what does STR stand for? how many base pairs long are they? what makes them so discriminatory?
short tandem repeats
2-7bp long
no. of repeats varies between individuals
what are the two types of STRs? what is the difference?
tetranucleotide- 4bp long
pentanucleotide- 5bp long
what is the allelic ladder?
a collection of known DNA fragments that represent common alleles found at different short tandem repeat loci
what are the contents of PCR amplification kits such as NGM detect?(4)
- master mix
- primer set
- allelic ladder
- DNA control
what is contained in the master mix? (6)
- DNA polymerase
- dNTPs
- buffer solution
4.MgCl2 - stabilisers + enhancers
6.nuclease free water
what is the purpose of DNA polymerase in the master mix?
it is an enzyme that synthesizes DNA strands from the template during PCR
what is the purpose of dNTPs in the master mix?
These are used to add to the DNA single strand. complimentary dNTPs are added to the strand once the DNA polymerase reads the DNA template. they are the building blocks for DNA synthesis.
what is the purpose of the buffer solution in the master mix?
maintains optimal pH and salt conditions for enzyme activity
what is the purpose of MgCl2 in the master mix?
cofactor required for DNA polymerase activity. without it polymerase wont function
what is the purpose of stabilisers and enhancers in the master mix?
improve the stability of enzymes during thermal cycling
what is the purpose of nuclease free water in the master mix?
used to bring the master mix to a desired volume
what does dNTP stand for? and what are they?
deoxynucleotide triphosphate. these are the building blocks of DNA. dNTP contains 3 parts: a nitrogenous base (A, T, C or G), deoxyribose sugar, 3 phosphate groups
what is the purpose of the DNA control in the contents of PCR kits
a known DNA sample used as a reference confirming whether the STR markers amplify properly+ if system can detect+ size alleles properly
what are the 5 types of stochastic variation
- stutter
- pull up
- dye blobs
- spikes
- split/shoulder peaks
what is stutter caused by? and how do you detect it?
caused by backward/forward slippage of taq polymerase during extension. it is 1 repeat smaller or bigger than mainpeak
what does the allele number tell us on a DNA profile?
the number of repeats at each position
what do the 3 numbers represent at each peak on a DNA profile?
- top no.= allele no.
- middle= relative fluorescence units
- base pairs
what is pull up and why does it occur?
it is a colour bleed from one spectral channel to another, caused by high RFU directly above or below the peak
how can you determine whether a peak is pull up?
if it is within 1bp
minor peaks in the wrong dye colour
what does OL mean on a DNA profile
means it is off the ladder, it is not within the grey bin TF off the allelic ladder
what are dye blobs + what is there cause?
theyre wide small peaks, caused by fluorescent dye tags breaking off from the DNA and going through the detector
what is spike and what is it caused by?
will see it through every channel, cuased by an air bubble or voltage spike
what is a split/shoulder peak and what is the cause?
two closely spaced peaks instead of 1/ not fully separated. caused by incompleye adenylation in the extension phase. 1bp smaller than true alele
what are the interpretation rules?
- any peak under 25RFU so not consider
- any homozygous peak must be higher than 250 RFU
- heterozygous peaks need to be within 50% of each other
- stutter peaks must be under 6% of each other, DS22-12%
