Download November 2010 Prof Angela van Daal Forensic DNA

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,
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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.
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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
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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.
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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.
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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
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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.
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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
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• 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.
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