand, multilocus fingerprinting typically provides more information per sample than single locus fingerprints. Examples of both types of fingerprinting follow.


	Example 1: Single Locus Fingerprinting Minisatellite fingerprinting to demonstrate kinship using mixtures of two or three single locus probes (probe sets 1 and 2). The loci detected in the child (C) are clearly a composite of those present in the mother (M) and father (F). Example 2: Multilocus Fingerprinting Microsatellite fingerprinting to establish parentage. The probe, (CAG)5, recognizes a large number of loci. Examine the bands detected in DNA from the child that are not detected with DNA from the mother. Which male is the biologic father of the child? Example 3: Multilocus Fingerprinting Multilocus fingerprinting to match trace evidence from a crime with suspects. Which suspect matches the specimen?


	Example 2


	Example 1


	Example 3


	Applications of DNA Testing -------------------------------------------------------------------------------- The purpose of DNA typing in forensic medicine is to match a sample from the crime site with a suspect. Technically, application of the techniques described above do not actually determine whether the sample came from the suspect.


	Rather, statistical analysis of the test results yield a probability that the sample did not come from the suspect, and with DNA typing, that probability can be so miniscule as to be certain. Importantly, DNA testing has proven to be as powerful for exonerating suspects as it has for convicting them. Indeed, about one in three cases reported by FBI laboratories, DNA testing proved that the current suspect could not have committed the crime, which in many cases was followed by apprehension and conviction of the true perpetrator. A great diversity of criminal detection has benefited from DNA testing, and it has been especially valuable in solving rape and murder cases. Additional examples include robbery, assault, kidnapping, car accidents, extortion and blackmail. It also has been successfully applied to parentage determination and useful in settling certain immigration disputes that hinges on proving family relationships.