Molecular Diagnositcs 3 Flashcards
What is an alternative approach to ASO hybridization
allele-specific PCR
What is MLPA
combining lots of PCRs together
What is the principle in allele-specific PCR
PCR primers
looking for allele-specific mutations (like ASO but primers )
If you use normal primer on mutatnt allele what kind of product?
no PCR product
If you use mutant primer on normal allele what kind of product?
no PCR product
If there’s a mismatch at the 3’ end what kind of product
no PCR product
What kind of primer do you use for reverse primer for allele-specific pCR
allele-specific reverse primer
What kind of product do you look at to interpret Allele-specific PCR
bands instead of drops
Can you use reverse or forward primer for allele-specific PCR?
you can use either, have to choose which one you want to be
What is multiplex PCR
method to scan commonly deleted exons. It can scan large regions of genome
It does lots of PCR reactions combined into one test tube, so multiple done at once
Why do we use multiplex PCR? Give an example
big deletions, like duchenne muscular dystrophy. DMD is too big to sequence normal way.
How many primers are used for multiplex PCR
multiple primers are used, you are doing a ton of reactions at once. You don’t know how big deletion is so you use a bunch of primers to get an idea of how big deletion is
What kind of method could be used to diagnose DMD?
multiplex PCR
What does MLPA stand for
multiplex ligation-dependent probe amplification
What method can be used to identify carrier mothers for DMD
MLPA
In typical MLPA how many primers can be used
up to 40
Instead of a single probe what do you use for MLPA
two primers that hybridize end to end
In MLPA when two primers hybridize what do they form in the middle
nic
How do you combine the nic formed from two primers hybridizing in MLPA
ligase
ligase only works if what (in MLPA)
template DNA is there
The amount of ligated product will be directly proportional to what in MLPA
amount of template used
The primers all have the same what in MLPA
PCR binding site
How do you distinguish b/w the different primers in MLPA?
stuffer sequence
What is stuffer seuence?
little pieces of DNA that changes the size
How do you distinguish the different products in MLPA
they all have a different size, can run a gel and they are all different size
Are primers in MLPA flerourescently labeled?
yes
Each primer set represents a different what in MLPA
exon
What’s the biggest difference b/w MLPA and multiplex pCR
MLPA can scan bigger region
can determine more clearly if pt is heterozygous carrier
MLPA is more quantitative
What are the advantages of MLPA
scanning large regions (can use 40 primers at a time)
more quantitative
What is the negative part of MLPA
only detects copy number, doesn’t detect chromosomal rearrangmeents
What does FISH stand for
fluorescent In-situ hybridization
How do you determine what approach to use for detecting deletion?
depends on how big deletion is
What do you use to detect small deletions
ASO or PCR
What do you use to detect medium deletions (100s of BP)
PCR, Southern blotting, MLPA, CGH
What do you use to detect large deletions (10’s of kb)
Southern blotting, MLPA, CGH
What do you use to detect VERY large deletions (500 kb - 3 mb)
FISH, CGH
What does In-situ mean
looking in a cell or tissues
What is the target in FISH
cellular chromosomal DNA
what is the probe in FISH
BAC
What does BAC stand for
bacterial artifical chromosomes
what is size of BAC
100-200 kb
What is BAC how is it a probe
vector used to carry large amounts of DNA
How can you visualize in FISH
fluorescent microscopy
If probe is larger than deletion, what will happen in FISH
it won’t discriminate - it will still hybridize to the one you don’t want it to, if’s so big it doesn’t matter that there is deletion
What is metaphse FISH
when cells are growing in culture synchonize and arrest them in metaphase
what is the advantage of metaphase FISH
can see chromosomes - can see location and if chromosome is there
what is interphase fish
don’t arrest cells
what is the advantage of interphase fish
don’t have to arrest them
what is the disadvantage of interphase fish
can’t see chromosomes or location
What is chromosome painting
FISH probes span the entire length of choromsome
In chromosome painting what is labeled
the entire chromsome
what is chromosmal painting good for detecting
specific, known chromosomal rearrangements
What does SKY stand for
spectral karyotyping
What is SKY
all chromsomal are painted different colors
what is the function of SKY
useful for identifying unknown chromosomal rearrangements
What does VSD stand for
ventricular septal defect
22q11 How would you use FISH
two different probes
a control probe
test probe specific for the region we are looking for
What does metaphase FISH test for
If part of chromosome is there or not
What is the other name of 22q11 Deletion syndrome
DiGeorge Syndrome
What is the mode of inheritance for DiGeorge?
AD
CATCH22 is mneominic for symptoms for DiGeorge, liste them
cardiac defects abnormal facies thymic hypoplasia cleft palate hypocalemia frmo parathyroid aplasia chromosome 22
What test can you use to confirm Down Syndrome?
FISH - can use interphase FISh
What probes would you use to test for Down Syndrome using FISH
one labeling chromosome 18
One labeling chromosome 21
What does CGH stand for
comparative genome hybridization
What can CGH be used for
looking for changes in copy number of specific sites
can scan entire genome
What are the limitations of CGH
doesn’t detect balanced rearrangeement, can only detect copy number. Can only detect deletions and insertions
Describe process of doing array-CGH
label test and control two different colors and hybridize them against microarray. if they both bind to a spot and one is red and one is green then it will be yellow
In array-CGH the ratio of red and green determine what
if pts DNA is over or udner represented
If there is a lot of green (green is control) what does that mean array-CGH
control DNA is present, test DNA is not, so there is deletion
If there is red (red is test) in array-CGH what does that mean?
duplication
If there is yellow in array-CGH what does that mean
no difference, same amount of test and control DNA
If spots go lower on CGH output what does it mean
deletion
If spots go higher on CGH output what does that mean
duplication
How do you find out where chromosomal abnormality is if you do not know
CGH
Spots going up on chromosome 12 means what
duplication at the location that the dots go up
What is DNA sequencing
the final solution for genetic testing.
can look at highest level and see where mutation is
What are the limitations to DNA sequencing
can only look at a specific area at a time
expensive
When would you use DNA sequencing
AD conditions
ex: marfan syndrome, osteogensis imperfecta
AR where the mutation is not common, where it’s highly variable b/w families
What is the basis of dideoxy sequencing
uses dideoxynucleotide - it stops where they are
what is the difference in dideoxynucleotide
doesnt have 2 or 3’ OH
in electrophoretic separation and you read gel from bottom to top, what diretion in sequence are you reading
5’ to 3’
The sequence of the template strand is what in comparison to the gel
complementary
When you read a gel what sequence are you reading
NOT the template strand, have to take complement of the gel you’re reading
What is dye-termination sequencing
the theory is the same as dideoxynucleotide, but don’t have to use four different reqctions, combined in a single reaction
How do you distinguish b/w different fragments in dye-termination sequencing?
can tell termination by color
What do you run DNA through in dye-termination sequencing
run through capillary electrophoresis coupled to a fluorescence detector
What do you run DNA through in dideoxy sequencing
acrilymide gel
What does a peak mean in dye-termination sequencing
peaks of specific colors correspond to terminations at specific bases
How do you read peak graph from dye-termination sequencing
read left to right 5’ to 3’
