2-2 Molecular Genetic techniques Flashcards
what are the mechanisms of disease?
- loss of function
- in Autosomal recessive
- PKU
- Gain of function
- in AD
- Haploinsufficiency
- normal protein but in LOW ammount (not enough to prevent disease)
- AD
- dominant negative
- a bad protein interferes with function despite a normal protein present
what is PCR?
technique to amplify DNA
1) DENATURE heat up DNA ( so strands separate) 94 degrees
2) ANNEAL primers - cool down (50 degrees)
3) extend primers (72 degrees celcius
each PCR doubles DNA.
when was PCR invented and who invented it?
In 1983 by Dr.Kary Mullis and Dr.Suzane Hart
what is the most common form of short-limbed dwarfism?
achondroplasia
remember dwarf but short extremities
what is the inheritance for achondroplasia?
what % of the cases have NEW mutations?
autosomal dominant
80% have new mutations
which is the gene for achondroplasia?
what is the inheritance?
are mutations new or passed along?
what AA change occurs in the mutation?
FGFR3 fibroblast growth factor receptor 3 (FGFR3)
autosomal dominant
80% have new mutations
the mutation is a p.Gly380Arg glycine for Arginine
are the achondroplasia mutations coming from mom or dad?
always from the father
which is the most mutable nucleotide ever?
the one in achondroplasia mutation.
what is a restriction enzime?
AKA?
- AKA endonucleases
- its an enzyme that cuts DNA at restriction
- restriction sites arespecific recognition sites
what is transversion and what is transition ?
- transition: point mutation that changes a
- purine for another purine (A-G) or
- pyrimidine to another pyrimidine (C-T)
- Transversion: point mutation that changes
- purine for pyrimidine
what do we use the restriction enzimes for?
- to cut DNA
- to digest DNA
- there are specific restriction enzymes depending on the mutation that you want to check for
what was the diagnostic technique before PCR?
whyit didn’t work?
was it similar?
Southern blot
took a lot of DNA
took a lot of time
what is ethidium bromide?
substance that intercalates between DNA strands
does everyone with sickle cell have the same mutation?
what kind of mutation is it?
what nucleotide is changed?
- yes everyone has the
- Glu-Val
- missense (changing AA)
what is complementary?
its a high poutput test
spots the mutatinos
has specific probes
what is ASO? what is it used for?
ASO
allele
specific oligonucleotides
used in Complementary technique diagnosis
which is the most common autosomal recessive disease in caucasians?
what is the distribution in othe rpopulations?
Cystic Fibrosis
1 in 25 caucasians
1 in 45 hispanics
1 in 62 AA
1 in 150 asians
- where is the mutation for Cystic fibrosis?
- is it a large gene?
- what does it encode for?
- how many mutations on this gene cause CF?
- 7q31
- CF gene has 27 exons (pretty large)
- encondes for transmembrane glycoproteins
- over 1500 mutations described!!!! (think that RP has ~200 mutaitons described)
interpret the following mutations:
- deltaF508
- p.W1282*
- 3120+1G
- deltaF508 is a NON-frameshift mutation (delta deleted 3 nucleotides)
- p.W1282 is a NONSENSE mutation (the * means stop codon created)
- 3120+1G IS SPLICING MUTATION
are de novo mutations more common in AD or AR?
they are more common in AD for rare in AR diseases
what is OLA?
oligonucleotide Ligation Assay
using OLA probes to diagnose genetic diseases by electorphoresis
what are the indications to do DNA testing in CF?
- phenotype-genotype correlation
- for example, there are some phenotypes with pancreatic cancer
- help with diagnosis when chloride sweat test is not conclussive
- allow prenatal diagnosis
- male infertility testing
if you see a boy in a wheel chair with hypertrophic leg muscles specially the calfs waht dg should you consider?
duchene’s muscular dystrophy
what duchene muscular dystrophy?
what is the inheritance
what % are de novo mutations?
which is the gene?
is it large or small gene?
- severe muscular dystrophy
- muscle waekness begins age of 4
- progressive
- first in the pelvis and upper legs
- later in arms
- unable to walk by age 12
- muscles are replaced ny FAT so muscles look boggy (que musculazos!)
- scoliosis is common
- x-linked disorder
- 1/3 are de novo mutation
- Gene is the Dystrophin gene
- HUGE hene 79 exons!
- large deletions
- 15%
what is another type of muscular dystrophy besides Duchene?
what are the similarities and differences?
Becker muscular dystrophy
- Becker and duchene
- affect skeletal muscles and cardiac muscle (both have cardiopathy)
- almost only in MALES
- SAME GENE (but different mutations)
- dystrophin gene (called DMD)
- Differences:
- duchene appears EARLIER and progresses FASTER
- appear in early childhood
- by teens ar wheelchair dependent
- Becker appears in teens and goes very slow
- Becker and Duchene have HUGE DELETIONS
- Becker’s deletions are “in frame” so you have abnormal but STILL FUNCTIONAL PROTEIN
- Duchene has out-of-frame deletions
- duchene appears EARLIER and progresses FASTER
- Affects 1 in 5K
- X-linked recessive
what is the genetic difference between duchene and becker muscular dystrophy if both are due to mutations in the same gene?
- Becker and duchene
- affect skeletal muscles and cardiac muscle (both have cardiopathy)
- almost only in MALES
- SAME GENE (but different mutations)
- dystrophin gene (called DMD)
- Differences:Duchene appears EARLIER and progresses FASTER
- appear in early childhood
- by teens ar wheelchair dependent
- Becker appears in teens and goes very slow
- Becker and Duchene have HUGE DELETIONS
- Becker’s deletions are “in frame” so you have abnormal but STILL FUNCTIONAL PROTEIN
- Duchene has out-of-frame deletions
A couple has a boy with duchenes dystrophy, they ask you what are the chances that the next offspring will have the disease. what do you tell them?
duchenes MD is an x-linked recessive disease
- it will appear only in males
- there is 50% chances to pass it on the next child
- but to be a male there is another 50%
- so the chance to have an affected boy is 1 in 6
- BUT remember this could have been a de-novo mutation
- 1/3 of x-linked disease are caused by de-novo mutations
what is LMPA?
multiplex ligation-dependent probe amplification (MLPA)
(separates amplification products
- compares with same sex control
- its the best for deletion/duplication analysis
what is myotonic dystrophy and
what kind of mutation happens on it?
- its a multisystem disorder that affects
- skeletal muscle
- smooth muscle
- heart
- endocrine system
- OCULAR
- ptosis
- ophthalmoplegia
- decreased vision
- CHRISTMAS TREE cataract
- maculopathy and pigmentary retinopathy
- mutation is a REPEAT mutation
- trinucleotide REPEAT
- autosomal dominant
what are the ocular manifestations of Myotonic dystrophy
OCULAR
- ptosis
- ophthalmoplegia
- decreased vision
- CHRISTMAS TREE cataract
- maculopathy and pigmentary retinopathy
what genetic testing can you do for myotonic dystrophy?
remember this is a nucleotide REPEAT mutation
what gene?
what chromosome?
what is the repeat?
how many repeats you see?
- gene is DMPK
- chromosome 19
- CTG repeat in 3’ untranslated region
- affected people have more than 50 repeats!!
what is anticipation?
what kind of mutation causes anticipation?
what does that mean?
- anticipation is when the disease appear at EARLIER age OR with MORE severity in the next generations
- when you see this is likely due to REPEAT expansion siorder or DYNAMIC MUTATION
- you can dg with Souther blot or if you want to go faster…. you can use:
- TRIPLET PRIMED PCR
what is the process to sequence DNA?
we need:
- DNA primer
- DNA template
- DNA Polumerase enzyme
- nucleotides
- put all in a pie and the in to the sequenzer
- laser detects created electrophorectogram
what is sanguer sequencing?
- its a method to sequence DNA
- uses selective incorportaion of chain-terminating nucleotides
- by DNA polymerase
- its a gold standard test but only for
- small deletions or duplications
- does not detect mosaicism
what technology is used for the smart panels?
next generation sequencing
what is whole exome sequencing?
what % of the genome is captured with this?
what % of exons?
- genetic test
- to sequence all protein coding genes (exomes)
- 1st selects all exons
- exons are 1-2 % n (30CM)
- then sequences them
- using high-throughput technology
- 1st selects all exons
- This approach is cheaper than checking all the genome
- WES is good for MENDELIAN DISEASES
what is a DNA microarray?
- its microscopic DNA attached to solid surface that has small containers
- each container has 10-12 picomoles of DNA sequence (known as probes)
- used to measure gene expression
what is picomole?
pico means one TRILLION
what is SNP array?
is a type of DNA microarray used to detect polymorphisms within a population.
SNP = single nucleotide polymorphism
There are 85 million SNPs identified
when to use SNP array vs WES?
- WES is good for
- rare mendelian diseases
- because it checks all genetic variants in one person
- SNP array
- good for common diseases
- detects genetic variants common in a population
what % of genome is EXONS?
just 1-2%
what % of exons are targeted in WES?
80%
what % of WES is successful in identifying the mutation?
about 30%
what % of WES are successful ?
what are the reasons of WES failure?
30% successful
- reasons for failure
- genes has not been identified
- exon has not been identified
- problem is in non-coding regions
*
what is super important to do before doing WES and why?
- DO genetic counseling because
- you may find surprises and you need to know if they want to know:
- carrier of AR condition
- mutations related with LATE-ONSET diseases
- susceptibility alleles
- non-paternity
- VOUS
- false positives and negatives
1) can you test a NORMAL minor for a late-onset disorder?
for ex, can you test a normal girl for the BRCA gene if there is strong family hx of breast cancer?
2) what if you do WES and you find a disease causing gene such as BRCA?
- NOOOOO
- only at age 18 they can decide
- so children only tested if they are diseases happening at the moment
- if something is found on WES you are supposed to tell them
what is the charcot-marie tooth disease?
what is the tooth problem?
- its a progressive distal weakness and sensory loss
- caused by degeneration of peripheral nerve
- there is no Tooth problem but it was described by Dr.Tooth
when and why are panels convenient?
for diseases that have multiple genes and multiple phenotypes
for example RP or the maculopathies
a patient has AR condition
parents deny consanguinity
gene sequence shows he is homozygoud, how to explain?
due to ISODIZOMY (one parent is carrier and gave two copies)
could be de-novo but that is unlikely in AR
when to use SNPs?
- in mendelian disorders with variable expresitivity such as ABCA4
- in linkage analysis
- determine location of gene associated with phenotype
are de-novo mutations more common in AR or AD?
in AD
AD-de-novo.
