Sso Probes And Linear Array Typing Assays

Methods for screening mtDNA variation (see Butler and Levin 1998 and Budowle et al. 2004). One of the most widely used screening assays for assessing mtDNA variation used to date are the sequence-specific oligonucleotide (SSO) probes originally designed by Mark Stoneking and colleagues in 1991. Rather than sequencing the entire HV1 and HV2 regions, the most polymorphic sites are examined through hybridization of PCR products to oligonucleotide probes designed to anneal to different variants....

Studies With Additional Ystr Markers

Within the last several years, a number of new Y chromosome STR markers have been characterized and new multiplex assays developed (Redd et al. 1997, Prinz et al. 1997, Gusmao et al. 1999, Bosch et al. 2002, Butler et al. 2002, Redd et al. 2002, Schoske 2003, Hanson and Ballantyne 2004). Information on additional Y-STR loci and assays is available on the NIST STRBase web site at A few population studies have been conducted that go beyond the minimal haplotype loci in order to assess the power...

Monitoring Needle Sharing

In yet another application of the capability to perform mixture detection with STRs, a laboratory method was described by Shrestha et al. (2000) using the CODIS STR marker D8S1179 (called D6S502 in the paper) to differentiate between single and multi-person use of syringes by intravenous drug users. D.N.A. Box 7.2 Natural mixtures and chimeric individuals In May 2002, the New England Journal of Medicine published a report of the genetic analysis of a phenotypically normal chimeric individual...

Software For Deciphering Mixture Components

Computer programs can be used to aid the process of deciphering mixture components and determining mixture ratios. A linear mixture analysis approach was reportedly able to derive estimated mixture ratios from quantitative STR peak information that were similar to known input mixture proportions (Perlin and Szabady 2001). Researchers at the University of Tennessee are also working on algorithms to decipher mixture components in an automated fashion (Wang et al. 2002). Expected peak profiles for...

Chromosomes Genes And Dna Markers

There are approximately three billion base pairs in a single copy of the human genome. Obtaining a complete catalog of our genes was the focus of the Human Genome Project, which announced a final reference sequence for the human genome in April 2003 (D.N.A. Box 2.1). The information from the Human Genome Project will benefit medical science as well as forensic human identity testing and help us better understand our genetic makeup. Within human cells, DNA found in the nucleus of the cell...

The World Trade Center Dna Identification Effort

The DNA identification efforts for the World Trade Center (WTC) victims have become arguably the world's largest forensic case to date. More than 19917 pieces of human remains were collected from a pile of rubble weighing over a million tons and extending more than 70 feet in height following the crushing collapse of the twin towers. The initial removal and sorting of human remains took place between September 2001 and May 2002. However, the DNA identification efforts went on for more than...

Indels Insertiondeletion Polymorphisms

Another form of a bi-allelic (or di-allelic) polymorphism is an insertion-deletion or indel. An indel can be the insertion or deletion of a segment of DNA ranging from one nucleotide to hundreds of nucleotides (such as is seen with an Alu insertion). The two alleles for di-allelic indels can simply be classified as 'short' and 'long'. From a certain perspective, STR markers can be thought of as multi-allelic indels since the different alleles are typically insertions or deletions of a tandem...

Crimes For Inclusion In A State Dna Database

As ofJune 1998, all 50 states in the United States had passed legislation requiring convicted offenders to provide samples for DNA databasing. Each state though has different requirements as to what types of offenses are considered for DNA sample collection. In many states these requirements are changing over time to include more and more criminal offenses. The requirements for having to donate a blood sample range from all felons to strictly sex offenses. The trend is for laws that require a...

Location And Structure Of mtDNA

The vast majority of the human genome is located within the nucleus of each cell (see Figure 2.3, Table 10.1). However, there is a small, circular genome Comparison of human nuclear DNA and mitochondrial DNA markers. Nuclear DNA (nucDNA) Mitochondrial DNA (mtDNA) Percent of total DNA content per cell Inherited from Chromosomal pairing Generational recombination Replication repair Unique Unique to individual (except identical twins) Described in 2001 by the Human Genome Project Not unique to...

The Polymerase Chain Reaction Dna Amplification

Out of a natural laziness, I always start with the easiest possible protocol and work from there. Better yet, I suggest that someone start from there, and I come back in a month to see how things worked out. Forensic science and DNA typing laboratories have greatly benefited from the discovery of a technique known as the polymerase chain reaction or PCR. First described in 1985 by Kary Mullis and members of the Human Genetics group at the Cetus Corporation (now Roche Molecular Systems), PCR has...

Dna Typing

DNA testing is to justice what the telescope is for the stars not a lesson in biochemistry, not a display of the wonders of magnifying glass, but a way to see things as they really are. (Barry Scheck and Peter Neufeld, Actual Innocence) In the darkness of the early morning hours of 26 August 1999, a young University of Virginia student awoke to find a gun pointed at her head. The assailant forced her and a male friend spending the night to roll over on their stomachs. Terrorized, they obeyed...

Ystr Haplotype Databases

The largest and most widely used Y-STR database was created by Lutz Roewer and colleagues at Humbolt University in Berlin, Germany and has been available online since 2000. The information in this database comes from 89 collaborating institutions located in 36 different countries (Roewer 2003). As of May 2004, more than 24 000 samples from greater than 224 populations Figure 9.7 Alleles present in (a) PowerPlex Y and (b) Y-PLEX 12 allelic ladders for the DYS385 a b locus. around the world can...

The Product Rule

The lack of population structuring with allele frequencies in Hardy-Weinberg equilibrium and linkage equilibrium (see Chapter 20)justifies the assumption that genotypes are independent at unlinked loci. With the assumption of independence, it then becomes possible to equate the overall match probability with the product of the locus-specific match probabilities. This combination of locus-specific match probabilities is referred to as the product rule. In other words, the match probability for...

Comparison Of Dna Typing Methods

Technologies used for performing forensic DNA analysis differ in their ability to differentiate two individuals and in the speed with which results can be obtained. The speed of analysis has dramatically improved for forensic DNA analysis. DNA testing that previously took 6 or 8 weeks can now be performed in a few hours. The human identity testing community has used a variety of techniques including single-locus probe and multi-locus probe RFLP methods and more recently PCR (polymerase chain...

Various Applications For mtDna Testing

Mitochondrial DNA variation is extensively studied in several other disciplines besides forensic science. Medical scientists have linked a number of diseases to mutations in mtDNA (see Wallace et al. 1999). Evolutionary biologists examine human mtDNA sequence variation relative to other species in an effort to determine relationships. A good example of this application is the determination that Neanderthals are not the direct ancestors of modern humans based on control region sequences...

General Match Probability

As noted in this entire section, profile probabilities need to be calculated for a variety of scenarios. Balding (1999) points out that there are five different sets of people and possible relationships to a suspect (1) the suspect's siblings, (2) his other relatives, (3) other members of his sub-population, (4) other members of his racial group, and (5) anyone else outside of his population (e.g., racial) group (see also Foreman and Evett 2001, Weir 2003). Example calculations with NRCII...

Determining The Number Of Contributors To A Mixture

Typically assumptions are made as to the number of contributors possible in a mixture. The vast majority of cases most laboratories face are two-person Mixture profile at the STR locus FGA from a three-person mixture. Genotypes and peak heights in relative fluorescence units are shown underneath each peak. The peak height variation observed can be used to tentatively associate alleles 20 and 22 as coming from the same individual, alleles 18.2 and 23 as coming from the second contributor, and...

Biology Related Artifact Peaks

Stutter products are the most common source of additional peaks in an electro-pherogram of an STR sample. When STR loci are PCR-amplified a minor product peak four bases (n 4) shorter than the corresponding main allele peak is commonly observed (see Chapter 6). Validation studies conducted in a laboratory help define maximum percent stutter for each locus. However, if the target allele peak is off-scale then the stutter product can appear larger than it really is in relationship to the...

Impact Of Stutter Products On Data Interpretation

Stutter products impact interpretation of DNA profiles, especially in cases where two or more individuals may have contributed to the DNA sample (see Chapter 7). CTAT CTAT CTAT CTAT CTAT CTAT (b) Insertion caused by backward slippage CTAT CTAT CTAT CTAT CTAT CTAT (c) Deletion caused by forward slippage

Impact Of Various Population Databases

From the three combined STR profile frequencies calculated in Table 21.2 it is apparent that different populations can yield different frequency estimates due to variations in allele frequencies in these populations. A calculation of the same STR profile as used in the previous examples against 97 different published population databases (including the three present in Appendix II) found that the cumulative profile frequency ranged from 1 in 3.43 X 1014 to 1 in 2.65 X1021 (D.N.A. Box 21.1). The...

Mixture Interpretation

Contoh Flowchart Membuat Kue

This next section will review the principles described by Gill et al. (1998b) and Clayton et al. (1998) for interpreting mixed forensic stains using STR typing results. Their six primary steps for interpreting mixtures are outlined in Figure 7.4. The interpretation steps will first be discussed and then an example mixture will be reviewed to put these steps into the context of a real sample. An understanding of how non-mixtures behave is essential to being able to proceed with mixture...

Evidence Collection And Preservation

The importance of proper DNA evidence collection cannot be overemphasized. If the DNA sample is contaminated from the start, obtaining unambiguous information becomes a challenge at best and an important investigation can be compromised (see D.N.A. Box 3.2). Samples for collection should be carefully chosen as well to prevent needless redundancy in the evidence for a case. The following suggestions may be helpful during evidence collection to preserve it properly (see page 38). Importance of...

Nonhuman Dna Testing And Microbial Forensics

We urge as rapid development of new systems as is consistent with their validation before they are put into general use. One's ideas must be as broad as Nature if they are to interpret Nature. (Sherlock Holmes, A Study in Scarlet) While the vast majority of forensic DNA typing performed for criminal investigations involves human DNA, it is not the only source of DNA that may be useful in demonstrating the guilt or innocence of an individual suspected of a crime (Sensabaugh and Kaye 1998)....

Kinship And Data Analysis Panel Kadap

The National Institute of Justice (NIJ) organized a panel of experts that convened every other month in the two years following 11 September 2001 to aid in reviewing data and providing guidance and recommendations to NYC OCME regarding statistical thresholds for reporting DNA matches with direct matches and kinship associations. The names and affiliations of the advisory WTC Kinship and Data Analysis Panel (KADAP) are listed in Table 24.2. KADAP gathered in NYC, Albany, Baltimore, and...

An Example Mixture

An example mixture will now be examined to demonstrate how the steps illustrated in Figure 7.4 may be used to interpret a mixture. Figure 7.5 shows a mixed sample that is a combination of male and female DNA, typical of what might be seen in a sexual assault investigation. The STR markers for the mixture are separated into three panels based on their dye label in order to visualize each STR locus more easily. The first thing that is obvious in this example is the presence of more than two peaks...

Dna Separation Methods Slab Gel And Capillary Electrophoresis

Capillary Electrophoresis Dna

It is a capital mistake to theorize before one has data. Insensibly one begins to twist facts to suit theories, instead of theories to suit facts. Sherlock Holmes, A Scandal in Bohemia A polymerase chain reaction PCR reaction in which short tandem repeat STR alleles are amplified produces a mixture of DNA molecules that present a challenging separation problem. A multiplex PCR can produce 20 or more DNA fragments that must be resolved from one another. In addition, single base resolution is...

Abi Prism 3100 Genetic Analyzer 16capillary System

3100 Genetic Analyzer

The multi-capillary ABI PRISM 3100 Genetic Analyzer became available in 2001 and offers a nice solution to higher throughputs with a very similar feel to the single capillary ABI 310 instrument. Both 96-well and 384-well plates of samples may be processed in the ABI 3100. With each run taking roughly 45-60 minutes, a 96-well plate can be analyzed in approximately 5-6 hours with six injections containing 16 samples each. Several features of the ABI 3100 versus the ABI 310 are compared in Table...

Commonly Used Short Tandem Repeat Markers And Commercial Kits

Str Marker Th01

Ever since their discovery in the early 1980s, the ubiquitous occurrence of microsatellites -also referred to as short tandem repeats STRs or simple sequence repeats SSRs -has puzzled geneticists Understanding STRs is important if we wish to understand how genomes are organized and why most genomes are filled with sequences other than genes. Eukaryotic genomes are full of repeated DNA sequences see Ellegren 2004 . These repeated DNA sequences come in all types of sizes and are typically...

Allelic Ladders

Str Markers

An allelic ladder is an artificial mixture of the common alleles present in the human population for a particular STR marker Sajantila et al. 1992 . They are generated with the same primers as tested samples and thus provide a reference DNA size for each allele included in the ladder. Allelic ladders have been shown to be important for accurate genotype determinations Smith 1995 . These allelic ladders serve as a standard like a measuring stick for each STR locus. They are necessary to adjust...

Str Multiplex Example

An example of DNA profiles obtained from two different individuals using STR markers is shown in Figure 1.3. In a single amplification reaction, unique sites Can a simple DNA test have the power to impact world events In 1998, DNA evidence and independent counsel Kenneth Starr was investigating allegations that U.S. Monica Lewinsky's President William Jefferson Clinton had a sexual relationship with a young blue dress White House intern, Monica Lewinsky. President Clinton had publicly denied...

Advantages And Disadvantages Of Snps

SNPs are appealing to the forensic DNA community for several reasons. First and foremost, the polymerase chain reaction PCR products from SNPs can be less than 100 bp in size, which means that these markers would be able to withstand degraded DNA samples better than STRs that have amplicons as large as Single Nucleotide Polymorphisms SNPs Occurrence in human genome General informativeness Marker type Number of alleles per marker Detection methods Multiplex capability Major advantage for...

Differential Extraction

Differential Extraction Sperm

Differential extraction is a modified version of the organic extraction method that separates epithelial and sperm cells Figure 3.2 . Differential extraction was first described in 1985 Gill et al. 1985 and is commonly used today by the FBI Laboratory and other forensic crime laboratories to isolate the female and male fractions in sexual assault cases that contain a mixture of male and female DNA. By separating the male fraction away from the victim's DNA profile, it is much easier to...

Slot Blot Quantitation

Probably the most commonly used method in forensic labs today for genomic DNA quantitation is the so-called 'slot blot' procedure. This test is specific for human and other primate DNA due to a 40 base pair bp probe that is complementary to a primate-specific alpha satellite DNA sequence D17Z1 located on chromosome 17 Waye et al. 1989, Walsh et al. 1992 . The slot blot assay was first described with radioactive probes Waye et al. 1989 but has since been modified and commercialized with...

Desirable Characteristics Of Strs Used In Forensic Dna Typing

For human identification purposes it is important to have DNA markers that exhibit the highest possible variation or a number of less polymorphic markers that can be combined in order to obtain the ability to discriminate between samples. As will be discussed further in Chapter 7, forensic specimens are often challenging to PCR amplify because the DNA in the samples may be severely degraded i.e., broken up into small pieces . Mixtures are prevalent as well in some forensic samples, such as...

Determining The Presence Of A Microvariant Allele

Suspected microvariants can be fairly easily seen in heterozygous samples where one allele lines up with the fragment sizes in the allelic ladder and one does not Figure 6.6 . In the example shown here, the sample contains a peak that lines up with allele 25 from the FGA allelic ladder and a second peak that is labeled Detection of a microvariant allele at the STR locus FGA. The sample in the bottom panel is compared to the allelic ladder shown in the top panel using Genotyper 2.5 software....

Anomalous Amelogenin Results

While amelogenin is an effective method for sex-typing biological samples in most cases, the results are not full proof. A rare deletion of the amelogenin gene on the Y chromosome can cause the Y chromosome amplicon to be absent Santos et al. 1998 . In such a case, a male sample would falsely appear as a female. It appears that this deletion of the Y chromosome amelogenin region is more common in Indian populations Thangaraj et al. 2002 than those of European or African origins. A study of...

Reduced Stutter Product Formation

Th01 Variant

The amount of stutter product formation may be reduced when using STR markers with longer repeat units, STR alleles with imperfect repeat units, and DNA polymerases with faster processivity. Several pentanucleotide repeat loci have been developed in an effort to produce STR markers that exhibit low amounts of stutter products to aid in mixture interpretation Bacher and Schumm 1998 . The first seven loci have been labeled Penta A through Penta G. Penta E has been incorporated in the GenePrint...

The 13 Codis Str Loci

In the United States, utilization of STRs initially lagged behind that of Europe, especially the efforts of the Forensic Science Service in the United Kingdom. However, beginning in 1996, the FBI Laboratory sponsored a community-wide forensic science effort to establish core STR loci for inclusion within the national DNA database known as CODIS Combined DNA Index System . Chapter 18 covers CODIS and DNA databases in more detail. This STR Project beginning in April 1996 and concluding in...

Peaks Outside The Allelic Ladder Range And Threebanded Patterns

Allelic Ladder

Occasionally new rare alleles may fall outside the allele range spanned by the locus allelic ladder. If these peaks fall between two STR loci in a multiplex set, they can be challenging to assign to a particular locus unless testing is performed with individual locus-specific primer sets or a different multiplex. These extreme 'off-ladder' alleles can be confirmed with singleplex amplification of the two loci in the multiplex bracketing the new allele. Alternatively the sample could be...