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FSCI6370 - DNA > Practical DNA Processing > Flashcards

Practical DNA Processing Flashcards

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1
Q

What is allelic drop-out?

A
  • failure to detect an allele in a sample or failure to amplify an allele during PCR
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2
Q

What level of proposition is it if the DNA cam from the POI?

A
  • sub-source
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3
Q

What are the basic steps in the sample retrieval of DNA samples?

A

1 - steps against contamination:
- PPE (facemask, mobcap, nitrile gloves, lab coat, second nitrile gloves)
- wipe down any items brought into lab with alcohol (not actually alcohol, it is the friction)
- all people entering lab must provide elimination DNA samples
- each bench has its own set of equipment which is logged so any contamination can be traced back

2 - sample retrieval
- bench number and scientist name noted
- evidence must be recorded, photographed and drawn with annotations (in pen, no correction fluid, initials after correction) - ensure complete log of item
- check inside out, pockets
- look for any obvious staining (blood, semen, saliva)
- test any staining to check it is that substance (noted on diagram using different coloured pencils)
- any damage caused during testing must be noted

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4
Q

What is order for recovery sequence?

A
  • done in order of increasing likelihood of damage
  • any hairs/fibres removed so no possibility of being destroyed/lost
  • blood tested using dry swab
  • greasy stains noted by eye and recorded
  • any areas with touch DNA are swabbed in case low level of DNA
  • wet tests (wetting exhibit may damage evidence)
  • saliva test
  • semen test
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5
Q

What is DNA extraction?

A
  • removal of DNA from cellular material
  • isolation or purification of DNA
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6
Q

How are cells harvested from sample?

A
  • sample is wetted to hydrate it
  • centrifuged so that supernatant can be produced
  • pellet of cells at bottom of tube and can be removed
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7
Q

What steps are taken when a bodily fluid is suspected to be semen before DNA analysis?

A
  • would need to be confirmed beforehand
  • the pellet would have a sample taken and examined under a microscope in order to see if any sperm heads are seen
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8
Q

What are the summary steps of DNA extraction

A

1 - cell lysis
- where the DNA is released into solution by denaturing the cell wall and breaking down peptide bonds to free DNA
- uses ATL (a tissue lysis buffer used for purification) or proteinase K (used for destruction of proteins in cell lysate) and involves adding reagents

2 - precipitation/isolation
- sodium ions neutralise the negatively charged DNA and then alcohol is added to precipitate the DNA (form a solid) and bind to silica column

3 - purification
- the DNA precipitate is washed with alcohol to remove any impurities and inhibitors
- some use phenol chloroform followed by ethanol precipitation
- elution buffer is added to break hydrogen bonds between silica column and DNA
- dissolved in water again for storage

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9
Q

What is the extraction negative?

A
  • a blank sample that is extracted using the same reagents and protocol as the samples on the extraction batch
  • used to identify contamination (from potentially contaminated reagents or cleanliness of person/instruments
  • if it produces zero peaks = extraction has been successful and no contamination
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10
Q

What are the four methods of DNA extraction?

What do the basic principles of DNA extraction revolve around?

A
  • choice of method depends on sample type, desired yield, and downstream applications:
    1 - organic extraction
    2 - solid phase extraction
    3 - epithelial extraction
    4 - differential extraction
  • basic principles of DNA extraction revolve around:
  • disrupting the cells
  • separating DNA from other components
  • purifying it for further analysis
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11
Q

Organic extraction:
- what is it

A
  • involves use of organic solvents to break down cells and isolate DNA
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12
Q

Solid phase extraction
- what is it
- what samples is this useful for
- how does it work

A
  • utilises specialised columns or magnetic beads to selectively bind DNA
  • useful for samples where it is likely that they will contain low levels of DNA and require an additional step
  • e.g. swabs of touch DNA, pieces of solid material (paper, cigarettes, fabric), swabs taken during sample retrieval process
  • sample is placed into piece of equipment and spun at high speed to release as much DNA from swab/material as possible
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13
Q

Epithelial extraction
- what samples does this deal with?

A
  • deals with highly sourced samples for example, direct amounts of skin, other tissue, bodily fluids
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14
Q

Differential extraction:
- what is it
- when is it used
- how does it work

A
  • used when mixed DNA samples require separation of male and female genetic materials
  • often used when sperm is present along with other types of cells
  • differentiates between male (XY) and females (XX) chromosomes
  • isolates male DNA and allows for individual identification of each contributor within the mixed sample
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15
Q

What are the four advantages of differential extraction?

A
  • improved DNA recovery (can distinguish between male and female)
  • augmented specificity
  • conservation of material evidence
  • adept at dealing with degraded or ancient samples
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16
Q

What are the limitations of differential extraction?

A
  • high time consumption
  • optimisation can be hard due to discrepancies in biological specimens
  • more expensive due to its specialised nature
  • limited scope - not suitable for all situations
17
Q

What are the steps in differential extraction?

A

1 - add SAK sample, epithelial lysis buffer (contains ProK and surfactant) and regents to centrifuge tube
2 - incubate then centrifuge to form sperm pellet
3 - remove supernatant to leave a sperm pellet
4 - sperm pellet washed using more buffer solution, centrifuging and removing supernatant (epithelial cells removed this way)
5- sperm pellet then reconstituted and incubated in a new buffer solution (sperm lysis buffer) to lyse sperm cells releasing DNA
6 - DTT (reducing agent) is added to reduce disulphide bonds within sperm cell head

18
Q

What is cell lysis?

A
  • dissolution of structures such as sperm head or a cell so that the components of the structure go into free solution
19
Q

What must happen before the DNA can be replicated?

A
  • it must first be quantified
  • to allow the calculation of exactly how much DNA is present
  • it is vital to know this in order to prepare the sample for electrophoresis
20
Q

What are the three types of quantifier that are often used?

What principle do these work on?

A
  • Quantfiler - measures human DNA
  • PicoGreen - not human specific but quick and easy
  • HY - gives an indication as to how much male DNA is present
  • by adding a fluorescent probe or dye to the sample and then comparing how much the sample fluoresces against control samples/standards already loaded in the equipment
21
Q

What are the advantages, disadvantages and applications of DNA Quantfiler?

A
  • advantages:
  • dependable and finely-tuned mechanism for calculating DNA samples
  • facilitates precise judgement of DNA concentrations across a variety of samples
  • highlights human DNA, shrinking contamination risks
  • rapid and proficient assay
  • disadvantages:
  • inability to differentiate between degradation and inhibition
  • primarily developed for single source samples (struggles with intricate mixtures)

applications
- aids in the identification and profiling of questionable individuals
- resolves paternity disputes

22
Q

What are the advantages, disadvantages and applications of PicoGreen?

A
  • advantages:
  • vast sensitivity
  • fluorescence nucleic acid has limited interference with contaminants
  • environmentally friendly and safer substitute (no need for radioactive materials)
  • disadvantages:
  • non-specificity to double-stranded DNA (impacts precision)
  • sensitivity to pH levels and temperature
  • applications:
  • DNA-protein interactions
  • DNA labelling
  • cell viability assays
  • can distinguish intact DNA from degraded fragments
23
Q

What is the process involved with the HY DNA quantifier?

A

1 - DNA specimens are procured from the assembled biological samples
2 - DNA conc is evaluated using a spectrophotometer
3 - a distinct Y-chromosome region undergoes targeted PCR amplifications
4 - amplified DNA fragments are then differentiated using capillary electrophoresis

24
Q

comparison of standard PCR and qPCR

A
  • standard:
  • doesn’t provide real-time monitoring of DNA amplification process
  • amplification is carried out for a set number of cycles after which the products are analysed
  • commonly used for research, diagnostics and forensics
  • qPCR:
  • provides real time data on amount of DNA present in sample as reaction progresses
  • allows for monitoring of amplification of a targeted DNA molecule during the PCR process
  • uses fluorescent dyes or probes that emit signals as DNA amplification occurs
  • allows for quantification of initial amount of DNA and detecting the presence of specific DNA sequences
  • can skip quantitation phase
25
Q

What are the advantages and applications of qPCR?

A
  • advantages:
  • allows for detection and quantification of DNA/RNA in actual time
  • heightened sensitivity
  • high specificity
  • applications:
  • clinical diagnostics
  • gene expression profiling
  • mutation identification
  • viral load measurement
  • detection of pathogens
  • studies on genetic variation
26
Q

What is the leading strand in DNA replication?

A
  • the leading strand is synthesised continuously in the 3’ to 5’ direction
  • template for continuous synthesis of new DNA
27
Q

What is the lagging strand in DNA replication?

A
  • the lagging strand is synthesised discontinuously in short fragments called Okazaki fragments
  • lagging strand requires addition steps:
  • synthesis of RNA primers and joining of Okazaki fragments by DNA ligase
28
Q

What can be said about comparison of DNA profiles produced using earlier forms of DNA profiling vs current forms of DNA profiling?

A
  • cannot be matched to current records
29
Q

What is positive and negative pressure in a lab?

A
  • pre-PCR positive pressure = pressure higher than the pressure outside the room (keep contaminants out)
  • post-PCR negative pressure = pressure lower than the outside (keeps everything in)
30
Q

What is denaturation and annealing?

A
  • denaturation - process of separation of DNA strands
  • annealing - process of two strands of DNA rejoining
31
Q

What are amplicons?

A
  • amplified DNA fragments
32
Q

What is the melting temperature and what is the annealing temperature?

A
  • Tm - temp at which DNA solution is heated sufficiently that the double stranded DNA unwinds and the hydrogen bonds that hold the two strands together weaken and break
  • Ta - temp approximately 5 degrees below the lowest melting point of base of primers used
33
Q

What can be said about the number of STR markers that are coded?

A
  • more STR markers = greater amount of data in resulting profile = more discriminating = more unlikely to get coincidental match
34
Q

What is PCR positive?

A
  • an application using known concentration input of DNA
  • used to show a successful PCR reaction has occurred
  • can be used to monitor the performance of the thermocycler
35
Q

How do we see which allele pair is present at each marker?

A
  • the fragments copies need to be separated
  • done by size/molecular weight using capillary electrophoresis
  • smaller fragments travel faster
  • a sensor identifies the strands as they go past detection window which results in a series of different colour fluorescent peaks of different sizes
  • computer than translates peaks into series of numbered alleles
36
Q

What is allelic ladder?

A
  • control sample
  • sample of all available types of allele at a locus is run through a genetic analyser
  • also called analytic ladder
37
Q

What is an off ladder (OL) allele?

A
  • those which size outside the categories represented in the ladder
38
Q

What are internal size standards?

A
  • specific DNA fragments of known sizes which are defined and used to size unknown fragments
39
Q

What is a locus?

A
  • a particular place on a chromosome or within the genome
  • may be defined as a single base pair at a particular point

FSCI6370 - DNA (16 decks)

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