Written Laboratory Protocol

An essential element of any clinical or forensic DNA typing method is a detailed written laboratory protocol. Such a protocol should not only specify steps and reagents, but also provide precise instructions for interpreting results, which is crucial for evaluating the reliability of a method. Moreover, the complete protocol should be made freely available so that it can be subjected to scientific scrutiny.

Procedure For Identifying Patterns

There must be an objective and quantitative procedure for identifying the pattern of a sample. Although the popular press sometimes likens DNA patterns to bar codes, laboratory results from most methods of DNA testing are not discrete data, but rather continuous data. Typically, such results consist of an image—such as an autoradiogram, a photograph, spots on a strip, or the fluorometric tracings of a DNA sequence—and the image must be quantitatively analyzed to determine the genotype or genotypes represented in the sample. Quantitation is especially important in forensic applications, because of the ever-present possibility of mixed samples.

Patterns must be identified separately and independently in suspect and evidence samples. It is not permissible to decide which features of an evidence sample to count and which to discount on the basis of a comparison with a suspect sample, because this can bias one's interpretation.

Procedure For Declaring a Match

When individual patterns of DNA in evidence sample and suspect sample have been identified, it is time to make comparisons to determine whether they match. Whether this step is easy or difficult depends on the resolving power of the system to distinguish alleles. Some DNA typing methods involve small collections of alleles that can be perfectly distinguished from one another—e.g., a two-allele RFLP system based on a polymorphism at a single locus. Other methods involve large collections of similar alleles that are imperfectly distinguished from one another—e.g., the hypervariable VNTR systems in common forensic use, in which a single sample might yield somewhat different allele sizes on repeat measurements.1 It is easy to determine whether two samples match in the former case (assuming that the patterns have been correctly identified), but the latter case requires a match criterion—i.e., an objective and quantitative rule for deciding whether two samples match. For example, a match criterion for VNTR systems might declare a match between two samples if the restriction-fragment sizes lie within 3% of one another.

The match criterion must be based on the actual variability in measurement observed in appropriate test experiments conducted in each testing laboratory. The criterion must be objective, precise, and uniformly applied. If two samples lie outside the matching rule, they must be declared to be either ''inconclusive" or a "nonmatch." Considerable controversy arose in early cases over the use of subjective matching rules (e.g., comparison by eye) and the failure to adhere to a stated matching rule.

Identification of Potential Artifacts

All laboratory procedures are subject to potential artifacts, which can lead to incorrect interpretation if not recognized. Accordingly, each DNA typing method must be rigorously characterized with respect to the types of possible artifacts, the conditions under which they are likely to occur, the scientific controls for detecting their occurrence, and the steps to be taken when they occur, which can range from reinterpreting results to correcting for the presence of artifacts, repeating some portion of the experiment, or deciding that samples can be reliably used.

Regardless of the particular DNA typing method, artifacts can alter a pattern in three ways: Pattern A can be transformed into Pattern B, Pattern A can be transformed into Pattern A + B; and Pattern A + B can be transformed into Pattern B. It is important to identify the circumstances under which each transformation can occur, because only then can controls and corrections be devised. For example, RFLP analysis is subject to such artifacts as band shifting, in which DNA samples migrate at different speeds and yield shifted patterns (A→B), and incomplete digestion, in which the failure of a restriction enzyme to cleave at all restriction sites results in additional bands (A→A + B).

Some potential problems can be identified on the basis of the chemistry of DNA and the mechanism of detection in the genetic-typing system. Anticipation of potential sources of DNA typing error allows systematic empirical investigation to determine whether a problem exists in practice. If so, the range of conditions in which an assay is subject to artifact must be characterized. In either case, the results of testing for artifacts should be documented. Empirical testing is necessary, whether one is considering a new method, a new locus, a new set of reagents (probe or enzyme) for a pre-existing locus, or a new device. Under some circumstances, even small changes in procedure can change the pattern of artifacts.

Once potential artifacts have been identified, it is necessary to design scientific controls to serve as internal checks in each experiment to test whether the artifacts have occurred. Once the appropriate controls are identified, analysts must use them consistently when interpreting test results. If the appropriate control has not been performed, no result should be reported. When a control indicates irregularities in an experiment, the results in question must be considered inconclusive; if possible, the experiment should be repeated. A well-designed DNA typing test should be a matter of standardized, objective analysis.

Sensitivity to Quantity, Mixture, and Contamination

Evidence samples might contain very little DNA, might contain a mixture of DNA from multiple sources, and might be contaminated with chemicals that can interfere with analysis. It is essential to understand the limits of each DNA typing method under such circumstances.

Examination of a Control Pattern

Every Southern blot procedure should be applied to a known DNA sample (in addition to the evidence samples in question), to verify that the hybridization was performed correctly. If this control sample does not yield a clean result that shows the correct pattern for a particular hybridization, the result of the test hybridization should be discounted.

Single-Band Patterns

Sometimes, only a single band will be detected when two distinct alleles are present. That might occur because the second allele is so small that it has migrated off the end of the gel, because the second allele is similar in size to the first allele and thus is not resolved, or because the second allele is much larger, and larger fragments are preferentially lost in partially degraded samples.

When only a single band is found, the interpretation should always include the possibility that a second band has been missed—i.e., that the pattern is actually of a heterozygote, not a homozygote. (For statistical interpretation, the frequency of a single-band pattern should be taken to be the sum of the frequencies of all patterns containing this band. This is approximately twice the allele frequency of the band.) In some cases, it could be important to interpret the absence of a second larger fragment—e.g., when two samples match in a smaller band, but the questioned sample lacks a second larger band. That could arise either because the samples are from different persons or because the samples come from the same person but the questioned sample is partially degraded. Ideally, to distinguish these alternatives, one should determine whether a second larger band could have been detected in the questioned sample by hybridizing the membrane with a single-copy probe that detects an even larger monomorphic fragment—i.e., one that is constant in all humans. In contrast, it would not be sufficient simply to estimate the degree of degradation from the ethidium bromide staining pattern of the sample.

Anomalous Bands

A sample might show more than two bands for various reasons. E.g., the hybridization conditions were improper and caused the probe to hybridize to incorrect fragments; the probe was contaminated with another sequence, which caused it to recognize other fragments; the membrane was incompletely stripped after a previous use, so a pattern seen on the previous hybridization is still being detected; the restriction digestion did not proceed to completion, so the region recognized by the probe is present in incompletely cut fragments of multiple sizes; or the sample actually contains a mixture of multiple DNAs. The last example is extremely important to recognize, because it can bear importantly on a case. Whenever extra bands are observed, their origin should be determined.

The following clues provide a partial decision tree:

If the hybridization conditions were improper or the probe contaminated, the pattern in the control DNA should be seen to be incorrect; the hybridization should be repeated.

If the membrane was improperly stripped, the extra bands will be in the same location as in the previous hybridization and might be present in the control sample; the hybridization should be repeated.

If the restriction digestion was incomplete, one should see additional bands, even with the use of monomorphic probes that typically give only a single constant band. To ascribe extra bands to incomplete digestion, one should therefore perform such a hybridization. If incomplete digestion has occurred, the sample ideally should be re-extracted and redigested, and a new Southern blot should be prepared. If that is not possible, because there is too little sample, it will usually be difficult to get a reliable result.

If the samples are mixtures from more than one person, one should see additional bands for all or most polymorphic probes, but not for a single-copy monomorphic probe. Mixed samples can be very difficult to interpret, because the components can be present in different quantities and states of degradation. It is important to examine the results of multiple RFLPs, as a consistency check. Typically, it will be impossible to distinguish the individual genotypes of each contributor. If a suspect's pattern is found within the mixed pattern, the appropriate frequency to assign such a "match" is the sum of the frequencies of all genotypes that are contained within (i.e., that are a subset of) the mixed pattern.

Another possible cause of extra bands is leakage between adjacent sample lanes or misloading of two samples in a single lane. Such an occurrence can be exceedingly difficult to detect and could result in an incorrect conclusion. It is therefore important to leave a blank lane between a suspect sample and an evidence sample, so that leakage can be detected and will not lead to false-positive results.

Reporting of Anomalies

Examiners should document their interpretations of samples thoroughly in writing. They should note all observed bands and any questionable densities that they do not consider to be bands. Anomalous bands should be explained on the basis of appropriate control experiments of the sorts described above.

Amplification Conditions

The quality and specificity of amplification with PCR depends on the amplification conditions: the amplification cycling program (temperatures, mixture (e.g., primer, nucleotide, polymerase, and magnesium concentrations), times, and number of cycles), the composition of the amplification and the amount and nature of the target DNA in the sample (single-stranded or double-stranded).7 In some cases, results can vary among thermocyclers from different manufacturers, among thermocyclers of a single manufacturer, and even among different sample wells in a single machine. It is therefore essential that precise conditions be established for each typing system and that the system be thoroughly characterized for its sensitivity to variations in these conditions. If an assay yields spurious or confusing results under particular conditions, it may be necessary to prescribe strict condition limits or to discard the assay altogether. No PCR assay should be used until it has been rigorously characterized in this way.

Qualitative and Quantitative Fidelity

Ideally, PCR amplification products would faithfully represent the starting material in the sample—both qualitatively and quantitatively. But that is not always the case.

PCR amplification is known to result in misincorporation of nucleotides at the relatively low rate of less than one per 10,000 nucleotides per cycle.8 Amplification is usually performed on a sample that contains a large number of molecules; if the misincorporation is random, the low frequency of random errors will not be detected in most systems and will pose no problem for the typing result. Difficulties arise for systems in which the misincorporation is not random. For example, DNA sequences that contain tandem repeat sequences—such as the dinucleotide (CA)n or some VNTRs—present serious problems. Apparently, the DNA polymerase can slip during amplification, introduce or delete copies of the repeat, and produce a heterogeneous collection of fragments, often making interpretation difficult. That drawback is unfortunate: such simple sequence repeats tend to be highly polymorphic in the human population and so would seem to be useful for forensics. Because there is no way to predict which PCR assays will be subject to this problem, each assay must be thoroughly characterized.

In some cases, PCR can be qualitatively faithful but quantitatively unfaithful, because some alleles amplify more efficiently than others. A sample might contain a 50:50 mixture of two alleles and yield an amplified product with a 90:10 ratio.9 Differential amplification can arise through several mechanisms. It has been observed in the amplification of allelic products of different sizes (larger products tend to amplify less efficiently than shorter products) and in the amplification of sequences that differ significantly in GC content (because of differing denaturation efficiencies). In some cases, faithful amplification occurs at some temperatures and differential amplification at other temperatures.8 The possibility of differential amplification needs to be addressed in the design and development of amplification protocols for each genetic-marker system. The safeguards to ensure that differential amplification does not occur should be defined and documented.

Quantitative analysis of mixed samples with PCR might be problematic. Suppose that PCR amplification reveals four alleles in a sample, and alleles 1 and 2 give a stronger signal than alleles 3 and 4. A conclusion that the two stronger alleles correspond to one contributor with genotype 1/2 and the two weaker alleles to a contributor with genotype 3/4 would be justified only if one had demonstrated that the amplification and detection process yielded signals that were directly proportional to the initial quantities of the alleles. If the locus were subject to differential amplification, the conclusion might be unjustified. This underscores the importance of characterizing possible differential amplification.

Ideally, primer pairs should amplify only the desired target locus. However, nonspecific amplification can be seen, if one amplifies for extended cycle numbers. Limits on the cycle number might be required as a safeguard against nonspecific products.

Amplification Inhibition

Some forensic samples contain factors that inhibit amplification, either by binding to the target DNA or by inhibiting the polymerase. In particular, amplification inhibition is often seen with DNA from older bloodstains. It can usually be remedied by re-extracting the DNA to remove the inhibiting factor, by diluting the offending DNA, or by increasing the concentration of polymerase. There is no evidence that any of those procedures affects typing adversely. Nevertheless, the nature of inhibiting factors and the mechanism of the inhibition effect deserve additional study. Each PCR system should be thoroughly characterized on a range of simulated and known forensic samples, to document any effect on reliability.

Contamination

One of the most serious concerns regarding PCR-based typing is contamination of evidence samples with other human DNA. PCR is not discriminating as to the source of the DNA it amplifies, and it can be exceedingly sensitive. Potentially, amplification of contaminant DNA could lead to spurious typing results. Three sorts of contamination can be identified, as set forth below; each has its own solutions.

• Mixed samples. Some evidence samples occur as mixtures, e.g., sexual-assault evidence, which often contains a mixture of semen and vaginal fluids. In mixed samples that contain semen, it is possible to extract the sperm DNA and the DNA of vaginal epithelial cells separately. That allows the genetic contribution of the male and female to be distinguished. However, there is one important caveat: if the sperm fraction shows a genotype that matches that of the victim, one cannot conclude that this represents the genotype of the perpetrator, inasmuch as it could be due to residual vaginal epithelial cells. The problem should disappear as PCR-based assays for more loci become available. For other mixtures, such separation is not possible. For example, it is not possible to separate the DNA contributed by different persons in mixed bloodstains or in sexual-assault samples that involve two or more perpetrators. Mixed samples are a reality of the forensic world that must be accommodated in interpretation and reconstruction. As a rule, mixed samples must be interpreted with great caution. Their interpretation should always be based on results from multiple PCR assays, so that one can check for consistency across various loci. Interpretations based on quantity can be particularly problematic—e.g., if one saw two alleles of strong intensity and two of weak intensity, it would be improper to assign the first pair to one contributor and the second pair to a second contributor, unless it had been firmly established that the system was quantitatively faithful under the conditions used.
• Contamination from handling in the field and laboratory. It is conceivable that DNA can be transferred to evidence samples or reaction solutions through handling, either from the person doing the handling or in transfer from other evidence samples. There are no hard data on the amounts of DNA transferred by physical contact, but there are anecdotal reports of experimenters who contaminated their PCR mixtures with their own DNA. It is difficult to assess the likelihood of this sort of contamination. Steps should be taken to minimize it, such as handling samples with gloves and preparing solutions and processing samples in separate areas. Contamination of solutions can be recognized with appropriate positive-control and blank-control amplifications, which should be used routinely. When a stain composed of blood, semen, or other biological material is analyzed with PCR, it is important to analyze unstained materials next to the stain with PCR as a control for contamination.
• PCR product carryover contamination. The most serious problem is contamination of evidence samples and reaction solutions with PCR products from prior amplifications. Such products can contain a target sequence at a concentration a million times greater, and even a relatively small quantity could swamp the correct signal from the evidence sample. Even the simple act of flipping the top of a plastic tube might aerosolize enough DNA to pose a problem.

Many research and diagnostic laboratories have been afflicted with the problem of PCR carryover. Contamination risks can be minimized by strict adherence to sterile technique; the use of separate work areas for sample processing, solution preparation, amplification, and type testing; the use of separate pipettes in each area (pipettes are a major source of carryover contamination); and maintenance of a one-way flow of materials from the evidence-storage area to the sample-preparation area to the type-testing area.

Those precautions focus primarily on preventing PCR carryover contamination. But it has become clear that carryover products from the PCR reaction to another must also be eliminated. One way is to use the nucleotide dUTP in place of dTTP in all PCR reactions.10 PCR products made in this manner can be selectively destroyed by the enzyme uracil N-glycolase (UNG), which excises dUTP. Accordingly, all evidence samples would be treated before PCR amplification with UNG, to destroy contamination from previous PCR reactions. The method holds promise, although it has not yet been extensively tested in practice. Methods of detecting and preventing contamination from one PCR reaction to another in forensic laboratories are generally still in their early stages, and additional development should be encouraged.

As with contamination due to handling, carryover contamination can be signaled by the appearance of product in blank controls and of mixed or inappropriate types in samples and positive controls. Such controls should be used rigorously. Moreover, it should be remembered that the controls are useful for monitoring general contamination in the laboratory, not the accuracy of a particular experiment. If a blank control is positive in one experiment, it indicates a potential problem not just for that experiment, but for any experiment performed at about the same time—even in a laboratory contaminated with PCR carryover, blank controls do not necessarily become contaminated on every occasion. It will be wise to repeat all work with samples that have never been exposed to the PCR-typing laboratory.

In view of the problem of contamination due to handling and carryover, laboratories must incorporate contamination control into their standard operating procedures. And outbreaks of contamination and the steps taken to correct the problem should be documented.

One of the best safeguards against contamination is to have DNA typing independently performed in two laboratories, each starting with a piece of the unprocessed evidence sample. Given the inexpensiveness of typing, serious consideration should be given to independent replication of results—at least during the early stages of this technology.

Reverse Dot Hybridization

In forensic analysis today, the single PCR-based kit available uses reverse dot hybridization to detect variation at the HLA-DQ locus.11 Reverse dot hybridization is based on a yes-no detection scheme. Theoretically, the absence of a signal in a dot means that the test allele is not present in the DNA sample, and a signal in a dot indicates the presence of the test allele. An intermediate signal in a dot—a signal that is considerably less intense than a second signal in the test hybridization—can result from a second DNA sample's contaminating the test sample, technical variation in the conditions of analysis (e.g., hybridization temperature), or true heterozygosity for the allele that produces the intermediate signal. Usually, such problems can be resolved through repeat experiments or by comparing results from a number of loci with many alleles (such loci could shed light on the nature of mixtures). If the DNA sample of a crime victim contains the allele in question, it would suggest contamination as the source. If repeat of the hybridization and washing procedures eliminates the intermediate signal, it would suggest its spurious nature. Rarely, the origin of an intermediate hybridization signal might remain unresolved; if the type of the sample is still questionable, the data should be discarded.

Other Detection Methods

When restriction-enzyme digestion of a PCR product is necessary to demonstrate alleles, the major pitfall is incomplete digestion. One can control the problem in the test sample by using a restriction enzyme for which a constant site that produces a fragment of constant size exists in the test fragment. For example, suppose that digestion of a 500-bp PCR fragment by HaeIII yields a constant 300-bp fragment and either a 200-bp fragment or 120-bp and 80-bp fragments; a poor yield of the 300-bp fragin a particular sample would suggest incomplete digestion of the PCR product.

Allele-specific amplification has been used in DNA diagnosis of genetic disease. Diagnosis can be based solely on absence of an amplification product, so it is a difficult technique to control adequately. Absence of a fragment can indicate either failure of the amplification procedure or absence of the allele in question from the test sample. If the former applies, a typing error would result. For this reason, the committee recommends that this method not be used for forensic analysis.

DNA sequence analysis of PCR products is commonly carried out either manually or with automation. Some ambiguity of nucleotide sequence at one or more positions is common and can signal DNA contamination or a technical problem in the analysis. Another important problem occurs when the DNA sequence of a fragment demonstrates variation at more than one position in the nucleotide sequence. Because both alleles at a locus are sequenced in the procedure, it is difficult to determine what fraction of the variation is contributed by each allele. For example, if heterozygosity is observed at two positions in a sequence, one cannot know, without further experimentation, whether one allele contains both variants or each allele contains one.

New methods of detection of PCR products will surely be devised. Well-controlled, extensive studies of the methods will be required before their use in forensic science, and the quality-assurance procedures described in Chapter 4 will be important to ensure their rigorous testing and reliability.

Use of Kits

One commercial kit for forensic PCR analysis has been marketed. Other such kits will probably be ready for commercial markets soon. The committee sees a potential for introduction of unreliable kits and the misuse of kits. The existence of a kit suggests ease of use and low chance of technical error. The committee believes that nonexpert laboratories will run a significant chance of error in using kits. We therefore recommend that a standing committee (discussed later in this chapter) consider the issue of regulatory approval of kits for commercial use in forensic DNA analysis. Even though no precedent exists for regulation of tests in forensic applications, we believe that it might be necessary for a government agency to test and approve kits for DNA analysis before their actual forensic use.