Forensic DNA Extraction and Analysis

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1
What are the major stages of a forensic DNA laboratory workflow?
Evidence assessment, sampling, DNA extraction, quantitation, amplification, separation and detection, interpretation, technical review, and reporting.
2
What is DNA extraction?
The process of releasing DNA from biological material and preparing it for subsequent analysis.
3
What is DNA purification?
The removal of proteins, inhibitors, and other unwanted substances from a DNA-containing preparation.
4
What is organic DNA extraction?
An extraction method traditionally using phenol-chloroform chemistry to separate proteins and other materials from nucleic acids.
5
What is Chelex-based DNA extraction?
A method using a chelating resin to bind metal ions and help prepare DNA for amplification.
6
What is silica-based DNA purification?
A method in which DNA binds to silica under suitable chemical conditions and is subsequently washed and eluted.
7
What is magnetic bead-based DNA extraction?
A purification method using particles that bind nucleic acids and can be separated from liquids using a magnetic field.
8
What is differential extraction in forensic DNA analysis?
A procedure intended to separate sperm-associated DNA from other cellular DNA in suitable mixed biological samples.
9
Does differential extraction guarantee complete separation of sperm and nonsperm DNA?
No. Incomplete separation and cross-contamination between fractions can occur.
10
What is cell lysis?
The disruption of cellular membranes or structures to release intracellular components, including DNA.
11
What is the purpose of proteinase K in many DNA extraction procedures?
To digest proteins and help release and protect DNA during sample preparation.
12
What is the purpose of dithiothreitol or DTT in certain forensic DNA extractions?
To reduce disulfide bonds, including those in sperm nuclear proteins, facilitating DNA release.
13
What is an extraction blank?
A negative control processed through DNA extraction without an evidentiary sample to monitor contamination.
14
What is a PCR negative control?
A reaction containing the amplification reagents without added target DNA, used to monitor contamination introduced during amplification setup.
15
What is a PCR positive control?
A reaction containing known DNA expected to produce a specified amplification result.
16
Why should DNA extraction and amplified DNA products be handled in separated laboratory areas?
To reduce the risk that concentrated amplified DNA contaminates unamplified evidence samples.
17
What is carryover contamination in forensic DNA testing?
Unintended transfer of DNA from a previous sample or analytical process into another sample or reaction.
18
What is cross-contamination between evidence samples?
The unintended introduction of biological material from one sample into another.
19
What is elimination DNA profiling?
Comparison with reference profiles from relevant personnel or others to help identify possible contamination sources.
20
What is the purpose of changing gloves between biological evidence samples?
To reduce unintended transfer of DNA and other material between samples.
21
What is DNA degradation?
The breakdown or fragmentation of DNA through chemical, biological, or environmental processes.
22
How can ultraviolet radiation affect DNA evidence?
It can produce DNA damage that may interfere with successful amplification or interpretation.
23
How can moisture and microbial activity affect biological evidence?
They can promote decomposition and DNA degradation, particularly when evidence is stored improperly.
24
What is a DNA degradation index?
A measure comparing amplification or quantitation signals from different-sized DNA targets to help assess fragmentation.
25
Why can shorter DNA targets be more successful in degraded samples?
Shorter intact fragments are more likely to remain available for amplification when DNA has been fragmented.
26
What is a mini-STR assay?
An STR analysis approach designed to amplify shorter DNA fragments, potentially improving results from degraded material.
27
What is DNA quantitation?
The measurement or estimation of DNA amount or concentration in a sample.
28
Why is DNA quantitation performed before STR amplification?
To select an appropriate DNA input amount and identify potential issues such as low quantity, inhibition, or degradation.
29
What is an internal PCR control or IPC?
A known amplification target included in a reaction to help assess amplification performance or inhibition.
30
What is a stochastic effect in low-template DNA analysis?
Random variation in amplification that may produce imbalanced peaks, allele dropout, or other unpredictable profile features.
31
What is peak height in an STR electropherogram?
The measured fluorescence intensity of a detected peak, commonly expressed in relative fluorescence units.
32
What does RFU stand for in forensic DNA electropherograms?
Relative fluorescence units.
33
What is an analytical threshold in STR interpretation?
A laboratory-defined signal level used to distinguish reportable peaks from background noise under specified conditions.
34
What is a stochastic threshold in traditional STR interpretation?
A validated signal threshold used to assess the risk that an associated allele may have dropped out.
35
What is heterozygote peak-height imbalance?
A difference in the heights of two allelic peaks at a heterozygous locus beyond or within expected analytical variation.
36
What is peak-height ratio in STR analysis?
A comparison of the heights of two peaks, often used to evaluate heterozygote balance or mixture characteristics.
37
What is pull-up in capillary electrophoresis?
A spectral artifact in which fluorescence from one dye channel appears in another because of imperfect color separation.
38
What is a dye blob in STR electrophoresis?
An artifact caused by fluorescent dye-related material that may produce a broad or atypical signal.
39
What is an off-ladder allele?
An observed STR allele that falls outside the allele positions represented in the allelic ladder and requires appropriate evaluation.
40
What is a tri-allelic pattern?
The observation of three allelic signals at a locus, which may reflect biological variation, a mixture, or an analytical issue.
41
What is the purpose of replicate testing in challenging DNA samples?
To assess the consistency of observed results and help evaluate stochastic effects or other uncertainties.
42
What is a DNA mixture contributor number?
An estimate of how many individuals contributed DNA to a mixed sample.
43
Why can contributor-number estimation be uncertain?
Allele sharing, dropout, peak imbalance, low-level contributors, and artifacts can obscure the number of sources.
44
What is kinship analysis in forensic genetics?
The statistical evaluation of genetic evidence to assess proposed biological relationships.
45
What is a paternity index?
A likelihood ratio comparing the genetic evidence under a specified paternity proposition with an alternative proposition.
46
What is a combined paternity index?
A combination of locus-specific paternity indices under appropriate genetic and statistical assumptions.
47
Why must DNA conclusions distinguish source-level from activity-level propositions?
A DNA result may support who contributed biological material without establishing how or when the material was deposited.
48
Scenario: An extraction blank produces a DNA profile matching an evidence sample. What should the laboratory investigate?
Potential contamination affecting the extraction batch and the reliability of the associated evidence results.
49
Scenario: A degraded sample produces alleles at short STR loci but no results at longer loci. What is a plausible explanation?
DNA fragmentation may have prevented successful amplification of longer targets.
50
Scenario: A DNA mixture has strong support for a person's contribution but the item was handled by several people. What limitation must be reported?
The genetic association does not independently establish the timing, mechanism, or criminal significance of the DNA transfer.
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