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November 2010 Prof Angela van Daal Forensic DNA Glossary Accreditation Accreditation is a process that assesses an organisation's overall compliance with systems and products standards. It does not assess technical competence. NATA accreditation is a peer review process. NATA assesses (1) the qualifications, training and experience of staff (2) that equipment is correctly calibrated and maintained and (3) that there are adequate quality assurance procedures in place. See Appendix 1 for NATA Section 5.4 Test and calibration methods and method validation Adenine One of the four bases in DNA. Adenine pairs with thymine. Allele Alternative form of a gene or DNA region. There are two alleles at each locus with one allele inherited from each parent. Allele Frequency The proportion of individuals in a population that has a particular allele. Allelic Ladder A mixture of the different STR alleles used to compare with the amplified samples. Amelogenin A gene located on the X and Y chromosome which is 6bp longer on the Y chromosome. This allows differentiation of DNA from males (two fragments of 212 and 218 bp) and females (one 212 bp fragment only). Amplification The process that exponentially copies a certain region of DNA. Autosome A chromosome not involved in sex determination. The diploid human genome contains 46 chromosomes, 22 pairs of autosomes, and one pair of sex chromosomes (the X and Y chromosomes). Base pair (bp) Two complementary DNA bases, one on each strand of the DNA molecule, held together by weak hydrogen bonds. Adenine always pairs with thymine and guanine always pairs with cytosine. Buccal Cells Cells on the inner lining of the cheek. These cells are present in saliva and are scraped from the inner cheek surface for collection of DNA reference samples. Cell The basic unit of structure, invisible to the naked eye, in all living things. The human body contains many different types of cells eg skin (epithelial), liver, bone, muscle. All cells except red blood cells contain a nucleus, in which the nuclear DNA resides. Cells also contain hundreds to thousands of mitochondria (the cell’s “energy factories”), which contain a small circular DNA molecule. Chromosome The long DNA molecule must be highly compressed to fit within the nucleus of the cell. This structure is called a chromosome. Humans have 46 chromosomes in all cells (except sperm and egg cells which have 23). The chromosomes are numbered from 1 to 22 in order of size, 1 November 2010 Prof Angela van Daal Forensic DNA Glossary with chromosome 1 being the largest. The sex chromosomes are called X and Y. Females have two X chromosomes and males have one X and one Y chromosome. Each parent contributes the equivalent of one chromosome to each pair, so children get half of their chromosomes from their mother and half from their father. Complementary sequences DNA sequences form a double-stranded helix structure by complementary (matching) base pairs. The complementary sequence to A-C-G-T is T-G-C-A. Confirmational Bias Tendency to search for information to confirm prior beliefs and to avoid information that may be contrary to those beliefs. Contamination The finding of extraneous or foreign DNA in a sample. Contamination can occur at the crime scene or in the laboratory. Controls in the laboratory should detect the latter but not the former class of contamination. Contextual Bias The use of existing information to reinforce a position or belief. Contextual bias is where other evidence is used to bolster the findings from the specific evidence being analysed. Cytosine One of the four bases in DNA. Cytosine pairs with guanine. Denaturation Creation of two single strands of DNA using heat to disrupt the hydrogen bonds that hold the two strands of the DNA double helix together Diploid All DNA containing cells in the body are diploid except the reproductive cells (sperm and egg). Diploid cells contain two complete genomes, one derived from the mother and the other from the father. DNA (deoxyribonucleic acid) DNA is a chemical made up of a linear sequence of millions of nucleotides. It is a doublestranded helical molecule. DNA is the genetic blueprint that contains the genetic information about an individual. It is found in all cells except red blood cells. DNA extraction The process by which DNA is isolated from the cells contained in the evidence sample. DNA Profile The DNA typing of an individual or evidence sample resulting from the examination of one or more polymorphic loci DNA replication The process by which double stranded DNA unwinds and makes an exact copy of itself. DNA sequence The order of the DNA base pairs in a fragment of DNA, a gene, a chromosome or the entire genome Double helix The shape that two linear strands of DNA assume when held together by hydrogen bonds. 2 November 2010 Prof Angela van Daal Forensic DNA Glossary Electropherogram The DNA profile pattern of fluorescent peaks produced by DNA fragments after capillary gel electrophoresis. Electrophoresis The process of separating different sized fragments of DNA in an electric field in an agarose or acrylamide matrix. Enzyme A protein molecule that functions to facilitate a biochemical reaction. Exclusion When the DNA from an evidence sample does not match that of a reference person sample. Familial Search Searching a DNA database for a non-exact match in order to identify not the source of the evidence DNA profile, but rather a close relative (usually parent or sibling). In other words it is the use of the DNA of a family member to identify a closely related suspect through a DNA database search when no exact match has been found. Flanking Region Flanking regions are the stretches of DNA outside the region of interest. For STRs for example, these sequences are the non-repeated DNA regions which, unlike the repeat regions, are are the same amongst individuals. The primer sequences are designed from DNA in the flanking regions such that they will amplify DNA from all individuals. Fluorescence Fluorescent molecules emit light at one wavelength upon excitation with a laser. DNA molecules can be tagged with different fluorescent molecules so that it is possible to detect and distinguish DNA fragments of similar size. Gene A gene is the basic unit of heredity. A gene is a region of DNA at a particular site on a chromosome which contains the information to make a particular product. Genetic markers Genetic markers include alleles of genes and DNA polymorphisms. There are several types of DNA markers: • microsatellites: short tandem repeat sequences (2 to 5 bp) • minisatellites: longer tandem repeat sequences (9 to 80 bp) • indels: insertions or deletions of DNA at particular locations on the chromosome. • SNPs: single nucleotide polymorphisms where one base is changed (eg A changed to G). Genetics The study of the patterns of inheritance of specific traits Haploid The reproductive cells of the body, sperm and egg, are haploid and contain only one complete genome such that when the sperm and the egg fertilise a diploid embryo is created Heterozygous An individual with two different alleles at one locus. For an STR locus this means two different 3 November 2010 Prof Angela van Daal Forensic DNA Glossary peaks are seen at that locus. Homozygous An individual with one allele at a locus. For an STR locus this means only one peak is seen at that locus. Hybridization The binding of single stranded DNA to its complimentary sequence. Inclusion When the DNA from an evidence sample is the same as that of a reference person sample. Inconclusive When it is not possible to conclude that the DNA from an evidence sample is the same or different from that of a reference person sample Junk DNA Regions of DNA that do not code for genes. It is also called non-coding DNA. Kilobase (kb) Unit of length for DNA fragments equal to 1000bp (base pairs) Linkage A measure of the association between two different loci. Loci that are near each other on the same chromosome are linked and are likely to be inherited together Low Copy Number (LCN) The analysis of very low levels of DNA template, usually less than 100pg, that results in exaggerated stochastic effects and consequential DNA profile interpretation problems. Locus (plural is loci) The position of a gene or a region of DNA on a chromosome. Match When the genotypes from two DNA profiles are the same. Microsatellite Repetitive stretches of short sequences of DNA repeated a number of times in tandem. Mitochondria Cells contain hundreds to thousands of mitochondria (the cell’s “energy factories”), which contain a small circular DNA molecule. Mitochondrial DNA (mtDNA) Mitochondrial DNA is maternally inherited i.e. it is passed from the mother to all her children. The genetic material of the mitochondria, the organelles that generate energy for the cell, is a small (16kb) circular molecule. mt DNA Typing mt DNA typing is typing is carried out by sequencing which is laborious and time consuming. The power of discrimination achieved using mtDNA typing is substantially lower than that provided by the battery of STR loci. For these reasons it is used only where nuclear DNA typing is not successful, usually with shed hair and bone samples. 4 November 2010 Prof Angela van Daal Forensic DNA Glossary Mixture A mixture refers to a sample containing DNA from more than one person. Mixture Interpretation Threshold (MIT) The MIT is used for mixture samples to circumvent the potential stochastic effects associated with low levels of a minor mixture component. MIT sets the minimum peak height required to confidently call peaks at a locus such that no alleles failed to be detected due to stochastic effects. Multiplexing PCR analysis of several loci in the same reaction. Mutation A heritable change in DNA sequence. A mutation can be a single base change to the sequence of the DNA or a change in the length of a particular region of the DNA. Nanogram (ng) One nanogram is one billionth of a gram. There are approximately 167 diploid cells in 1ng DNA. NATA National Association of Testing Authorities. See accreditation. Nuclear DNA Typing The Y chromosome and the autosomal STR loci are contained in the nucleus. STR and Y STR typing are both nuclear DNA typing methods. Nucleotide The molecular unit (building block) of DNA that contains one base. Nucleotides join together to make a DNA molecule. Nucleus The structure in the cell that contains the chromosomes. Off-Ladder Alleles An allele that is not included in the allelic ladder. These can be true, rare alleles or artefacts Partial Profile A profile where genotypes were obtained only from a subset of the loci amplified. PCR (polymerase chain reaction) A technique that generates millions of copies of a particular region of DNA through repeated cycling of a thermal reaction. The region copied is defined by two small primer sequences, forward and reverse, that flank the fragment copied. Peak Amplitude Threshold (PAT) The minimum peak height that can reliably assign a peak as an allele. PAT takes into account instrumental “noise” and sets the lowest peak height value for which a laboratory will call a DNA fragment an allele rather than instrument background. Phenotype The physical appearance of an individual at one or more loci. An individual’s genotype for eye colour may have one allele for brown eye colour and one for blue eye colour. The genotype is Bb. The phenptype is brown eyes. 5 November 2010 Prof Angela van Daal Forensic DNA Glossary Picogram (pg) One picogram is one thousandth of a nanogram. One diploid human cell contains approximately 6pg DNA. Polymerase The enzyme that copies DNA. It uses an existing DNA strand as a template to make the complementary strand. Polymorphic A genetic locus that is highly variable in a population. DNA markers that are very polymorphic have many different alleles that distinguish individuals. Primer A short DNA sequence (18-25bp) designed to flank the region of DNA that is copied in PCR. Prosecutor’s Fallacy The prosecutor’s fallacy states that the frequency of the evidence DNA profile that matches the defendant is the probability that the defendant is innocent. Pull-up An artefact that occurs when there is too much fluorescence, usually because too much DNA has been used in the PCR reaction. The pull-up is a result of an intense peak in one colour channel, detected as a minor peak in another colour channel. Quality Assurance (QA) QA refers to a program for the systematic monitoring and evaluation of the various aspects of a service to ensure minimum standards of quality are provided. Quality Control (QC) QC involves a system of standard technical activities to measure the quality of a method and its results. QC provides regular checks to ensure data integrity and correctness and also to identify any errors. Relative Fluorescence Units (rfu) An arbitrary scale from 0-6,000 used to measure the relative amount of fluorescent signal. Replication See DNA replication. Secondary Transfer The transfer of material from one source to another (eg blood from a victim to the offender’s hands) is referred to as primary transfer. Secondary transfer refers to the subsequent transfer of the material (eg the victim’s blood on the offender’s hands to a door knob). Spike An artefact peak seen in all colour channels of an electropherogram. It is caused for example by dust or an electrical disturbance. STR (Short Tandem Repeat) STRs are short (2-5 bases), tandem repetitive sequence elements. STRs are polymorphic such that the number of repeat elements varies between individuals. The alleles are size separated by electrophoresis. Stutter A common artefact of STR analysis in which a peak is seen at the allele position one smaller 6 November 2010 Prof Angela van Daal Forensic DNA Glossary than the true allele. This stutter allele produces a signal that up to 20% of the signal of the true allele peak. Tandem repeat The end-to-end duplication of genomic DNA sequences. Taq polymerase An enzyme that catalyses the polymerase chain reaction at high temperatures. It is isolated from bacteria that live at high temperature (in thermal springs). Template Genomic DNA that is added to the PCR reaction. Threshold A minimum value, usually 50 - 200 rfu, used to determine when a peak can reliably be called as an allele. See Mixture Interpretation Threshold and Peak Amplitude Threshold. Thymine One of the four bases in DNA. Thymine pairs with adenine. Validation Definition taken from the NATA Forensic Science FAD document (http://www.nata.asn.au/publications/category/4-nata-accreditation-requirements). “Validation is the developmental process used to acquire the necessary information to assess the ability of a procedure to obtain a result reliably, to determine the conditions under which such results can be obtained and to determine the limitations of the procedure. The validation process identifies critical aspects of a procedure which must be carefully controlled and monitored.” See Appendix 1 for NATA Section 5.4 Test and calibration methods and method validation X chromosome One of the two sex chromosomes, X and Y. An individual with two X chromosomes is female. Y chromosome One of the two sex chromosomes, X and Y. An individual with a Y chromosome is male. The Y chromosome is paternally inherited i.e. it is passed from father to son. 7 November 2010 Prof Angela van Daal Forensic DNA Glossary Appendix 1 NATA Validation Requirements (http://www.nata.asn.au/publications/category/4-nataaccreditation-requirements ) 5.4 Test and calibration methods and method validation 5.4.1 All test/examination methods and related procedures (eg. sample procurement) must be documented and readily available to the analysts/examiners. In addition to a description of the steps involved in the analysis/examination, documentation of methods and procedures must include, where appropriate: • description of the sample/item to be tested/ examined; • parameters or quantities to be determined; • equipment/instrumentation required; • descriptions of sample preparation methods, controls, standards and calibration procedures; • a discussion of precautions, possible sources of error or limitations of the procedure; • criteria for the rejection of suspect results; • data/observations to be recorded and method of analysis and presentation; • literature references. Note: The availability of documented methods will give the analyst/examiner the necessary resource material to support written conclusions and expert testimony. Note: AS 2929 provides guidance on the format and content of test methods. In general, the level of detail is expected to be sufficient for a new staff member with basic scientific training in the relevant area to be able to perform the procedure. 5.4.2 a) Test/examination methods and procedures used must be generally accepted in the fi eld or supported by data gathered and recorded in a scientific manner. Note: Since a variety of scientific procedures may appropriately be applied to a given problem, standards and criteria for assessing procedures need to remain fl exible. In forensic science, well established procedures are often scattered throughout peer reviewed literature as well as in less formal documents obtained from conference proceedings and in-house laboratory manuals. Furthermore, minor modifications to improve published methods can be implemented by a laboratory as appropriate to the particular need. The important point is that the procedures used be demonstrably capable of producing valid results. b) i. Where a laboratory introduces a new (validated) method, it must first demonstrate the reliability of the procedure in-house against any documented performance characteristics of that procedure. As a minimum, the method must be tested using known samples (eg. proficiency test samples, samples from an external agency). It is recommended that the method also be tested using non-probative samples. ii. Records of performance verifi cation must be maintained for future reference. c) i. Where a test can be performed by more than one method, there must be documented criteria for method selection. ii. Where appropriate, the degree of correlation between the methods must be established and documented. d) When destructive tests are necessary, procedures must ensure that as much material as possible is retained for reanalysis, if necessary. e) i. The quality of the standard samples and reagents must be adequate for the procedure used. ii. Lot/batch numbers of standards and critical reagents must be recorded. iii. All critical reagents must be routinely tested for their reliability. iv. Standards and reagents must be labelled with: • name of the reagent/standard; • concentration, where appropriate; • preparation date; • identity of the preparer. Where necessary, the following must also be included on labels: • expiry date; • storage conditions; • hazard warning. Note: 5.4.5.1 Validation is the developmental process used to acquire the necessary information to assess the ability of a procedure to obtain a result reliably, to determine the conditions under which such results can be obtained and to determine the limitations of the procedure. The validation process identifies critical aspects of a procedure which must be carefully controlled and monitored. Note: Methods may be validated by comparison with other established methods using certifi ed reference materials (where available) or materials of known characteristics. In validating test methods, the following issues (among others) may need to be determined, as appropriate: • matrix effects • interferences • sample homogeneity • concentration ranges 8 November 2010 Prof Angela van Daal Forensic DNA Glossary • specifi city • stability of measured compounds • linearity range • population distribution • precision • measurement uncertainty Note: Validation studies can be conducted by the scientific community (as in the case of standard or published methods) or by the forensic science laboratory itself (as in the case of methods developed in-house or where significant modifications are made to previously validated methods). Note: NATA Technical Note 17 provides guidance on procedures for validation and verification of analytical test methods. f) For audio and video signal processing, the method employed to copy a primary image for archive should be validated to ensure that the process produces a binary copy. g) For audio and video signal processing, the choice of capture device, output device or image compression should be validated to ensure the purpose and requirement of the image are met or exceeded. 5.4.5.2 Records of all in-house validations must be maintained for future reference and available for review at assessment. 5.4.6 Estimation of uncertainty of measurement where results of tests are not numerical (eg. pass/fail, positive/negative, detected/not detected or other qualitative data) will not be required at this stage. However, laboratories are encouraged to have an understanding of the variability of all their results where this is possible. Additional information is available in both national and international forums and available on the NATA website as well as the ILAC (www.ilac.org) and APLAC (www.aplac.org) websites. 9