11 DNA Profiling, Forensics, and Other Applications Brief Chapter Outline I. Satellite DNA: A. Repetitive DNA B. Microsatellites C. Minisatellites D. Macrosatellites

11 DNA Profiling, Forensics, and Other Applications Brief Chapter Outline I. Satellite DNA: A. Repetitive DNA B. Microsatellites C. Minisatellites D. Macrosatellites II.Population Genetics and Alleles III.Multilocus Minisatellite VNTR IV. Single-Locus VNTR for STR V. Restriction Fragment Length Polymorphisms VI. Methods of DNA Profiling VII.Technical Considerations: 1. DNA digestion2. Gel electrophoresis3. Probe selection VIII. Polymerase Chain Reaction IX. Digital DNA Typing X. Population Comparisons XI. Admissibility of Scientific Evidence in a Court of Law:A. Frye StandardB. Daubert Standard XII. Databases A. Cause for Concern? What About Privacy? XIII. Other Methods of Profiling DNA and Their Applications A.Random Amplification of Polymorphic DNA B.Amplified Fragment Length Polymorphism C.Single-Strand Conformation Polymorphism D. Single Nucleotide Polymorphism E. Mitochondrial DNA and the Y Chromosome: 1. Mitochondrial Eve2. Y Chromosomal Adam XIV. Forensic Archaeology A. Biotech Revolution: DNA Profiling and Human Remains

I.Introduction and History of DNA Profiling. A. Since the late 1960s, protein polymorphisms such as the ABO blood groups and MHC antigens have been used to determine genetic differences among individuals. B. Because protein tests have limited variability, DNA testing was developed in 1985 by Alec Jeffreys in England. Testing helped to solve an immigration dispute in late 1985. C. Jeffreys testing system was used to solve rape and murder cases in 1986, and in 1987 DNA fingerprinting was admitted as evidence in both the UK and the United States. D. The US Office of Technology Assessment (OTA) determined that DNA fingerprinting was a scientifically valid way of determining individuality. E. In 1992, the US National Research Council confirmed the scientific validity of DNA testing and issued guidelines for its use. DNA evidence is now admissible in court. F. Many commercial laboratories provide DNA fingerprinting services for forensic and parentage analysis, such as Orchid Cellmark.

II. Satellite DNA. A. Repetitive DNA. 1. In two major classes: a) Tandemly repetitive sequences of satellite DNA (about 10% of the genome). b) Interspersed repetitive DNA makes up 520% of the genome. They are scattered throughout the genome and are further subdivided: (1) SINES (short interspersed elements) are sequences of fewer than 500 bp. (2) LINES (long interspersed elements) are sequences of 500 bp or more. 2. Tandemly repeated DNA comprise long macrosatellite near the centromeres and shorter minro- and mini-satellite DNA scattered within the the genome. Only found in eukaryotic organisms, without a known biological function. 3. Variable Number of Tandem Repeats (VNTRs) (Figure 11.1). a) Made of a short sequence, or motif, called a core sequence, which is repeated many times in a row. b) Inherited from the parents. c) Core sequences range from two to six bp to over 300 bp long. d) The number of times the core sequence is repeated is variable. e) A VNTR refers to a single location with different alleles, characterized by differences in the number of times the core sequence is repeated. f) Can have hypervariable loci where a locus may has many different alleles varying in the number of repeats. g) Jeffreys was able to use DNA probes for VNTRs by using Southern blotting to yield a DNA fingerprint. h) Can generate by restriction enzyme digestion or PCR. 4. Sattelite DNA is found only in eukaryotes. Suggested biological function of sattelite DNA include providing sequences necessary for the pairing of homologous chromosomes during meiosis and site of recombination.

B.Microsatellites. 1.Also called simple tandemly repeated sequences or short tandem repeats (STRs). 2.Very short, with a repeated sequence of about 2-5 bp. 3.Most common repeated motif is (CA)n-(GT)n, with n meaning the number of times the sequence is repeated. 4.The total length of the repeat is less than one kb (usually 70-200 bp). STR are randomly distributed throughout the genome. The variation in length between alleles of the same locus is due to replication slippage. 5.Because of their hypervariability (multiple alleles/locus), STRs are more valuable than RFLPs which usually have two alleles/locus. 6.Often several loci are examined simultaneously by PCR (multiplexing) to generate a fingerprint. 7. The FBI uses a set of thirteen well-characterized STR loci for DNA profiling.

C. Minisatellites. 1. Located near the ends of chromosomes (the telomeres) and vary due to recombination of alleles. 2. Share a core sequence of about 20 bp units, although the rate of mutation can be very high. A minisatellite core unite can be repeated up to a total length of one to 30 kb. 3. The number of alleles can also be very high, which means that it is hypervariable (greater than the microsattelites). 4. Loci may number in the thousands, and the sequences are used to detect length polymorphisms scattered throughout the genome (called multilocus profiles).

D.Macrosatellites. 1. Located near the centromeres and telomeres. 2. Megabases in length, and need a special type of electrophoresis called pulsed-field gel electrophoresis. 3. Length makes them subject to breakage, so they are not used in forensic analysis especially that collected samples may already be somewhat degraded.

III.Population Genetics and Alleles. A.Because humans are diploid organisms, each individual has two alleles per locus. B.Individuals could be: 1. Homozygoustwo copies of the same overall length, even though the DNA sequence may be different. 2. Heterozygoustwo copies of different overall length. C. Many alleles exist in a population with the maximum number of alleles being two times the number of people in the population. D. Some DNA regions can be hypervariable (usually used in forensic analysis), while others are not as variable. E. Allelic polymorphisms in a population are maintained by genetic drift or natural selection.

IV.Multilocus Minisatellite VNTR (Figure 11.2). A. Supported by genetic and population data, and the data provided by the probes are reliable and are used in paternity (Figure 11.3) and immigration cases (Figure 11.4). B. More difficult to interpret than single-locus patterns for several reasons: 1. The large number of bonds usually generated. 2. Incomplete cutting of the DNA. 3. DNA degradation. 4. Low DNA recovery. 5. Identification problems with mixed DNA samples from more than one individual. C.DNA fingerprinting has still been successful in forensic cases and evaluating the genetic diversity of plants and animals. D.Usually detected using Southern blot hybridization.

V.Single-Locus VNTR for STR. A.Generate only one DNA fragment (homozygous) or two fragments (heterozygous). B.If more than one location is used, the more informative the analysis (Figure 11.5). C.Is technically less difficult because it eliminates the possibility of co-migration of alleles, and it is less likely to produce artifacts (Figure 11.6). D.The method of choice today is using PCR, by using at least four or five single-locus primer sets in separate PCR reactions or in the same reaction (called multiplexing). E.Some potential problems with multiplexing that need to be overcome: 1. Primers from one locus can sometimes complex with those of other loci. 2. One locus may not amplify as well as another locus. 3. The optimization of PCR conditions can present a challengeannealing temperature of primers and primer concentrations must be determined and a uniform annealing temperature may be difficult to determine. 4. If the problems are not resolved, the absence of a specific STR allele may not actually represent the individuals genotype. An alternative may be to recover the DNA from one reaction, and use it in another reaction (called sequential multiplex amplification). F. Can determine the frequency of alleles at different loci, as well as the frequency for combinations of alleles (Table 11.1).

G. The FBI has selected thirteen tetrameric STR loci to make up the core of the Combined DNA Index System (CODIS). Having this system has the following benefits: 1. The adoption of this system by forensic DNA analysts, allowing standardization of methods. 2. STR alleles are now readily available using commercially available kits. 3. STR alleles have been well characterized and act according to known principles of population genetics. 4.Laboratories worldwide are contributing data to the database so that the STR allele frequency in many different human populations can be determined. 5. The data are digital (see later discussion on digital DNA typing) and therefore ideal for computer databases. 6. The frequency for each of the thirteen loci can be combined to calculate the frequency of an individual profile.

H.STR-based DNA typing is the method of choice because: 1. STR analysis is much less labor intensive than RFLP analysis. 2. DNA profiles from badly degraded DNA can be obtained. 3. Very small amounts of DNA are requiredless than 0.20 ng of target DNA can be used, although 0.5-1.0 ng of DNA is optimal. 4. Small amounts of contaminants will not yield nonspecific PCR products. 5. Mixed DNA samples from other people can be resolved. 6. Automated fluorescent detection of amplified STR fragments can be conducted using DNA sequencers.

VI. Restriction Fragment Length Polymorphisms (RFLPs). A. Alleles can differ in sequence by a single base, which can alter where a restriction enzyme cuts a DNA sequence. This can cause a difference in restriction enzyme patterns, which can be generated in two ways: 1. Agarose gel electrophoresis, followed by DNA hybridization with a probe. 2. PCR amplification of a DNA fragment, followed by restriction digestion of the PCR product, and agarose electrophoresis. B. DNA sequence polymorphisms are common in animals and can be used as inherited markers (Figures 11.17, 11.8a and 11.8b), which allows for paternity and maternity testing. C. Can also be used for disease detection, such as sickle cell anemia. D. More than 100 genes have been mapped using RFLPs, and are beneficial when alleles are linked to mutated genes that can encode genetic diseases, because the locations of these genes can be made (Figure 11.9). E. Individuals can be screened for RFLPs that are linked to a disease-causing gene.

VII. Methods of DNA Profiling. A. Methods used in DNA fingerprint analysis include: 1. Southern blot hybridization which includes restriction enzyme digestion, gel electrophoresi, and finally autoradiography. 2. PCR. B. The goal of DNA profiling is to exclude suspects or to determine a possibility of a match between samples collected from a suspect.

VIII. Technical Considerations. A. Why make technical considerations? 1. If methods are not followed closely, then DNA profiles may yield incorrect results. 2. The following considerations must be made to maintain consistency: a) Preserve the integrity of DNA. b) Completely digest the DNA with restriction enzymes. c) Standardize hybridization methods. d) Select appropriate probes that are stably inherited. 3. Errors may occur from: a)Contamination of the sample. b)DNA degradation. c) Difficulties in interpreting the bands on the x-ray film. Artifacts (for example, extra bands, missing bands, band shifting) might provide false information. d) Statistical misinterpretation of a match.

B. DNA. 1. Need to make the following considerations to have high-quality DNA: a) Tissue samples need to be collected and stored on ice. b) DNA should be extracted as soon as possible. c) Samples may be contaminated by bacterial DNA or DNA from other people. d) Sometimes DNA might be fragmented if it comes from places such as mummies, fossilized plants, or frozen humans. 2. DNA Digestion. a) Incomplete digestion of DNA can create inaccurate DNA profiles. b) DNA may be methylated and will not allow the enzyme to cut it. c) Enzymes may cut in incorrect spots if there are less than optimal enzyme conditions. 3. Gel Electrophoresis. a) Using the correct gel matrix, such as polyacrylamide for very small DNA fragments like STR fragments. b) The correct percentage of agarose or polyacrylamide is needed to more optimally separate specific pieces of DNA. c) The correct voltage is neededhigh voltages do not separate large pieces well, and low voltages do not separate small pieces well. d) Thinner gels allow better separation of DNA pieces. e) DNA may diffuse out of the gel if the gel is submerged too deeply in the gel running buffer. f) DNA samples loaded in the middle wells migrate more quickly than samples loaded at the edges of the gel. 4. Probe Selection. a) Synthetic oligonucleotide probes are used to cover all sequence variants. b) DNA should also be hybridized with a bacterial probe (usually a bacterial ribosomal RNA gene) to detect bacterial sequences.

IX. Polymerase Chain Reaction. A. PCR is easy to conduct, results are obtained quickly, and very small amounts of DNA from as little as a single cell can be used. B. Even degraded DNA can be analyzed with PCR, however cross-contamination is a potential problem because contaminating DNA can be amplified. C. Usually PCR products are transferred to a membrane and hybridized with a labeled probe or with the PCR product by the process of dot blot hybridization. D. Ideal marker allele for efficient amplification should have between 100 and 500 bp, so that the pieces can be amplified from even degraded DNA. E. All samples should be analyzed with electrophoresis and hybridization. F. Commercial biotechnology companies market forensic kits that use things such as probe strips that allow for subtyping of samples and comparison of results.

X.Digital DNA Typing. A. Also called repeat coding, this is when the variation within one location is measured, and not the number of repeat units in specific locations. B. The locus is amplified by PCR and then cut with a restriction enzyme. The enzyme may cut the DNA or it may not cut the DNA. C. If the DNA is cut or not (or both), a number is assigned to the repeat, and since several loci are used, a numerical readout called a barcode is generated. D. The barcode is used to compare samples with others, eliminating the need for electrophoresis of each sample because the DNA pieces do not need to be sized.

XI.Population Comparisons. A. Two major challenges in forensic analysis are: 1. Calculate probability of coincidental matches by comparing with a reference population. 2. Find the criteria for determining what constitutes a relevant reference population. B. Individual DNA polymorphisms must be used in the context of population data in order to determine the probability that a match can occur by chance alone: 1. The individuals data must be compared to an ethnic group relevant to the individual. 2. Identical patterns between two individuals or an individual and a sample must be established. 3. The probability that a DNA banding pattern is present in another person should be extremely low. 4. Answer the question What is the probability that a DNA match is random? This question requires thorough and careful study of the population. 5. Need to assume that the probes for VNTR loci separate independently and are not linked, although this requires the sampling of a large population, which may not be possible in small ethnic groups. Many laboratories (as well as CODIS) are establishing local population databases.

XII. Admissibility of Scientific Evidence in a Court of Law. A. About DNA evidence and how it is submitted in courts: 1. Trial courts make preliminary determinations of admissibility of evidence through pretrial hearings where the judge evaluates all evidence. 2. Expert witnesses testify with their opinions on the evidence. 3. Laws based on the Federal Rules of Evidence, which were signed into law in 1975. Rule 702 governs the use of scientific evidence and requires that all scientific testimony be relevant, reliable, and grounded in scientific methodology. 4. States set the standards by which DNA evidence is allowed, with the two major standards being the Frye and Daubert standards. 5. Parties in a case may have to submit to a Frye or Daubert hearing in order to show that a scientific principle is well-established and can be used as evidence. This hearing is usually requested by the defense team to see if there is any potential weakness in the scientific methods. 6. Rejection of DNA evidence is usually based on factors such as methods used to collect DNA and the possible contamination of samples.

B.Frye Standard. 1. Sometimes referred to as the general-acceptance rule, limits acceptable scientific evidence to evidence which has been generated by methods that are reliable, well-established, accepted by the scientific community, and supported by scientific principles. 2. A Frye test is used to prevent the presentation of invalid opinions based on invalid scientific procedures to a jury. 3. Witnesses who present scientific data must be experts in the field and possess the academic and professional credentials that allow them to understand the scientific principles used to generate the evidence. 4. Proof of reliability of a method must also be established, and a manual called Technical Working Group on DNA Methods Analysis has been published and used by many commercial laboratories.

C. Daubert Standard. 1. Evolved from a 1993 decision involving a claim that the antinausea drug Benectin caused birth defects. The Supreme Court rejected the Frye standard in this case. 2. In the Daubert case, Rule 702 took precedence over the Frye standard. 3. The Daubert standard established six questions that needed to be answered when considering the evidence: a) Is the hypothesis presented by the scientific expert testable? b) Has the theory or method been subjected to peer review and publication? c) What is the known or potential error rate of the method? d) What are the experts qualifications and stature in the scientific community? e) Does the method rely on the special skills and equipment of one expert or can it be replicated by other experts elsewhere? f) Can the method and the results be explained in such a way that the court and the jury can understand and evaluate the evidence? 4. Scientific knowledge must be produced using the scientific method and supported by peer review, forcing the experts testimony to be subject to scientific validation

XIII. Databases. A. How databases are made and used. 1. Information provided to data banks comes from forensic collections, tissue samples, blood samples, neonatal screenings, and members of the armed forces. 2. Examples of DNA databases include CODIS, the Department of Defense, and the state of Virginia. 3. May be very useful, but may also infringe on our privacy. B. Cause for Concern? What About Privacy? 1. Genetic information can be stored in a database and could be subject to misuse. 2. Specific regulations need to be in place where donors must be told exactly what data are stored and must consent to the use of their biological samples. 3. Employers and insurers must not base any decision on genetic information.

XIV.Other Methods of Profiling DNA and Their Applications. A. Random Amplification of Polymorphic DNA (Figure 11.10). 1. Used to detect variability or polymorphisms in PCR priming sites. 2. Uses between eight and ten-nucleotide primers, and will generate DNA fragments of different sizes and cause differing patterns. Patterns will not be the same for every member of a population. 3. Some features of RAPD include: a) RAPD methods are simple and can be performed in a short time. b) Because they are polymorphic, they can be used as genetic markers. c) They are dominant, and do not distinguish between homozygous and heterozygous allele states. d) RAPDs closely linked to a particular gene can be used by animal and plant breeders to ascertain whether they have transferred the desired trait. e) Useful for determining genetic variability within and between populations of bacteria, plants, or animals.

B. Amplified Fragment Length Polymorphism (Figure 11.11). 1. Used in a variety of fields, from forensic analysis to plant and animal breeding. 2. Essentially a PCR-amplified RFLP, it assesses the entire genome instead of just specific locations. It is an effective tool to use in population genetics studies. 3. Abundant, randomly distributed, and inherited in a Mendelian fashion. 4. The method is as follows: a) Genomic DNA is digested to completion with the enzymes MseI and EcoRI. Combinations of DNA pieces will be generated: those cut with only MseI, those only cut with EcoRI, and those cut with both enzymes. b) The ends of the resulting DAN fragments are ligated to specific 25-30 bp adaptor sequences that act as priming sites for select primers. c) Preselective PCR is performed, using primers that have extra nucleotides (one to three) on their 3 end. Only a small amount of the fragments cut with both enzymes will be amplified. d) A second PCR, called selective PCR, is performed using the PCR products from the previous step as a template. The primer that anneals to the EcoRI adaptor sequence is labeled with fluorescent or radioactive nucleotides. e) The PCR products are analyzed by capillary gel or agarose gel electrophoresis. 5. 100-200 bands are generated, and only a small number will be polymorphic.

C. Single-Strand Conformation Polymorphism. 1. Single-strand DNA can change mobility based on its nucleotide sequence. 2. Asymmetric PCR is done where one primer is in higher amount than the other. 3. When the low-amount primer is used up, the PCR reaction continues, generating single-stranded pieces of DNA. 4. Pieces are separated with native gel electrophoresis (does not denature DNA). 5. Features include: a) The mobility of single-strand DNA depends on intramolecular base pairing. This base pairing can form loops, stems, and other secondary structures, altering their mobility. b) The exact sequence and location of the polymorphism can be unknown, and only the mobilities are important and reflective of the DNA polymorphism. c) Most SSCP methods analyze single DNA loci of individuals. d) Useful for detecting individual genetic variation in populations, detecting mutations in genomic DNA, and serving as molecular markers.

D.Single Nucleotide Polymorphism. 1. Most are nucleotide substitutions, and are useful to detect mutations in genes that might be related to disease development. 2. Many new methods have been developed involving hybridization of allele-specific probes that fluoresce when they bind to the target sequence. 3. Can be applied to comparing genotypic variation to phenotypic variation, ecological research, pharmacogenomics, and the expansion of SNP databases by data mining.

E. Mitochondrial DNA and the Y Chromosome. 1. Mitochondrial Eve. a) Mitochondrial DNA studies have shown that: (1) Recent ancestors of modern humans originated in Africa, and therefore, had a recent African origin. (2)Modern humans appeared in one founding population. (3) Our closest ancestors evolved approximately 171,500 years ago. It is believed that all modern humans arose from a few females at about this time, and all different mitochondrial sequences coalesced into one. (4) Anatomically modern humans migrated to other parts of the world to replace other hominids. b) Mitochondria have a genome of about 16,500 bp, containing 27 genes. c) There are advantages to using mitochondrial DNA: (1) Mitochondrial DNA undergoes mutations at a higher rate than nuclear DNA. Differences between closely related individuals can be resolved. (2) Mitochondria are inherited only from the maternal line so that a direct genetic line can be traced without the need to separate two different lineages (father and mother). (3) Mitochondrial DNA does not undergo recombination, so there are not combined male- female lineages to decipher.

2. Y Chromosomal Adam. a) A record of paternal inheritance and it is believed that all men can be traced back to one ancestral male who lived between 35,000 and 90,000 years ago. b) Believed to have originated in Africa, along with mitochondrial Eve. c) Y-chromosome profiling shows that males moved south from Africa into Australia and Eurasia. d) Other applications of Y-chromosome studies include: (1) The evolutionary history of the Lemba, a South African Bantu-speaking population of possible Jewish ancestry. (2) Finding that islands of Jewish priests called the Cohanim have been maintained in populations of Eastern European Jews.

XV. Forensic Archaeology. A. The use of forensic science to examine and make conclusions about archaeological discoveries. B. They are also used to solve old crimes. C. Can even use very small amounts of DNA that may be damaged (such as a mummy). D. Examples of forensic archaeology include: 1. Identification of individuals. 2. Studying human and animal evolution. 3. Tracing the migration of humans and animals, including extinct groups. 4. Tracing origins and relationships of different ethnic groups around the world. 5. Determining family relationships of ancient remains, such as the pharaohs of Egypt.

E. Biotech Revolution: DNA Profiling and Human Remains. 1. Two of the most famous cases dealing with ancient human remains are: a) Romanov family: (1) Nine skeletons were discovered in Ekaterinburg, Russia, in 1991. (2) Tests on remains included STR testing, mitochondrial DNA, and sex chromosome testing. (3) Found the czar, his wife, and three of his children among the nine. b) Otzi, the Ice Man discovered in the Alps in 1991: (1) Believed that he died about 5200 years ago. (2) Found that he probably died of a violent fight. (3) Found the blood of four other people on his knife, coat, and arrow.

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11 DNA Profiling, Forensics, and Other Applications Brief Chapter Outline I. Satellite DNA: A. Repetitive DNA B. Microsatellites C. Minisatellites D. Macrosatellites