Why wouldn't an alteration be detected via sequence analysis? (3)
- alteration in region not tested for - alteration due to large deletion - pt does not have mutation
Mutation scanning involves physically testing _____ to confirm presence of mutation before sequencing used to determine exact mutation
exons, reduces amount of DNA that needs to be sequenced
Targeting mutation analysis is used to test for three things. What are they
- presence of specific mutation - specific type of mutation (nucleotide expansion in HD) - set of mutations (panels for CF-microarray)
If targeting mutation analysis is negative, what does this mean?
Pt may still have mutation, should do sequence analysis or mutation scanning
CF exhibits allelic heterogeneity. What percentage of patients have mutation in delta F508?
70%, 28% have 96 common mutations 2% have rare mutations
Quantitative PCR must be used to detect what three things?
1) hetero deletion mutations 2) carriers of auto recessive disorder 3) carriers of X-linked disorder
3-5% of individuals with autism have other chromosomal abnormalities including ___________________, inversions, deletions and duplications
balanced and unbalanced translocations
Which of these would not be used for direct DNA testing? a) Protein electrophoresis b) Southern Blotting c) PCR d) Northern Blotting
a + d
Which of these would not be indicated for use in detecting hemaglobinopathies? a) Northern Blotting b) Southern Blotting c) Protein electrophoresis d) Biochemical assay
c - detects protein changes in charge or size
Which of these methods is not employed in protein detection (changes in protein and structure)? a) Western Blot b) ELISA c) Protein electrophoresis d) Immunohistochemistry
Which of these would not be used in assessing changes in chromosomal number and structure? a) karyotyping b) FISH c) biochemical assays
What would you use to detect promoter defects?
RNA and protein assays
What has northern analysis larger been replaced with?
RT-PCR and chip microarrays
Southern blots uses _________ digestion
True or false - Both Northern and Southern blots require specific labeled probes and can only distinguish LARGE differences
Alleles of VNTR polymorphisms are different sizes. How can these be detected?
What is the most common disease causing mutation in DMD?
2/3 of DMD patients have a deletion of one or more exons (X-chromosome, XLR)
How is disease detected in affected males?
Looking at mutant dystrophin protein via immunofluorescence
Can PCR be used to detect carrier females of DMD? What can be used?
No, can use FISH or Quant PCR. Point mutations can be detected via sequence analysis.
How is allele specific oligonucleotide (ASO) testing used to screen for sickle cell mutation?
ASO labeled (single BP difference causes SCA - Glu6Val) then normal /SCA DNA visualized If + for both normal and SCA gene then carrier
What is the single BP difference that causes sickle cell anemia? What percentage of AAs are carriers? Africans?
Glu6Val in B-globin 10%, 30%
Describe the amino acid changes in SCA and Sickle-C disease
Normal amino acid = Glutamine = negatively charge (travels furthest down gel) SCA = Valine = no charge Sickle-C = Lysine = + charge (travels least)
Thalessemias are caused by globin chain imbalances. Differentiate between the two types
ALpha - no alpha globin Beta- no beta globin
In alpha thalessemia trait, __ (number) copies are lost, and there is a reduced _____ but person is asymptomatic
2, RBC size
In Hemoglobin H disease there is moderate to marked _____ and loss of ___ copies
In alpha thalessemia there is a loss of ___ copies and _______ occurs
all 4, death/hydrops fetalis
In beta thalessemia minor, the heterozygote is asmpytomatic and has ___ mutant allele
In beta thalessemia trait, there are ____ mutant alleles and transfusion is/is not required
In beta thalessemia major, there are ___ mutant alleles and transfusion is/is not required
Hemoglobin lepore occurs when allele of B thalessemia is caused by _________ and is functionally active but expressed at low levels due to fetal promoter