Practical DNA Processing Flashcards
Define allele
a different form of a gene at a particular locus
Define allelic drop-out
failure to detect an allele in a sample or failure to amplify an allele during PCR
define amplicon
amplified DNA fragments
Define AFLP
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
Define Bayesian probability
system of probability based on beliefs in which the measure of probability is continously revised as available information changes
What equipment is used to collect DNA from bone samples?
Freezer mill
Detergents are added to dissolve proteins, to free DNA in this step?
Lysis
What is a singke peak on an EPG called?
Homozygous
What level of proposition is it if the DNA came from the person of interest address?
Sub source
What steps should be taken for sample retrieval of DNA samples?
- 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
What is differential extraction?
- 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
How does differential extraction work?
- 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
What are the advantages of differential extraction?
- improved DNA recovery
- augmented specificity
- conservation of material evidence
What are the disadvantages of differential extraction?
- 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
What are the steps in differential extraction?
- add SAK sample, epithelial lysis buffer and reagents to centrifuge tube
- incubate and then centrifuge
- remove supernatant to leave a sperm pellet
- sperm pellet washed for about 15-30mins
- add sperm pellet, sperm lysis buffer and reagents and then incubate to form buffer containing sperm DNA
What is cell lysis?
the dissolution of structures such as sperm head or a cell so that the components of the structure go into free solution
What is PCR?
the process of taking a small sample of DNA and copying it many times
What is the leading strand in DNA replication?
the DNA strand that is synthesised continuously in the 5’ to 3’ direction
* template for the continous synthesis of new DNA
What is the lagging strand in DNA replication?
- 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
Why is the lagging strand only synethesised in fragments?
Nucleotides cannot be added to the phosphate (5’) end because DNA polymerase can only add DNA nucleotides in a 5’ to 3’ direction
What is real-time PCR?
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
What is standard PCR?
- 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
How does qPCR work?
- 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
What is the role of DNA polymerase in DNA replication?
- 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
What are the steps against contamination?
- 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
What steps are taken when a bodily fluid is suspected to be semen before DNA analysis?
sample pellet is taken and examined under a microscope in order to see if any sperm heads are seen
What is DNA extraction?
removal of DNA from cellular material; isolation or purification of DNA
How are the cells harvested from the sample?
- 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
What is organic extraction?
involves the use of organic solvents to break down the cells and isolate the DNA
What is solid-phase extraction?
uses specialised columns or magnetic beads to selectively bind DNA
What are the steps in DNA extraction?
- cell lysis
- DNA isolation
- DNA purification
What is cell lysis?
- where the DNA is released into solution by denaturating the cell wall
- uses ATL or ptoteinase K and invloves adding reagents
What is ATL in cell lysis?
a tissue lysis buffer used for purification
What is Proteinase K used for in cell lysis?
destruction of proteins
What is DNA purification?
release of DNA from the silica column
What is the extraction negative?
- 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
What is quantification?
- allows for the calculation of exactly how much DNA is present
What are the three types of quantifier that are often used?
- quantifiler - measures human DNA
- PicoGreen - not human specific but quick and easy
- HY - indicates how much male DNA is present
What are the advantages of DNA quantifiler?
- 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
What are the disadvantages of quantifiler?
- inability to differentiate between degradation and inhibition
- primarily developed for single source samples - struggles with intricate mixtures
What are the applications of DNA quantifiler?
- aids in the identification and profiling of questionable individuals
- resolves paternity disputes
What are the applications of PicoGreen?
- DNA-protein interactions
- DNA labelling
- cell viability assays
- can distinguish intact DNA from degraged fragments
What are the advantages of PicoGreen?
- vast sensitivity
- fluorescence nucleic acid has limited interference with contaminants
- environmentally friendly and safer substitute - no need for radioactive materials
What are the disadvantages of PicoGreen?
- non-specificity to double-stranded DNA - impacts the precision
- sensitivity to pH levels and temperature
What is the process involved with the HY DNA quantifier
- DNA specimens are procured from the assembled biological samples
- DNA conc is evaluated using a spectrophotometer
- a distinct y-chromosome region undergo targeted PCR amplification
- amplified DNA fragments were then differentiated using capillary electrophoresis
What is positive and negative pressure?
- 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)
What is denaturation?
The process of separation of the DNA strands
What is annealing?
the process of two strands of DNA rejoining
What is the annealing of primers?
- 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
What are the advantages of qPCR?
- detection and quantification of DNA or RNA in actual time
- heightened sensitivity
- high specificity
What are the applications of qPCR?
- clinical diagnostics
- gene expression profiling
- mutation identification
- viral load measurment
- detection of pathogens
- studies on genetic variation
What is a PCR positive?
- 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
How do we see which allele is present at each marker?
- 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
What is an allelic ladder?
- control sample
- made up of DNA fragments that represent common alleles at a locus
What is a locus?
a particular place on a chromosome or within the genome
What is an off ladder allele?
alleles which size are outside the categorises represented in the ladder
What are internal size standards?
specific DNA fragments of known sizes which are defined and used to size unknown fragments
