Microsatellites and Individual ID Flashcards
What is CODIS 20 Loci?
a loci with many varients which has lot of power to individualize.
how many loci do wildlife cases use?
at least 10
for muscox and bison how many loci are used? Why?
20-40, they have little variation (very inbred)
what are the applications of microsatelites?
- individual identification
- relatedness
- population assignment
- much more variation than markers for species ID.
- no “universal” primers
- sometimes used to delineate b/w very closely related species and their hybrids or breeds.
How often are STR or VNTR scattered in a genome?
~1/10,000 base pairs
where do primers go on STR regions? Why?
flank them on either side, for subsequent amplification of locus and incorporating fluorescent tags during PCR
what is the name of ACATACATACAT?
tetranucleotide
T/F: CCACCC is an uninteruppted microsatellite.
false, this is interupted
what kind of repeats are most common?
di and tetra nucleotides
T/F: STRs have high discriminating power, at >0.8 per locus.
false, it is >0.9 per locus
STRs have a high percentage of what?
heterozygotes
how do you avoid linkages of loci?
separating chromosomal locations
T/F: STRs amplify well in degraded samples and in combination with other microsatellites.
true
What are the applications of STRs?
- low stutter characteristics
- low mutation rate
- allele range b/w 100 and 500 bp
how large are microsatellite profiles?
7 locus profile for each individual, 14 numbers = genotype
once a microsatellite profile is created what do you do with it?
evaluate rarity of genotype in frequency of alleles in population
what are challenges with genotype interpretations?
- stutter
- non-template addition
- microvariants
- null alleles
- mutation
- allelic dropout
what are stutters?
peaks smaller than allele by one or more repeats
how do stutters occur?
from DNA pol slipping during PCR
which kind of nucleotide has more stutters?
dinucleotides, tetra have some not a lot.
when do stutters happen more frequently?
repeated sequences
what is backward slippage?
in the reverse strand which is an insertion causing the backwards slippage resulting in shorter STR
what is forward slippage?
Occurs when the DNA polymerase slips forward on the template strand, leading to an extra repeat unit being added to the amplified sequence.
This results in a longer PCR product than expected.
What are the complications from stutter peaks?
its hard to discern stutter from alleles when numerous sources contribute to DNA profile because stutter has same size as potential alleles
when is stutter especially problematic?
when there is a minor contributor
What does Taq pol do?
often adds an extra A to the 3’ end of PCR product
what can influence the interpretation of alleles?
the non-template addition of A
what is the suggested quantity of template DNA?
2 ng
what are null alleles?
mutations in primer binding sites resulting in primers not annealing and no product for that allele especially when mutations are at the 3’ end of the primer
when are null alleles a huge problem?
when amplifying markers across geographically isolated populations or species and parentage
what do null alleles lead to?
false exclusions
what is the solution for null alleles?
- primer redesign or if it is recurrent then drop the locus
- re amplify with lower annealing temp
- verify locus for nulls based on parentage
what is the ultimate source of polymorphisms?
fixation of mutation in a population
when STR mutations lead to a new allele what is the thought of cause?
it is from replication slippage and/or defective replication repair
are mutations a problem for individual ID? Why/why not?
no, because the other mutations should be the same in all cells of an individual
what is the cause of allelic dropouts?
- often matter of low template amount
- can also be an issue with degraded DNA and larger alleles
- often an issue with a few hairs/scat, run PCR several times
T/F: exclusions are straight forward; inclusions require statistical evaluation.
true
what is the amelogenin gene?
this is a sex-linked tooth enamel gene on both X and Y chromosomes.
where are primers placed on amelogenin gene?
they flank the deletion in intron 1 of the amelogenin gene on X homologue
apart from amelogenin, how can you tell sex?
Zfx/Zfy
what uses the X-O system? What is female/male?
bugs, male: X; female: XX
what uses the Z-W system? What is female/male?
birds, male: ZZ; female: ZW
how are bees different for sex determination?
the males have half the amount of chromosomes than females, male: 16; female: 32
what is a microvarience?
non-perfect microsatellite where there might be a poly A or something between the 4 repeats.
Small populations lead to what?
less gene flow, more local databases are needed
large populations means what?
more gene flow and less structured and similar frequencies
how do you create a database?
decide on the number of samples and grouping -> gather samples -> analyze samples at desired genetic loci (summarize DNA types) -> determine allele frequencies for each locus -> perform statistical tests on data
how many regions are needed to get a database?
20-30
what do you want to avoid at each population?
sample bias
empirical evidence suggests that 20-30 samples is a good balance of what?
accurate frequencies and expense of running samples
what is the equation for minimum allele frequency?
5/2N
explain the MAF equation
you need to observe allele 5X in population to avoid bias in statistical elements, and the 2N is because 2 chromosomes per individual
Why is non-random mating important?
for parentage assignment statistics
genetic diversity is linked to what?
the number of markers you use
what do population assignments help with?
- monitor illegal animal releases
- track source populations for disease outbreaks
- ensure animals/meat not moved between regions with and without disease
how is an individual is assigned to a population?
which its genotype has the highest probability of occurring.
