Topic 3 - How to Generate Profiles ' Flashcards
before DNA 17 what were the three types of test used to generate DNA profiles in order of invention
RFLP
DQ Alpha
Automated STR
what does RFLP stand for
when were these tests used
restriction fragment length polymorphism
used in the 80s and 90s
how long did RFLP take to generate a profile
one month or more
what were the profiles generated by RFLP called
auto-radiographs
these resembled an X-Ray plate and look like bar code patterns
what was good about RFLP
it was very discriminating
what was the name of the case that first used DNA in the conviction
Colin Pitchfork
in DQ alpha, what showed us the DNA molecules in the sample
the presence and absence of blue dots on test strips
what was DQ Alpha the first type of
what was this also called
DNA profiling method based on PCR
also called poly-marker
what were the 2 benefits of DQ Alpha over RFLP
could work with much smaller samples (more sensitive)
results generated quicker - in half a day rather than a month or more
what are known as the genetic scissors
restriction enzymes
what is meant by discrimination power
the ability to differentiate between two things
what is meant by an advantacious match
a random match that occurred by chance due to using a low number of loci
in 1985 what was the name of the DNA profiling method used by Sir Alec Jeffrey
what were the 3 things wrong with this method
MLP = multi locus probe
lengthy, needed a lot of sample, results were complex
in 1987 what was the process called that was used in DNA profiling
how was this an improvement from MLP (2)
what was still lacking with this method (3)
Single Locus Probe (SLP) or VNTR = variable number tandem repeats
easier to interpret results, quicker
lacked sensitivity, poor results with degraded samples, difficult to resolve mixtures
how many controls were run in SLP
2
did RFLP or DQ Alphas have higher discriminative value
RFLP
what match probabilities could be assigned with
a) RFLP
b) DQ Alpha
a) 1 in 1 million
b) 1 in a few hundred or few thousand
DQ Alphas was less discriminative but more sensitive
what is meant by sensitivity
the amount of starting material or the size of the stain that can be worked with
what is meant by automated STR profiling and why was this beneficial
the use of automated systems to analyse the STR regions in DNA samples
increased efficiency, accuracy and throughput of DNA samples. also higher discriminative power and sensitivity
what are the 6 criteria used to assess whether a profiling method is good or not
discriminating power
sensitivity
ability to deal with artefacts
speed
ability to deal with mixtures
ability to conduct a database search
is analysis using mitochondrial DNA or automated STRs more sensitive and why
using mitochondrial DNA is more sensitive because each cell contains the genetic material
but each cell also has thousands of mitochondria and each of these have copies of mitochondrial DNA
what is the volume range we typically deal with in DNA analysis
10-20 microlitre
using micropipettes
what can be used in electrophoresis to reduce the human interaction with samples
an autosampler
what is the type of profiling that focuses on DNA that is paternally inherited
briefly explain this method
Y-STR profiling
STR markers that reside on the human Y chromosome are considered
Y chromosomes are paternally inherited so male relative are likely to have the same = less discriminative
what are the two downfalls of using mitochondrial DNA rather than nucleic DNA
mitochondrial DNA is only inherited from the mother - making it less discriminative as many maternal relative are likely to share this genetic material
this type of DNA can be small compared to nucleic DNA so less info is contained within it
everyone has the same mitochondrial DNA as their…
mother
when is Y-STR testing beneficial
when you have a mixed profile with a male and female contributor and you want to focus on the male profile
e.g sexual offence cases
what is an allele
a region of DNA that varies between people - different forms of the same gene
two alleles are seen at each loci
one allele inherited from each parent
how much DNA is recommended for the new DNA 17 multiplex
what does this mean about this method compared to previous ones
200-500 pg - cant be seen with the naked eye
more sensitive
what amount of DNA do previous profiling kits recommend using
500-1000 picograms
100-200 cells
what is meant by a polymorphism
regions of DNA that differ between individuals
what are the regions of DNA that can be the same between individuals
coding regions - these code for things like eye colour or hair colour
not good to be used in profiling
what is a
a) loci
b) locus
a) multiple locus
b) a specific location on a chromosome
what are amplified fragments of DNA called
amplicons
what is the purpose of the elution buffer in DNA extraction and purification
to remove the DNA bound to the silica column by breaking the hydrogen bonds
in electrophoresis how are the DNA fragments measured
in RFUs - relative fluorescent units
a measure of light intensity picked up by a camera in the detector at different points in time
what are the first peaks typically seen in PCR - these peaks look much larger than the amplified DNA peaks
primer residue
how many coloured fluorescent markers are typically used in electrophoresis
4-6 - this is dependent on the type of multiplex being used
how many peaks in an electropherogram signify a mixed profile
three or more peaks at any loci
where are allele drop out peaks normally seen if they are present
the end of the EPG where the bigger fragments are as these are more susceptible to degradation
which loci represents the biological gender of the sample
amelogenin
what does the height of an allele peak represent
the relative amount of the allele
are smaller or larger DNA fragments on the left side of the EPG
smaller
what can we learn from an EPG (5)
biological gender
DNA profile - alleles present at each loci (homozygous or heterozygous)
single source or mixture
degraded sample - larger fragment allele peaks may be missing
peak height = relative amount of DNA
what is SGM+
previously used automated STR multiplex looking at 10 loci
what are the 5 hypotheses that could be put forward for observing a DNA match
same source
coincidence
contamination
analysis error or error in handling after collection
a mistake - could be deliberate or accidental
how is the chance of an accidental match ruled out
with the use of statistics giving random match probabilities
