Abi Prism 3700 And 3730 Genetic Analyzers 96capillary Systems
The ABI 3700, which is a 96-capillary array system, offers even higher potential throughput and automation than the ABI 377. Sample injection of 96 samples is performed in parallel. In a 24-hour period of unattended operation, more than 750 samples can be injected. Thus, approximately 190 000 sample injections can be theoretically processed per year. The ABI 3730 is widely used in human genome centers for DNA sequencing and offers even higher throughput compared to the ABI 3700 even through...
Dna Structure And Definitions
Nucleic acids including DNA are composed of nucleotide units that are made up of three parts a nucleobase, a sugar, and a phosphate Figure 2.1 . The nucleo-base or 'base' imparts the variation in each nucleotide unit while the phosphate and sugar portions form the backbone structure of the DNA molecule. The DNA alphabet is composed of only four characters representing the four nucleobases A adenine , T thymine , C cytosine , and G guanine . Basic components of nucleic acids a phosphate sugar...
Dna Separation Methods Slab Gel And Capillary Electrophoresis
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
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...
The Y Chromosome Markers Examined
DNA samples from each of the 19 blood specimens gathered by Dr. Foster were carefully extracted by a pathologist at the University of Virginia Murray and Duffy 1998 . The DNA samples were coded by Dr. Foster and then taken to England where researchers at Oxford University examined them. Eventually the team of scientists involved expanded to include researchers from the University of Leicester in England and Leiden University in the Netherlands. A variety of tests were run independently at these...
Reference Samplesnist Srm 2395
The use of reference samples will aid in obtaining calibrated and consistent results among laboratories performing Y-STR testing. The U.S. National Institute of Standards and Technology NIST released Standard Reference Material SRM 2395 in July 2003 that serves as a human Y chromosome DNA profiling standard. Five male DNA samples are provided with SRM 2395, each of which has been sequenced at over 20 different Y-STR loci including the common core loci present in all commercially available kits.
Commonly Used Short Tandem Repeat Markers And Commercial Kits
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...
Amplitaq Gold Dna Polymerase
AmpliTaq Gold DNA polymerase is a chemically modified enzyme that is rendered inactive until heated Birch et al. 1996 . An extended pre-incubation of 95 C, usually for 10 or 11 minutes, is used to activate the AmpliTaq Gold. The chemical modification involves a derivitization of the epsilon-amino groups of the lysine residues Innis and Gelfand 1999 . At a pH below 7.0 the chemical modification moieties fall off and the activity of the polymerase is restored. The Tris buffer in the PCR reaction...
Realtime Pcr Approaches To Dna Quantification
The primary purpose in performing a DNA quantification test is to determine the amount of 'amplifiable' DNA. A PCR amplification reaction may fail due to the presence of co-extracted inhibitors, highly degraded DNA, insufficient DNA quantity, or a combination of all of these factors. Thus, a test that can accurately reflect both the quality and the quantity of the DNA template present in an extracted sample is beneficial to making decisions about how to proceed. 'Realtime' PCR assays provide...
Isolation And Types Of Str Markers
In order to perform analysis on STR markers, the invariant flanking regions surrounding the repeats must be determined. Once the flanking sequences are known then PCR primers can be designed and the repeat region amplified for analysis. New STR markers are usually identified in one of two ways 1 searching DNA sequence databases such as GenBank for regions with more than six or so contiguous repeat units Weber and May 1989, Collins et al. 2003, Subramanian et al. 2003 or 2 performing molecular...
Allelic Ladders
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...
The 5 Nuclease Assay Taqman
TaqMan probes are labeled with two fluorescent dyes that emit at different wavelengths Figure 4.4 . The probe sequence is intended to hybridize specifically in the DNA target region of interest between the two PCR primers Ong and Irvine 2002 . Typically the probe is designed to have a slightly higher annealing temperature compared to the PCR primers so that the probe will be Schematic of TaqMan 5' nuclease assay. 5' Polymerization and Strand Displacement Probe Cleavage release of reporter dye...
Dna Quantities Used In Fluorescencebased Str Typing
PCR amplification is dependent on the quantity of template DNA molecules added to the reaction. Based on the DNA quantitation results obtained using either the slot blot procedure or some other test, the extracted DNA for each sample is adjusted to a level that will work optimally in the PCR amplification reaction. Most commercial STR typing kits work best with an input DNA template of around 1 ng. A quantity of 1 ng of human genomic DNA corresponds to approximately 333 copies of each locus...
Use Of Positive And Negative Controls
As noted by Melton and Nelson 2001 the two primary goals in mtDNA testing are 1 to protect the integrity of the evidence by preventing contamination at any stage in the testing and 2 to collect the maximum amount of available mtDNA data inherent to any sample. Control samples that are processed in parallel with evidentiary samples through each step of the process serve to monitor performance and assess one's success with the two goals noted above. Contamination assessment is performed with...
Stochastic Effects From Low Levels Of Dna Template
Forensic DNA specimens often possess low levels of DNA. When amplifying very low levels of DNA template, a phenomenon known as stochastic fluctuation can occur. Stochastic effects, which are an unequal sampling of the two alleles present from a heterozygous individual, result when only a few DNA molecules are used to initiate PCR Walsh et al. 1992 . PCR reactions involving DNA template levels below approximately 100 pg of DNA, or about 17 diploid copies of genomic DNA, have been shown to...
HOW MANY SNPs ARE NEEDED
Since each SNP locus typically possesses only two possible alleles, more markers are needed to obtain a high discriminatory power than for STR loci that possess multiple alleles. Computational analyses have shown that on average 25-45 SNP loci are needed to yield equivalent random match probabilities as the 13 core STR loci Chakraborty et al. 1999 . Another study predicted that 50 SNPs possessing frequencies in the range of 20-50 for the minor allele can theoretically result in likelihood...
c 1
One strand of PCR product is labeled with fluorescent dye allelic ladders that are labeled with the same fluorescent dye so that differences in dye mobilities do not impact allele calls. A laser strikes a fluorophore dye that is attached to the end of a DNA fragment. The fluorophore absorbs laser energy and then emits light at a lower energy higher wavelength . Filters are used to collect only emitted light at a particular wavelength or range of wavelengths. Photomultiplier tubes or...
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...
PRIVATE LABORATORIES CONDUCTING FORENSIC mtDNA CASEWORK
Several private laboratories in the United States have validated mtDNA procedures and offer mtDNA testing on a fee basis. These laboratories include Mitotyping Technologies, LLC State College, Pennsylvania , ReliaGene Technologies, Inc. New Orleans, Louisiana , Bode Technology Group Springfield, Virginia , Orchid Cellmark Dallas, Texas , the University of North Texas Health Sciences Center DNA Identity Lab Ft. Worth, Texas , and Laboratory Corporation of America Research Triangle Park, North...
Endpoint Pcr For Dna Quantification
A less elegant and less expensive approach for testing the 'amplifiability' of a DNA sample is to perform an end-point PCR test. In this approach a single STR locus Kihlgren et al. 1998, Fox et al. 2003 or other region of the human genome, such as an Alu repeat Sifis et al. 2002, Nicklas and Buel 2003a , is amplified along with DNA samples of known concentrations. A standard curve Pyrophosphorylation and luciferase light production can be generated from the samples with known amounts to which...
Whole Mitochondrial Genome Sequencing
The first description of a methodology for sequencing the entire mtGenome was by Deborah Nickerson's group at the University of Washington Rieder et al. 1998 . They used 24 pairs of primers to amplify PCR products ranging in size from 765 bp to 1162 bp. These primer pairs provide on average almost 200 bases of overlap between the various PCR products spanning the mtGenome. Ingman et al. 2000 used the Nickerson laboratory sequencing strategy to launch the era of mitochondrial population genomics...
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...
Other Applications Of Y Chromosome Testing
Several uses of Y chromosome testing are listed in Table 9.1. The Y chromosome has become a popular tool for tracing historical human migration patterns through male lineages Jobling and Tyler-Smith 1995, Jobling and Tyler-Smith 2003 . Anthropological, historical, and genealogical questions can be answered through Y chromosome results. For example, as will be discussed later in the chapter, Y chromosome results in 1998 linked modern day descendants of Thomas Jefferson and Eston Hemings leading...
Differential Extraction
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...
Direct Observation Of Sperm
Most forensic laboratories like to observe spermatozoa as part of confirming the presence of semen in an evidentiary sample. A common method of doing this is to recover dried semen evidence from fabric or on human skin with a deionized water-moistened swab. A portion of the recovered cells are then placed onto a microscope slide and fixed to the slide with heat. The immobilized cells are stained with a 'Christmas Tree' stain consisting of aluminum sulfate, nuclear fast red, picric acid, and...
Aluquant Human Dna Quantitation System
The Promega Corporation has developed a human DNA quantitation system that enables sensitive detection of DNA Mandrekar et al. 2001 . The AluQuant assay probes Alu repeats that are in high abundance in the human genome see Chapter 8 . Probe-target hybridization initiates a series of enzymatic reactions that end in oxidation of luciferin with production of light. The light intensity is read by a luminometer and is proportional to the amount of DNA present in the sample. Sample quantities are...
The Meaning Of A Y Chromosome Match
Due to the fact that the Y chromosome is passed down unchanged except for mutations from father to son, the observation of a match with Y-STRs does not carry the power of discrimination and weight into court as an autosomal STR match. Peter de Knijff 2003 discusses some of the challenges of presenting Y-STR results in court. He concludes that information from the Y-STR Haplotype Reference Database http www.yhrd.org should be seen as qualitative rather than quantitative because this database...
Ampflstr Identifiler Kit Innovations
Applied Biosystems introduced two new technologies with their AmpFlSTR Identifiler kit when it was released in 2001. The first, and most obvious, AmpFlSTR Identifier allelic ladders Applied Biosystems . A total of 205 alleles are included in this set of allelic ladders used for genotyping a multiplex PCR reaction involving 15 STR loci and the amelogenin sex-typing test. Comparison of represented alleles in commercially available STR allelic ladders Comparison of represented alleles in...
Examining Human Population Diversity
Whole genome scans with 377 autosomal STR loci Marshfield set 10 were used to genotype 1056 individuals from 52 populations in order to study human population structure Rosenberg et al. 2002 . Studies with this same set of data have identified particular STR loci that are effective indicators of ancestral origin Rosenberg et al. 2003 . Analysis of Y chromosome STRs see Chapter 9 and mitochondrial DNA see Chapter 10 have also been used for genetic genealogy studies Helgason et al. 2003 . Both...
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...
Minimal Haplotype Loci
The number of Y chromosome short tandem repeat Y-STR loci available for use in human identity testing has increased dramatically since the turn of the century and the availability of the human genome sequence. In the 1990s only a handful of Y-STR markers were characterized and available for use. At the beginning of 2002, only about 30 Y-STRs were available for researchers Butler 2003 . Since that time more than 200 new Y-STRs have been deposited in the Genome Database GDB http www.gdb.org...
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...
The Future Of Y Chromosome Testing
The field of Y chromosome analysis and its application to human identity testing has undergone rapid improvements in recent years. New markers and population groups are being characterized Kayser et al. 2004 . Commercial kits are now available to enable forensic practitioners to use core loci in male-specific amplifications. Validation studies and inter-laboratory studies have demonstrated that Y-STR typing is reliable. Internet-accessible databases house thousands of Y-STR haplotypes. Multiple...
SNaPshot A PRIMER EXTENSION ASSAY CAPABLE OF MULTIPLEX ANALYSIS
Minisequencing, sometimes referred to as SNaPshot, involves allele-specific primer extension with fluorescent dye-labeled dideoxynucleotide triphosphates ddNTPs to help visualize the results. There are three primary steps in performing minisequencing amplification, primer extension, and analysis Figure 8.1 . First, the region around each SNP locus is amplified using PCR. Amplicons can be pooled following singleplex PCR or simultaneously generated using multiplex PCR. The remaining dNTPs and...
Reduced Stutter Product Formation
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...
Impact Of Mutation Rates On Paternity Testing
Low mutation rates are especially critical for paternity testing see Chapter 23 . This is because links are being made between the child and the alleged father based on the assumption that alleles remain the same when they are passed from one generation to the next. Parent-offspring allele transfer information tests for germ-line mutations. Additionally, genotypes from different kinds of tissues from the same individual are examined to demonstrate that no somatic mutations occur. Mutations have...
Solutions To Null Alleles
If a null allele is detected at a STR locus, there are several possible solutions. First, the problem PCR primer could be redesigned and moved away from the problematic site. This approach was taken early in the development of the D7S820 primers for the Promega PowerPlex 1.1 kit Schumm et al. 1996 and more recently with a D16S539 flanking region mutation Nelson et al. 2002, see Chapter 5 . However, this solution could result in the new primer interfering with another one in the multiplex set of...
Short Tandem Repeat DNA Internet Database
These data are intended to benefit research and application of short tandem repeat DNA markers to aid human identity testing. The authors are solely responsible for the information herein. Purpose of Database This database has been accessed I www.digits.com - see disclaimer. times since 10 02 97. Counter courtesy Created by John M. Butler and Dennis J. Reeder NIST Biotechnology Division . with invaluable help from Christian Ruitberg and Michael Tung Site creators' curriculum vitaes available...
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...
Sporadic Contamination Of Pcr Tubes
The Forensic Science Service recently noted that sporadic contamination of consumables used in DNA testing, such as the small tubes in which the PCR amplification is performed, can introduce extraneous DNA profiles Howitt et al. 2003 . The FSS observed 11 casework-contaminating DNA profiles in running over one million samples in a three-year period. These contaminating profiles were identified in negative controls and quality control testing of select tubes examined to indicate general batch...
Realtime Pcr Analysis
There are three distinct phases that define the PCR process geometric or exponential amplification, linear amplification, and the plateau region Bloch 1991 . These regions can be seen in a plot of fluorescence versus PCR cycle number Figure 4.5 . During exponential amplification, there is a high degree of precision surrounding the production of new PCR products. When the reaction is performing at close to 100 efficiency, then a doubling of amplicons occurs with each cycle see Table 4.1 . A plot...
Thermal Cyclers
The instrument that heats and cools a DNA sample in order to perform the PCR reaction is known as a thermal cycler. Precise and accurate sample heating and cooling is crucial to PCR in order to guarantee consistent results. There are a wide variety of thermal cycler options available from multiple manufacturers. These instruments vary in the number of samples that can be handled at a time, the size of the sample tube and volume of reagents that can be handled, and the speed at which the...
Peaks Outside The Allelic Ladder Range And Threebanded Patterns
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...
Picogreen Microtiter Plate Assay
As higher throughput methods for DNA determination are being developed, more automated procedures are needed for rapid assessment of extracted DNA quantity prior to DNA amplification. To this end, the Forensic Science Service has developed a PicoGreen assay that is capable of detecting as little as 0.25ng mL of double-stranded DNA in a 96-well microtiter plate format Hopwood et al. 1997 . PicoGreen is a fluorescent interchelating dye whose fluorescence is greatly enhanced when bound to...
Fta Paper
Another approach to DNA extraction involves the use of FTA paper. In the late 1980s, FTA paper was developed by Lee Burgoyne at Flinders University in Australia as a method for storage of DNA Burgoyne et al. 1994 . FTA paper is an absorbent cellulose-based paper that contains four chemical substances to protect DNA molecules from nuclease degradation and preserve the paper from bacterial growth Burgoyne 1996 . As a result, DNA on FTA paper is stable at room temperature over a period of several...