Molecular Diagnostics Flashcards
All DNA technologies rely on what?
DNA & hybridization
If you heat DNA molecule, what will happen to DNA strand?
Denature
what happens to a denatured DNA molecule if you cool it down?
Renature
What is nucleic acid hybridization?
Heat and denature, cool and renature, can combine two different DNA strands
Name a few different types of lables
radioactive, fluorescent, enzymatic, etc.
What is the function of the probe?
Detect target DNA with sequence we are interested in
One strand of probe will bind to what in hybridization assay?
one strand of target DNA (they are complementary to each other)
What is non-productive in Hybriization?
two probes reanneal with each other and two DNA strands reanneal
What do you want to maximize in hybridization?
probe-target binding
What is hybridization Stringency
how “strict” you are when you allow hybridization to occur. the degree we tolerate or don’t tolerate mismatches
the higher the stringency, the:
fewer the mismatches we tolerate
when would you want to use low stringency?
finding genes part of gene family, that are similar but no identical
Usually in diagnostics, what kind of stringency is used
high
how do you control stringency?
temperature, salt concentration, denaturing agents
The higher the temperature
the higher the stringency
the lower the salt concetnration
the higher the stringency
the higher the concentration of denaturing agents
the higher the stringency
Why is DNA preferred as probe over RNA
more stable than RNA
main application of northern blotting
gene expression
why is southern blotting used more than western for MDs?
for western have to get protein sample - ex: if they have liver problem difficult and invasive to get liver sample
Detect DNA bands of a ____ _____ and determine their size
specfici sequence
Detect DnA bands of a speficif sequence and determine their ___
size
What does semi-quantitative mean?
the intensity of the band refelcts the amount of the target DNA prsent
Southern blotting can tell us what about the DNA (generally?)
size & how much DNA
DNA samples from different tissues, skin & liver and ran on gel, would southern blot show differences b/w the tissues?
No, genomic DNA doesn’t change from tissue to tissue
Would you use Southern blotting to look at differences b/w tissue?
No, genomic DNA doesn’t change b/w tissues
What are southern blots used for?
discriminate b/w alleles - get genotype of pt
How is DNA separated on for southern blot?
size
RFLP stands for what
restriction fragment length polymorphism
What is an RFLP
polymorphism in a restriction site in the genomic DNA
what is a polymorphism
sequence variant b/w individuals (at least 1% of pop. must have variant for it to be polymorphism)
What does SNP stand for
single nucleotide polymorphism
What is a SNP
single base change b/w people
vast majority of polymorphisms are what?
SNPs
Most RFLPs are a SNP within what?
a palindrome
Southern blotting has largely been replaced by what
PCR
When you get a map, pay attention to where the probe
hybridizes to
When do you do southern blot on alleles you will always see
two - we all have two alleles
Before you use a RFLP with a mutatnt you have to know what?
where the mutation is
What is pedigree specific when you are looking at RFLP?
where the mutation is. it will be different b/w different families and pedigrees
Just because husband and wife has the same allele does not mean
they both have the disease
When doing a RFLP problem pay special attention to where what is and why?
probe - that will be the only part that will appear on the blot. so even if restriction enzymes cut multiple sites, the only sites that will be shown are where the probe binds
For males how many alleles would they have for x linked diseases?
1 - males only have one x chrom.
a deletion or insertion b/w two restriction sites can cause an
RFLP
a chromosomal rearrangement can cause a
RFLP
Name all the ways you can get RFLP
a chromosomal rearrangement can cause a ,
a deletion or insertion b/w two restriction sites can cause an
creation or loss of restriction site due to SNP or mutation
What is cephalohematoma
lump under the skin on scalp
What are the two main types of hemophilia?
A & B
What is the most common type of hemophilia?
A
Where is the mutation in Hemophilia A?
Factor VIII gene (F8 gene)
Where is mutation in hemophilia B?
Factor IX (F9 gene)
What is mode of inheritance for hemophilia?
X-linked
what is the ultimate cause of hemophilia? what causes lack of blood clotting
fibrin isn’t produced
Symptoms: prolonged bleeding times, easy brusing, hemorrahge into joins and muscle is what disease?
hemophilia
What does hemophilia mild peresnt as?
Not as bad, not as much bleeding
What does hemophilia severe present as?
bleeding all the time, need blood transfusions
In the milder form of hemophilia, why is it more mild?
residual protein function
In the severe version of hemophilia, why is it more severe?
little or no protein function
What kind of mutation would lead to severe hemophelia?
nonesense, frameshift, complete deletion
What kind of mutation would lead to milder form of hemophilia?
missense (single aa changes)
What is the treatment for hemophilia A?
transfusion of purified/recombinant F8
40% of severe hemophilia A cases are cuased by what?
inversion
How do inversions occur usually in hemophilia?
homologous recominbation b/w repetitve lements proximal to the F8 gene
Why can you not use a PCR to detect the inversions that cause hemophilia?
All the exons are still present
Why does southern blot give different fragement sizes for hemophilia?
inversion changes distances b/w restriction sites, so sizes in fragemtns are different
If blots are fainter on southern blot, what does that say?
less DNA
complete deletion mutation - they only have one copy of gene compared to other individuals
Less target DNA results in a:
weaker signal (bands will be lighter)
The lane at the bottom of southern blot is used as a
control
ensures they loaded equal amount for each pt, shows that if there are differences on DNA they are real differences
We use RFLPs to track muation ins:
family
What is used to track muations in a family?
RFLPs
What is the principle idea for RFLP analysis?
linkage - the part of mutation will be inherited together
The probe will hybridize to ___ alleles
BOTH
with and without mutation
The RFLP is present in _____ at large
population
RFLPs are always what?
family specific
