DNA Profiling Flashcards
What is DNA profiling also known as and what is it?
DNA fingerprinting
It is the process of determining an organism’s genetic makeup and comparing it to others.
What percentage of the human genome is identical across all individuals?
More than 99%
What are polymorphic regions in the context of DNA profiling?
Regions that vary significantly among individuals, allowing for unique DNA profiles
They are crucial for establishing identity except in the case of monozygotic twins. But still, early mutations are common.
What are the 5 processes of DNA profiling?
Sample
Extract
Copy
Size
Match
What types of samples can be used for DNA profiling and what type of cells do they all contain?
- White blood cells
- Semen
- Hair roots
- Body tissue and fluids
All contain epithelial cells
Epithelial cells: a specialised cell that covers the skin, lines the internal organs + cavities + blood cells → protection, secretion, absorption
Where is DNA found in a cell?
In the nucleus
What are the steps for Extraction?
Lysis, Filtration, Precipitation
What is the purpose of lysis in DNA extraction and what are the steps?
To mechanically disrupt cells and release DNA
- Cut tissue into small pieces to break cell walls + membranes
- Salt solutions (detergents + enzymes like proteinase K) free proteins & DNA
DNA is - due to phosphate groups (- O atoms in them). Causes DNA to repel + remain dissolved. When Na+ ions are introduced they interact with the - phosphate groups, neutralising the charge repulsion between the DNA strands → reducing overall electrostatic repulsion and stabilising DNA in a less soluble form.
What is the function of filtration in DNA extraction?
To clear unseparated cells and fibers
This is done using filter paper or centrifuge.
What is the purpose of precipitation in DNA extraction?
To separate DNA from cellular debris.
DNA becomes visible by floating to the top when alcohol (ethanol/isopropanol) is added, as it is insoluble in alcohol.
What does PCR stand for?
Polymerase Chain Reaction
It is used to copy small amounts of DNA for forensic analysis.
What technologies are used in the Copy step of DNA profiling?
STR Analysis, PCR, NGS
What is a PCR Machine and what does it do?
A labratory instrument/machine that amplifies small segments of a specific DNA or RNA sequence, producing millions of copies from a single sample (target DNA).
What is the PCR machine solution made up of?
- Target DNA
- Primers
- Nucleotides
- DNA polymerase
- Buffer (creates optimum pH for the enzyme)
- MgCl2 (needed for the enzyme to work)
What are the steps for PCR?
- Denaturation - temp > 90°C → breaks H bonds → causes 2 strands to come apart/denature
- Annealing - temp lowers 55°C - 65°C → primers attached/annealed to DNA → match the specific sequence of DNA used to start the process of making new DNA.
- Extension - Temp increased again (72°C) - polymerase enzyme makes new matching DNA strands that were separated using spare nucleotides.
What are Short Tandem Repeats (STR)?
Regions of non-coding DNA containing repeats of the same nucleotide sequence.
They are used to build genetic profiles.
Where are STRs found?
Different genetic loci.
Loci is usually on different chromosomes: same STR + location is so rare
What are the steps for STRs?
- PCR amplification
- Flourescent tagging: STR regions labelled with dyes
- Capillary electrophoresis: DNA fragments separated by size, flourescent signals are analysed
How are STR regions amplified and why?
PCR increase the amount of DNA available for analysis
This allows for higher discrimination in profiling.
What is the role of fluorescent tagging in STR analysis?
To label STR regions for detection
This enables the analysis of fluorescent signals.
What is capillary electrophoresis used for?
To separate DNA fragments by size. The longer fragments are heavier and are thus closer to the wells.
It allows for the analysis of fluorescent signals from the separated fragments.
What are the uses of Sanger Sequencing?
- routine sequencing
- Validating NGS data
What is a high-fidelity DNA-dependent polymerase?
DNA polymerase enzyme that has a very low error rate during DNA replication, meaning it accurately replicates DNA sequences with minimal mistakes.
What is a single-stranded DNA template?
A single strand of DNA that serves as a blueprint for creating a new DNA or RNA strand
What is a primer?
A short, single-stranded DNA sequence designed to bind specifically to a particular region of a single-stranded DNA template through complementary base pairing.
What is DNA synthesis?
A.k.a. DNA replication. It’s where a cell creates an exact copy of its DNA, ensuring that each new cell has the same genetic information
Where does DNA synthesis happen?
From the primer 3’ end.
What gets added to the primer?
Random floating nucleotides in a way that’s complementary to the template DNA.
How are di-deoxynucleotides different from normal nucleotides?
- Don’t have a 3’ hydroxyl group which is needed for extension, therefore terminating the chain
- Have a fluorescent dye which makes it easy to be identified/detected
How does the order of the bases get identified?
The process of the ddNTP terminating the chain for the same DNA template gets repeated again and again. After some time, all of the nucleotide positions would have been terminated.
Then they get electrophoresed!
What are the uses of Next Generation Sequencing?
- Massively parallel sequencing reactions
- Millions/billions at the same times
What are the 3 steps for NGS?
Sample preparation, sequencing machines, data output
What does it mean for a sample to be library obtained?
It has to be prepared for sequencing
How can a sample to be library obtained?
Fragmentation: Long strands are too complex to sequence accurately, so fragmentation is needed to make them manageable. Endonucleases can usually be used to cleave the sample at the desired lengths. Then comes amplification, PCR, to make copies so that there are enough copies for reliable analysis.
Sequencing adapters: Uses ligase to attach sequencing adapters to DNA fragments → they are binding sites for sequencing primers, which is needed to initiate the sequencing process
How are the library fragments amplified?
Library fragments are amplified on a solid surface with DNA linkers on the surface that are covalent to the ends of the fragments. The ends bind (or hybridise) to their respective covalent points (actually called flow cells or beads) and the process continues until there are enough copies.
There are now clusters of DNA (which all originated from a single library fragment). When one of the new DNA fragments comes into contact with an emulsion bead (which is coated in the same primers as used in the previous method), many more copies are made
How is the raw data presented in NGS?
Data chips: in columns and rows
Rows represents a specific genetic marker, columns represent either the chromosome number, position on the chromosome, or the genotype.
How does gel electrophoresis work?
- DNA is negatively charged due to phosphate
- There is an electrical charge running through the agarose gel, where the wells are negative
- Therefore the DNA travels away from the wells
- Longer DNA pieces have a higher molecular weight and takes more time, being closer to wells
What are the separated DNA fragments called and what way are they read?
The DNA bands are read from shortest to longest
What is the purpose of restriction enzymes in DNA analysis?
To cut DNA into specific fragments
They cleave DNA at specific sequences for further analysis.
What’s an example of an endonuclease?
EcoR1: does not cut straight through the stands, but instead sticky ends - results from a jagged cut → therefore left without a complementary partner → easy to attach other DNA molecules that have been cut by the same RE together
What is the advantage of cleaving DNA jaggedly?
If there’s a mutation - will not cleave the molecule at that point
What is a DNA ladder?
Sample that has known fragment sizes/lengths. Use it to give estimates of how long the other fragments are by running them alongside each other.
How does a semi-log graph work?
What do the X and Y axis represent?
y-axis is logarithmic → separation between the ticks is proportional to the logarithm of numbers
x-axis is linear → ticks are evenly spaced, the combo useful for graphing exponential functions such as DNA bands.
Therefore, it is always linear.
What is Southern blotting?
A technique to detect specific DNA sequences in a complex mixture
It allows scientists to identify the presence and size of DNA fragments.
What does DVI stand for?
Disaster Victim Identification
It is the process used to identify victims of mass casualty incidents.
What is a major benefit of DNA analysis in DVI?
Reliable and efficient identification of bodies or body parts
It can identify remains even when severely fragmented or decomposed.
What are some challenges faced in DVI?
- Decomposition and fragmentation
- Commingling of remains
- Logistical challenges
These issues complicate the identification process.
What is one application of DNA profiling in forensic science?
Give some examples
Identifying criminals by matching biological evidence to suspects
9/11 World Trade Center attack.
Indian Ocean Tsunami.
Trenggalek shipwreck case.
How is DNA profiling used in medical contexts?
- Paternity testing
- Family-based immigration
- Identifying genetic disorders
- Organ transplant compatibility
It helps establish biological relationships and medical suitability.
What are the benefits of DNA profiling?
There are 2 main ones.
Accuracy
* Low error rates - very precise genetic identification
* Solves crimes by matching suspects with biological evidence → justice
* Exonerate innocent
Efficiency
* Small sample requirement - works with minute biological traces
* Rapid processing - takes hours for crimes and days for other uses e.g. paternity testing.
* Can be stored for decades if needed - DNA properly encapsulated with a salt remains
What are the limitations of DNA profiling?
Cost and accessibility
* Expensive - Requires advanced technology and expertise, which may not be accessible in underfunded regions or organisations.
* Wealthier individuals and countries benefit disproportionately, while marginalised groups face challenges in accessing the same resources.
Errors + Misuse
* Human error - Mishandling or contamination of samples can lead to inaccurate results. Have to label samples correctly.
* Frame innocent people - criminal can take DNA and plant it at the crime scene
* Database gaps - Not everybody’s DNA has been profiled - e.g. skin found and its profile, and if it is not databased and not from a suspect, then of no use
These factors can affect the reliability and fairness of DNA analysis.
What is the definition of Social Issues?
Challenges that affect society as a whole, such as privacy, discrimination, and inequality.
What is the definition of Ethical Issues?
Moral dilemmas about the rightness or wrongness of DNA profiling practices.
What does lack of autonomy do for social and ethical?
Social: Lose control over how their DNA is used. e.g. suspects giving their DNA without consent
Ethical: Strips people of their right to decide for themselves
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What does lack of confidentiality do for Social and Ethical?
Social: Misuse. E.g. data systems of hospitals hacked leading to identity theft + blackmail
Ethical: Failure to protect sensitive DNA data undermines trust and violates the right to keep genetic information private.
What does lack of privacy do for social and ethical?
Social: using genetic data without clear consent. E.g. Golden state killer case when relative’s submission of DNA led to matching the DNA with the killer
Ethical: DNA should not be exploited for purposes the individual did not agree to E.g. sharing it with law enforcement
What does lack of equity do for Social and Ethical?
Social: deepen social inequalities. Wealthier individuals have better access to resources to prove innocence, while lower-income groups may face barriers.
Ethical: Discrimination. E.g. refusing opportunities based on genetic predispositions.
What does GINA stand for?
Genetic Information Nondiscrimination Act
It aims to prevent discrimination based on genetic information.
What does bias in DNA databases do for Social and Ethical?
Social: Overrepresentation of certain ethnic groups. E.g. disproportionately including DNA samples from minority communities, leading to over-policing and wrongful accusations
Ethical: Systemic racism. Perpetuates inequalities, Rraises questions about justice and fairness in law enforcement practices
