L11 Molecular Diagnosis II Flashcards
What is a very good use of PCR (what type of disorders)?
detection of triplet repeat expansion disorders
- huntingtons disease - AD
- myotonic dystrophy - AD
- fragile X syndrome - X linked inheritance
in get electrophoresis, which fragments move faster? Which fragments move slower?
faster = small
slower = large
what is used to diagnose DMD in affected boys?
multiplex PCR
after the PCR reaction, the product is outside of the linear range which makes it difficult to discern if the pt is…
heterozygous for the mutation or homozygous for the normal allele
carrier are difficult to ID
what does MLPA stand for
multiplex Ligation Dependent Probe Amplification
What are the 2 key points of MLPA?
- only ligated probes will be amplified - probe sets designed so each pair creates a unique product
- less cycles of PCR amplification are allowed - allows ROBUST quantitative analysis of PCR product
*allows hundreds of sites to be tested and identified in a single reaction
what can MPLA detect?
insertions, deletions and single bp (ASO) changes
____ is used to amplify all ligated probe pairs (X,Y) by DNA polymerase = limited number of cycles
universal primer
the _____ of each probe has a unique length and can be separated in a column
amplification product
the primer has a _____, so it may be detected after separation
flourescent label
what was karyotype analysis used for
many major chromosomal abnormalities ID like aneuploidy, large translocations, insertions , deletions
what is karyotype analysis unable to do
unable to resolve chromosomal changes = microdeleltions
because G banding has low resolution
what is the most common type of standard karyotype analysis?
G banding
in G band, what are the dark regions of chromosomes?
A-T regions
in G band, what are the light regions of chromosomes?
G-C regions
what does G banding require?
highly experienced cytologists that are able to differentiate between chromosomes and recognize subtle changes from the normal banding pattern
what does FISH stand for
fluorescent in situ hybridization
what are the two general types of FISH analysis
- chromosome-specific unique sequence probes or gene specific probes
- SKY (spectral karyotyping) or M FISH (multiplex FISH)
what is DiGeorge syndrome better detected with
FISH instead of G banding
what are the advantage of FISH when compared to G banding
allows better resolution
what is the major disadvantage of FISH compared to G banding
the geneticist must know what they are looking for
what does SKY FISH allow
automated cytogenetics - reduces the training needed for an individual to practice and interpret cytogenetics
what does CGH mean
comparative genomic hybridization
what is array CGH
molecular cytogenic technique that allows detection of the DNA sequence copy number changes throughout the genome in a single hybridization
