DNA Technology in Forensic Science (1992) / Chapter Skim
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2 DNA TYPING: TECHNICAL CONSIDERATIONS
2 DNA TYPING: TECHNICAL CONSIDERATIONS
Pages 51-73
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From page 51... ...
There is no scientific dispute about the validity of the general principles underlying DNA typing: scientists agree that DNA varies substantially among humans, that variation can be detected in the laboratory, and that DNA comparison can provide a basis for distinguishing samples from different persons. However, a given DNA typing method might or might not be scientifically appropriate for forensic use.
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From page 52... ...
Instead, our main goal is to provide a general framework for the evaluation of any DNA typing method. ESSENTIALS OF A FORENSIC DNA TYPING PROCEDURE Scientific Foundations The forensic use of DNA typing is an outgrowth of its medical diagnostic use analysis of disease-causing genes based on comparison of a patient's DNA with that of family members to study inheritance patterns of genes or with reference standards to detect mutations.
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From page 53... ...
We outline below the principal issues that must be addressed for each DNA typing procedure. Written Laboratory Protocol An essential element of any clinical or forensic DNA typing method is a detailed written laboratory protocol.
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From page 54... ...
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)
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From page 55... ...
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.
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From page 56... ...
Without the benefit of open scientific scrutiny, some testing laboratories initially used methods (for such fundamental steps as identifying patterns, declaring matches, making comparison with a databank, and correcting for band shifting) that they later agreed were not experimentally supported.
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From page 57... ...
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.
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From page 58... ...
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.
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From page 59... ...
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.
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From page 60... ...
The committee considers it desirable for all samples to be tested for band shifting by hybridization with monomorphic probes that cover a wide range of fragment sizes in the gel. That approach will eliminate the rare production of a match by shifting of bands in an evidence sample to the same positions as in a suspect sample.
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From page 61... ...
For the present, several laboratories have decided against attempting quantitative corrections; samples that lie outside the match criterion because of apparent band shifting are declared to be "inconclusive." The committee urges further study of the problems associated with band shifting. Until testing laboratories have published adequate studies on the accuracy and reliability of such corrections, we recommend that they adopt the policy of declaring samples that show apparent band shifting to be "inconclusive." The committee recommends that all measurement data be made readily available, including the computer-based images and records.
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From page 62... ...
For the calculation to be valid, the same match criterion must be applied in screening the population databank and in comparing the forensic samples. Some testing laboratories originally used less stringent rules for declaring a match between forensic samples and more stringent rules for determining the frequency of matching alleles in the databank; the effect was an overstatement of the probability of obtaining a match by chance.
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From page 63... ...
Accordingly, testing laboratories should measure DNA samples before analysis (with accurate devices, such as fluorometers, as well as with ethidium-stained "yield" gels) and should use only the quantity of DNA required for reliable Southern blot analysis.
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From page 64... ...
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.
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From page 65... ...
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
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From page 66... ...
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.
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From page 67... ...
Issues Related to Detection of Amplified Product Variation after PCR amplification can be detected in several ways. The most popular detection schemes for nonforensic analyses are reverse dot hybridization, analysis of PCR products for size variation with gel electro
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From page 68... ...
Differential amplification could result in the typing of a true heterozygote as a homozygote, or the low level of hybridization of the second allele could suggest the presence of a contaminating DNA in the test sample. A repeat PCR amplification and analysis might be successful and pinpoint a problem in the first amplification procedure.
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From page 69... ...
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.
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From page 70... ...
Considerable advances in the use of PCR in forensic analysis can be expected soon; the method has enormous promise. NATIONAL COMMITTEE ON FORENSIC DNA TYPING Forensic DNA typing is advancing rapidly.
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From page 71... ...
NCFDT would consist primarily of molecular geneticists, population geneticists, forensic scientists, and additional members knowledgeable in law and ethics. Its charges would be to provide guidance about the power and limitations of DNA typing methods, to identify potential problems and their solutions, to provide guidance about whether new technologies are ready for practical use in the forensic laboratory, and to provide advice concerning the regulation of kits for forensic DNA typing.
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From page 72... ...
-Before a new DNA typing procedure can be used, it must have not only a solid scientific foundation but also a solid base of experience. · Regarding RFLP-based typing, the committee makes a number of technical recommendations, including specific recommendations about the choice of probes, the use of ethidium bromide in gels, controls for anomalous bands, measurement of fragment sizes, controls for band shifting, match criteria, and sample retention.
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From page 73... ...
eds. Forensic DNA technology.
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