Molecular diagnostics and Unstable expansion disorders Flashcards
Southern blots are used to detect
large deletions, large insertions and point mutations using RFLPs ( restriction fragment polymorphism- a very special case)
point mutations/RFLPs/Southern blot
allow for the detection of point mutations via RFLPs , which destroy (or create) a particular restriction fragment site, by using a Southern blot to recognize individuals who have the mutation and those who don’t have the mutation
ASO analysis is used to detect what kind of mutations
are used to detect single base pair mutations, small insertions and deletions, put the genomic DNA down, and a single probe for mutant allele and for the wild type allele to determine via hybridization if the mutation is present or not.
how does ASO analysis actually work?
- design 2 short probes that precisely match either the normal DNA sequence or the mutant DNA sequence, 2. do a DOT or a SLOT blot to apply the genomic DNA to the membrane,
- Add probes which hybridize to patient’s DNA and see which will bind 4. hybridization is determined by whether you get fluorescence or not
what is a dot blot or a slot blot?
a dot blot/slot blot - mechanism for putting genomic DNA on the membrane, genomic DNA is blotted on to tight spaces on a membrane (its not separated by size) and stuck to it
Other techniques used to detect point mutations
PCR with RFLP - using PCR rather than Southern Blot, know that the point mutation will create a new RsaI site, and will allow for you to detect sizes as well as Sequencing
what’s qPCR, real time PCR?
qPCR allows you to measure and quantify product in the sample in real time at its being made, i.e. as the PCR is running, establish the number of cycles required to reach the threshold from both samples to know the amount of DNA in the test tube, fewer cycles to reach the same threshold, more DNA in the sample
what’s standard PCR?
run the PCR to saturation, about 30 cycles , and then run the samples out on a gel to look at them
a premutation in unstable repeat expansion disorder
a non-disease causing expansion size, which is capable of expanding in the next generation, and causing disease. anyone w/a premutation is at risk of passing the disease
anticipation is
earlier onset and increased severity as you go from generation to generation, younger kids get it and it’s more and more severe
How does the repeat expands as it passes from generation to generation?
through slipped mis-pairing mechanism, the replicating strand detaches inappropriately from the template strand during DNA replication, and due to the many number of repeats the replicating strand slips from its proper alignment, it later re-attaches the newly synthesized strand contains extra repeats
