How to generate DNA profiles Flashcards
What is RFLP?
Restriction fragment length polymorphisms
* first DNA testing used
* took about a month to generate profiles
* separated by size
* very discriminating
* used radiation to mark where the DNA bands are
What is DQ Alpha?
- first PCR-based test
- look for the absence of blue dots - would show what sort of DNA molecules are present
What are the benefits of DQ Alpha over RFLP?
- faster
- more sensitive
What is MLP?
Multi-Locus Probe
* lengthy
* requires a large amount of sample to produce a profile
* results were complex to interpret
* only good for comparison
What is SLP?
Single Locus Probes
* easier to interpret
* intial results obtained in a few days but took over a week to prepare a full profile
* lacked sensitivity
* poor results with degraded samples
* difficult to resolve mixtures
What is sensitivity?
The amount of starting material or size of the stain we can begin working with
What is discriminating power?
The power to distinguish between one thing and another thing
What is the six criteria used for judging the benefits of a DNA profiling system?
- distriminating power
- sensitivity
- ability to deal with artefacts
- speed
- ability to deal with mixtures
- ability to conduct database searches
What is mitochondrial DNA?
- more sensitive than automated STRs
- every cell within it contains two copies of all of our genetic material
- relatively small compared to the amount of DNA that is present in the nucleus so not much information is contained within
What is another drawback of mitochondrial DNA?
- it is maternally inherited
- will be many more maternal relatives who will share the genetic material
What is Y-STR?
- tests on Y STR markers which reside on the human Y chromosome
- can give us insight into the male DNA profile without seeing contributers from the female
- helpful when there is a mixture
How much DNA do we need for DNA17?
500pg
What are polymorphisms?
- non-coding part of DNA
- differ greatly from person to person
- the parts that are in DNA profiles
Primers in the PCR process?
- forward and reverse primers
- attached to one primer is a fluorescent dye
What does taq polymerase do?
- is thermally stable
- synthesises new DNA strands by extending primers along the template DNA
- binds to the template strand at the start of the area for amplification
- doesnt proceed until the temperature is increased (optimised temperature)
- extension phase where the DNA polymerase synethesises new DNA strand by extending the primers along the whole amplification area
What is the function of dNTPs?
- building blocks for the new DNA strand
- in the extension step, DNA polymerase adds free dNTPs from the reaction mixture to the template DNA
What are STRs?
Short Tandem Repeats
* small pieces of the genetic code which are repeated
What is the vertical axis and horizontal axis represent in the raw data produced from capillary electrophoresis?
- Vertical axis = relative fluorescence units, measures of the intensity of light picked up by the camera in the detector window at different points in time
- Horizontal axis = minutes or seconds
What are the first peaks in the raw data from capillary electrophoresis?
primers left over from the PCR process
Where are the allelic drop out and drop in seen in an EPG?
at the end because they are the bigger bits so take longer to travel through and are more susceptible to degradation
What is on the left and right side of the EPG?
- left side: small fragments
- right ride: large fragments
What can be determine from the peak height in EPGs?
- proportional to the amount of DNA that gave rise to that particular peak
During the process of purification, where are the impurities to be found after centrifugation?
Pellet
What is heterzygous?
two different alleles at a locus
What are STRs and what are they also known as?
Short Tandem Repeats - mini/micro-satellites
* small pieces of genetic code that are repeated
