Molecular Diagnostics 4 Flashcards
Can you sequence genomic DNA directly?
no
what do you do first to sequece genomic DNA
isolate genomic DNA
PCR amplify
What do you use at template for sequencing DNA
PCR amplified DNA
What do you sequence for DNA
both alleles simultaneously
what is the issue with sequencing both alleles simultaneously for DNA sequencing
if there is mutation on one allele, whe you run a gell there will be two bands at the position there is a mutation
What is the mode of inheritance for beta thalassemia
AR
what regions does beta thalassemai have high incidence rate
mediterranean and africa (same as sickle cell)
Pt has hypochromia, osteoperosis abrnomalities, abnormalities of skull and bones, iron overload in liver, what is the disease
beta thalassemia
why is there iron overload in beta thalassemia
secondary to blood transfusions
what kind of mutation causes beta thalassaemia
null mutations
What kind of protein is produced with null mutation
no protein
If premmature stop codon is instroduced what will hapepn to protein
No protein
What is next generation sequencing
high-throughput, massively parallel. just a new technology that sequences very quickly, there are multiple kinds
What are the advanatges of next gen sequecing
very fast
less expensive
useful for whole genome resequencing
what are the disadvantages of next gen sequencing
not useful for de novo sequencing
high error rate
not targeted so can’t sequence a single gene
What techniques can be used to identify known mutations
RFLP
ASO
PCR
What techniques can be used to find changes in copy number (deletions, duplications)
FISH
CGH
MLPA
What techniques can be used to identify novel mutations
sequencing
If you don’t know anything about gene, where it is, where mutation is
allele-tracking
What is allele tracking
follow family pedigree and can follow how disease goes through pedigree
What are VNTRs
variable number of tandem repeats. also called short tandem repeats
what type of polymorphic marker is most used in allele tracking
CA-rpt marker, a type of VNTR
The higher the degree of polymophism, the what to eh population (about the marker)
the better the marker
How do you determine which of the 2 chromosomes is maternal or paternal in origin, etc. when doing allele tracking
use polymorphic markers
why do we use polymorphic markers in allele tracking
polymorphic & known location in genome
paternal relationships b/w a man and infant can be best determined by the technique commonly referred to as dna fingerprinting. which of the following sequencing is most conveniently analyzed in a DNA fingerprint
microsatellite tandem repeats (STRs) AKA VNTR
what is another name for VNTR
microsatellite tandem repeats
how do we analyze the VNTR
can measure the number of repeats by PCR amplifying across the repeat region & measuring size of product
what is the name of short tandem repeats
VNTR
What can you use to analyze VNTR
capillary or gel
Why do you want more highly polymorphic markers
the more allelles that are found in the population (the more polymorphic) the more likely they will be different
explain the M&M explanation
you want to find two different alleles, so the more alleles you have available the more likely it will happen you’ll pick a different one
is the genotype itself diagnositc
no
mutations are what regarding family
family specific
what are the 4 steps for gene tracking
- determine mode of inheritance
- determine which family member must be informative and whether or not they are
- establish the phase
- type the consultand
you are considered informative if you have what
two different alleles: heterozygous
what does it mean to establish the phase
determine which marker allele is on the same chromosome as disease mutation
who is the consultand
the child, the one you are trying to figure out if they are carrier or affected by disease
