Biology 3594A Lecture Notes - Lecture 11: Snp Genotyping, Phenotype, Neurodevelopmental Disorder
13 May 2018
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There are certain mutations seen across an individual’s karyotype
Individuals with this disease have a neurodevelopmental disorder
Graphical abstract is trying to describe model, observations they say, and go back and describe when they think these events happen
Mutator phenotypes –because of copy number variance in this instance
They found DNA repair genes that a person inherited, they were all aberrant
No pathway of repair, mutation frequency went up
Mutation state is too many mutations –looking at not the source but the product -> too many mutations because of lack of repair
Make sure probe is single stranded, working under denaturing situation, hybridize this
Listed from simple to more difficult
FISH is older, genetic, nice for in situ, can see where the fluorescent probes have hybridized on the metaphase chromosomes
From genic to genomic down the list (first two are genic?)
NGS is most expensive –need good bioinformatics if you want to detect CNVs
Had to learn to read the fluorescence from the NGS, normalizing it over of the genome and see where they get more or less DNA than they expect (to detect
knockouts or CNVs?)
Genetic = smol Genomic = da whole ting
FISH can be M-FISH, can move to be more genomic
Hybridization –get a piece of DNA to form a complementary match with target, H-bonds between nucleotides
Need to control temp and salt –want probes to bind but then you want only the specific matches to remain and wash everything else off
Doing CGH, SNP, Southern, FISH –want to capitalize on DNA complementarity and H bonding –work with known sequences and have probe that is perfect match
Do it in steps –start with low stringency so that your solution of probes and template will hybridize
Then raise the stringency so the non-specific will wash off and leave behind only your perfect match
Don’t go so high that everything comes off the sample, but go high enough that other stuff goes away
Can determine melting temperatures, we know how many H bonds are there
Millions of probes = all reactions that you’re expecting to go perfect -> it doesn’t, sometimes you get false positives
You get a lot of data but then you have to validate it
Array has row and column, produced by industries
In situ –do with a karyotype of chromosomes
In silico –a platform with probes
See how much hybridization we get
If we have probes that rep more of the genome, increasing the resolution we can look at –often probe sampling system, maybe every 1000 nucleotides you make a
probe
Lower resolution –10 000 nucleotides between loci that you look at for example
Genome wide –the sampling will include all the chromosomes, sometimes add mitochondrial chromosomes
Rows or columns identify what region of the genome
Red = reference
Yellow = equal proportion of two, sample matches our reference
Green = more sample than reference
Massively parallel hybridizations occurring
SNP genotyping probes –designed originally to figure out what the polymorphic loci looked like in an organism
Probes for common single base pair subs
Mouse Diversity Genotyping Array –most powerful for mice that we have
Use fluorescence intensities to figure out CNV
If you try running with cancer samples it’s crazy – genomes have chromosomal and base pair instability –not enough of normal fluorescence intensity across the array
Probes are 25-33 nucleotides
CNVs –recognize by how many bind
Redundancy across the array –have it spotted more than once, look at average intensity you gather from probes and those sites
Fluorescence intensity will dip from normal intensity = deletion
There is a solution of probe on the array, probes are not saturated
CEL file –shows fluorescence intensity
Depends on amount of template DNA that bound
Template DNA is labeled
Human is higher resolution because it has 1.8M sites in the genome that it’s working from, mouse has about 900 000
Karyotype of a mouse –19 autosomes
Working with person in CS
Learned that copy number variations might relate to having adaptations to the environment
Lec 11 Mutator Phenotype II
May 13, 2018
2:38 PM
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