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DNA Analysis & Interpretation > Practical DNA Processing > Flashcards

Practical DNA Processing Flashcards

1
Q

Define allele

A

a different form of a gene at a particular locus

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2
Q

Define allelic drop-out

A

failure to detect an allele in a sample or failure to amplify an allele during PCR

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3
Q

define amplicon

A

amplified DNA fragments

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4
Q

Define AFLP

A

Amplified Fragment Length Polymorphisms
* a highlly sensitive method for detecting polymorphisms in DNA
* DNA first undergoes restiction enzyme digestion, and a subset of DNA fragments is then selected for PCR amplification and visualisation

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5
Q

Define Bayesian probability

A

system of probability based on beliefs in which the measure of probability is continously revised as available information changes

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6
Q

What equipment is used to collect DNA from bone samples?

A

Freezer mill

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7
Q

Detergents are added to dissolve proteins, to free DNA in this step?

A

Lysis

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8
Q

What is a singke peak on an EPG called?

A

Homozygous

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9
Q

What level of proposition is it if the DNA came from the person of interest address?

A

Sub source

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10
Q

What steps should be taken for sample retrieval of DNA samples?

A
  • turn garment inside out and look inside pockets
  • look for obvious staining
  • all evidence which comes into the lab must be recorded, photographed and drawn with annotations before samples are taken
  • any damage caused during testing must be noted
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11
Q

What is differential extraction?

A
  • used when there are mixed DNA samples require separation of male and female genetic materials
  • often used when sperm is present along with other types of cells
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12
Q

How does differential extraction work?

A
  • focuses on the selective rupture of male cells, thereby isolating their DNA and facilitating the individual identification of each contributor within the mixed samples
  • male genetic code comprises two copies of the X and Y chromosomes
  • female has double-X chromosome
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13
Q

What are the advantages of differential extraction?

A
  • improved DNA recovery
  • augmented specificity
  • conservation of material evidence
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14
Q

What are the disadvantages 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
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15
Q

What are the steps in differential extraction?

A
  1. add SAK sample, epithelial lysis buffer and reagents to centrifuge tube
  2. incubate and then centrifuge
  3. remove supernatant to leave a sperm pellet
  4. sperm pellet washed for about 15-30mins
  5. add sperm pellet, sperm lysis buffer and reagents and then incubate to form buffer containing sperm DNA
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16
Q

What is cell lysis?

A

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

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17
Q

What is PCR?

A

the process of taking a small sample of DNA and copying it many times

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18
Q

What is the leading strand in DNA replication?

A

the DNA strand that is synthesised continuously in the 5’ to 3’ direction
* template for the continous synthesis of new DNA

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19
Q

What is the lagging strand in DNA replication?

A
  • the DNA strand that is synthesised discontinuously during DNA replication
  • synthesised in short, discontinuous fragments called Okazaki fragments
  • these fragments are later joined together by DNA ligase to form a continuous strand
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20
Q

Why is the lagging strand only synethesised in fragments?

A

Nucleotides cannot be added to the phosphate (5’) end because DNA polymerase can only add DNA nucleotides in a 5’ to 3’ direction

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21
Q

What is real-time PCR?

A

quantitative PCR
* allows for the monitoring of amplification of a targeted DNA molecule during the PCR process
* provides real-time data on the amount of DNA present
* ideal for quantifying DNA and detecting the presence of specific sequences

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22
Q

What is standard PCR?

A
  • doesnt provide real-time monitoring of the 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
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23
Q

How does qPCR work?

A
  • use fluorescent dyes or probes that emit signals as the DNA amplification occurs
  • allows for the quantification of the inital amount of DNA
  • can skip that quantitation phase
24
Q

What is the role of DNA polymerase in DNA replication?

A
  • enzyme responsible for synthesising new DNA strands during replication
  • adds nucleotides to the growing chain in a complementary fashion
  • uses existing DNA strand as a template
25
Q

What are the steps against contamination?

A
  • all people who enter the lab must provide elimination DNA samples
  • protective clothing: facemakr, mobcap, first pair of gloves, lab coat, second pair of gloves
  • gloves, equipment and benches are wiped with alcohol between each sample
  • each bench has its own set of equipment whihc is logged so that any contamination can be traced back
26
Q

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

A

sample pellet is taken and examined under a microscope in order to see if any sperm heads are seen

27
Q

What is DNA extraction?

A

removal of DNA from cellular material; isolation or purification of DNA

28
Q

How are the cells harvested from the sample?

A
  • sample is wetted to hydrate
  • centrifuged so that the supernatant can be produced
  • pellet of cells will be at the bottom of the tubes and can be removed
29
Q

What is organic extraction?

A

involves the use of organic solvents to break down the cells and isolate the DNA

30
Q

What is solid-phase extraction?

A

uses specialised columns or magnetic beads to selectively bind DNA

31
Q

What are the steps in DNA extraction?

A
  1. cell lysis
  2. DNA isolation
  3. DNA purification
32
Q

What is cell lysis?

A
  • where the DNA is released into solution by denaturating the cell wall
  • uses ATL or ptoteinase K and invloves adding reagents
33
Q

What is ATL in cell lysis?

A

a tissue lysis buffer used for purification

34
Q

What is Proteinase K used for in cell lysis?

A

destruction of proteins

35
Q

What is DNA purification?

A

release of DNA from the silica column

36
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
  • if it produces 0 peaks it suggests that extraction has been successful and not be affected by contamination
37
Q

What is quantification?

A
  • allows for the calculation of exactly how much DNA is present
38
Q

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

A
  • quantifiler - measures human DNA
  • PicoGreen - not human specific but quick and easy
  • HY - indicates how much male DNA is present
39
Q

What are the advantages of DNA quantifiler?

A
  • dependable and finely-tuned for calculating DNA samples
  • precise DNA concentrations across a variety of samples
  • highlights human DNA, shrinking contamination risks
  • rapid and proficient assay
40
Q

What are the disadvantages of quantifiler?

A
  • inability to differentiate between degradation and inhibition
  • primarily developed for single source samples - struggles with intricate mixtures
41
Q

What are the applications of DNA quantifiler?

A
  • aids in the identification and profiling of questionable individuals
  • resolves paternity disputes
42
Q

What are the applications of PicoGreen?

A
  • DNA-protein interactions
  • DNA labelling
  • cell viability assays
  • can distinguish intact DNA from degraged fragments
43
Q

What are the advantages of PicoGreen?

A
  • vast sensitivity
  • fluorescence nucleic acid has limited interference with contaminants
  • environmentally friendly and safer substitute - no need for radioactive materials
44
Q

What are the disadvantages of PicoGreen?

A
  • non-specificity to double-stranded DNA - impacts the precision
  • sensitivity to pH levels and temperature
45
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 undergo targeted PCR amplification
  4. amplified DNA fragments were then differentiated using capillary electrophoresis
46
Q

What is positive and negative pressure?

A
  • Pre-PCR positive pressure = pressure higher than the pressure outside the room (keep contaminants out)
  • Post-PCR negative pressure = lower pressure than the outside (keeps everything in)
47
Q

What is denaturation?

A

The process of separation of the DNA strands

48
Q

What is annealing?

A

the process of two strands of DNA rejoining

49
Q

What is the annealing of primers?

A
  • small pieces of DNA that provide the free ends needed for DNA replication to begin
  • starting point of DNA synthesis
  • forward and reverse primers due to anti-parallelism
  • DNA polymerase directed extension from primer binding sites
  • one primer will attach to the top strand and the other primer to the bottom strand at the other end
50
Q

What are the advantages of qPCR?

A
  • detection and quantification of DNA or RNA in actual time
  • heightened sensitivity
  • high specificity
51
Q

What are the applications of qPCR?

A
  • clinical diagnostics
  • gene expression profiling
  • mutation identification
  • viral load measurment
  • detection of pathogens
  • studies on genetic variation
52
Q

What is a PCR positive?

A
  • application using known concentration input of DNA
  • used to show how successful PCR reaction has occurred
  • can be used to monitor the performance of the thermocycler
53
Q

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

A
  • the fragments copied need to be separated
  • done by size/molecular weight using capilary electrophoresis
  • smaller fragments travel faster
  • a sensor identifies the strand as they go past which results in a series of different colour fluorescent peaks of different sizes
54
Q

What is an allelic ladder?

A
  • control sample
  • made up of DNA fragments that represent common alleles at a locus
55
Q

What is a locus?

A

a particular place on a chromosome or within the genome

56
Q

What is an off ladder allele?

A

alleles which size are outside the categorises represented in the ladder

57
Q

What are internal size standards?

A

specific DNA fragments of known sizes which are defined and used to size unknown fragments

DNA Analysis & Interpretation (17 decks)

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