Molecular Diagnostics 2 Flashcards
What does ASO stand for?
Allele-Specific Oligonucleotide Hybridization
What is the point of ASO hybridization?
to detect small, sequence variants. Usually single base, but can be very small like a three base mutation. Normally point mutations
When doing a ASO hybridization you have to know what about mutation
where it is
ASO hybridization can be useful for screening for ____ mutations
frequent
for ASO you need how many probes are needed?
one for wild type
one for mutation
What do you do first to amplify the DNA for ASO
PCR amplify the product
Is there separation on basis of size for ASO?
No
What are you looking at in ASO hybridization, if not size
if there is a mutation there or not
What is a dot blot?
How you see if mutation is there in ASO hybridization
dark blot on normal probe tells you want
pt has the normal alelle
Why are short probes used for ASO hybridization
for specificity
Sickle cell disease is caused by what kind of muatation
single point mutation, A to T
E6V means what
Glutamate to valine
What is the specific mutation for sickle cell disease
E6V
How many probs d you need to ASO to detect sickle cell disease
2 - one for normal one for mutatatn
The probes for ASO are very small and also have what characteristic
Very specific - so the mutant probe will only bind to mutant and normal will only bind to normal
What does PCR stand for
polymerase chain reaction
What does PCR do at its basic level
exponentially amplify DNA
PCR is a _____ reaction that doubles the amount of DNA
cyclical
PCR uses what kind of polymerase
DNA polymerase
What are 2 of the negative sides of PCR?
need at least some sequence info to make primers
susceptible to contaminiation due to high sensitivity
What polymerase is most often used for PCR?
taq polymerase
PCR does DNA synthesis by a thermostable:
DNA polymerase
What is one downside of taq?
no proofreading
besides taq what DNA polymerase is often used that has proofreading
Pfu
Which is more accurate, Pfu or Taq?
Pfu
How much DNA is made each cyce of PCR
doubling of DNA
What are the steps of PCR for one cycle
- Heat to denature → single strand
- Cool - Primers anneal to 3’ end of one strand & to 5’ end of another strand
- Heat to 72 C and allow polymerization → double stranded DNA
Which molecules are expoentially amplified?
only the target sequence, the products with the extra “grey” bits at end do not get exponentially amplified
What is the temperature to denature in PCR
95 C
What is the temp to anneal primers in PCR
55-60 C
What are the components of PCR
template DNA 2 primers all 4 dNTPs Buffer, Mg 2+ thermostable DNA polymerase (Taq)
Any reaction that uses ATP you need what ion
magnesium
What is the temp to allow polymeriztion
72 C
What is a thermocycler
machine that does the PCR heating and cooling process, computer controlled
What is the normal thermocycing sequence
#1: 95 C #2: 55 C #3: 72 C
Is PCR versatile
yes
Can PCR be used for RFLP
yes
How is PCR used in sequencing
used first to amplify DNA before sequencing
What is the upper limit of fragment amplification of PCR
1kb (pretty small)
can you analyze large DNA regions with PCR
No, limited to 1kb max
How many primers are needed in PCR
2
Each primer primes what in PCR
synthesis of a different strand, across region of interest
synthesis is always what direction in PCR
synthesis is always 5’ to 3’
what direction are templates read in PCR
3’ to 5’
What are the names of the two primers in PCR that ROdda uses
foward primer
reverse primer
how long does primer need to be
about 20 nucleotides long
how do we know what primer is going to be for pcr?
have to know the sequence
the forward primer is what
identical to 5’ end of region to be amplified of the given strand
reverse primer is what
complementary to the 3’ region of interested of given strand
Draw out how primers look on PCR, where they are on 3’ and 5’
pg 40 in ppt
What are 5 major applications of PCR?
DNA sequencing genotyping diagnostics forensics quantifying gene expression
How can you use PCR for genotyping
identifying mutations or polymorphisms in indiviuals or families
how can you use PCR for diagnostics
detection of viral infections
how can you use PCR for forensics
amplificatino of DNA for DNA fingerprinting
how can you use PCR to quantify gene expression
measure mRNA levels (need reverse transcriptase)
Preparation fo genomic and cDNA clones can be done via what
PCR
What is RT-PCR
reverse transcriptase PCR
What is RT-PCR used for?
RNA virus (like HIV) amplify mRNA of expressed genes (amplifies a cDNA)
what is qPCR
Real Time/Quantitative PCR
What is the function of qPCR?
it measures how much DNA is being produced in real time, it has lasers that can detect how much DNA is there after each cycle (DNA is fluorescently labeled)
What can be used to measure microsatellites
PCR
What are microsatellites
dynamic mutations
How does PCR measure microsatellites
primers go outside of area of amplification, how big the product is that is amplified shows how big the repeat is
Symptoms: painful swelling of fingers, splenomegaly, low hemoglobin, high reticulocytes, hemolytic anemia, blood film showing sickled erythrocytes is what disease
sickle cell disease
low hemoglobin and high reticulocytes are consistent with what
hemolytic anemia
what is hemolytic anemia caused by
early death of RBC
what ethniciites are most affected by sickle cell?
african american, hispanic (but more rare)
What mutation causes sickle cell disease?
E6V
What specific mutation causes sickle cell disease
A→T in the HBB encoding beta globin
missense
What codon is mutated in sickle cell disease
codon 6
what is edema
swelling in hands and feet
HbS is what
abnormal hemoglobin
what is HbA
normal adult hemolobin
What is the heterozygote advantage for sickle cell
heterozygotes with sickle cell survives better from malaria, so there are more mutant alleles where there are high incidents of malaria (like Africa and mediterranian)
In E6V mutation, what is the difference in amino acids after mutation?
acidic to hydrophobic
what is the trait of valine
hydrophobic
what is the trait of glutamic acid (glutamate)
acidic
what is mode of inheritance for sickle cell
AR
What is sickle cell trait
heterozygote carriers can have symptoms
What is pleiotropy
a genetic phenomenon where a single gene mutation leads to a variety of different systems or phenotypes throughout the body in seemingly unrelated systems
how does sickle cell show pleiotropy
single base mutated → abnormal hemoglobin S → sickling
sickle cells destroyed in spleen → anemia → abnormal skull shape, weakness, splenomegaly
infarctions in blood vessels, clumping → ischaemia, thrombiss, infarction → gut, spleen, extereities, brain, kidney, lung, heart
what is the class genetic example of pleiotropy?
marfan syndrom
PCR can be used to do what analysis for sickle cell
RFLP
The A→T mutation in sickle cell falls within what
restriction site -
how can you do RFLP analysis for sickle cell
the mutation falls within restriction site
If there is mutation what will Dde1 do?
It will not cut the restriction site
What are the advantages of PCR over southern blotting
faster
less genomic DNA required
no labelled probe required
less expensive
If there is no mutation what will Dde1 do?
will cut at AT location (where the mutation would be if there was one)
What disease is HFE gene mutated in?
hereditary hemocrhomatosis
Most cases of Turner syndrome have what mode of inheritance
mosaicsism
What is genotype of turner
45X
If a pt has turner syndrome and is mosiaic and gonads have 46XY what are they are risk for
cancer
how do you see if a turner pt has 46XY?
check for Y specific sequences via PCR
What effect does the mutation have on the restriction sites in the HFE locus?
The mutation introduces a restriction site. (pg 56)
