Molecular Pathology Flashcards
What is a loss of function mutation?
A single base or multiple bases that are either mutated or deleted.
Where are loss of function mutations found?
Frequently within the coding region but also within splice sites or gene promoter regions.
Does a mutation always have an effect?
No. Mutations may sometimes represent a polymorphism and have no effect on the function of the encoded protein.
What does a mutation in one of two alleles lead to?
Haplotype Insufficiency and a Dominant Negative Effect
What are DNA changes associated with?
Loss of function mutations, usually very heterogeneous. Recessive Diseases
What are gain of function mutations usually associated with?
Dominant Phenotypes and an increase in number of tri-nucleotide repeats
What are the key attributes of micro satellite repeats?
Highly Polymorphic
Where are micro satellite repeats usually found?
Throughout the genome, fairly evenly spaced along each chromosomal arm
What form do micro satellite repeats usually take?
(CA)n or (CGG)n
What is gene tracking?
A method for determining the inheritance of a particular gene in a family?
How is gene tracking carried out?
RFLP’s situated in or near the locus of interest and are identified using gene probes.
When would a marker not be clinically useful?
When everyone has the same heterogeneous genotype
What are the features of the Duchenne Muscular Dystrophy gene?
79 Exons
2400kb Genomic DNA
X-linked Recessive
Passed on to males but females can only be carriers
Why are micro sattelite markers special in Duchenne Muscular Dystrophy?
They lie within the gene itself
What are the major mechanisms for loss of function in Duchenne Muscular Dystrophy?
Deletion of one or more exons
What is linkage disequilibrium?
Linkage disequilibrium is the non-random association of alleles at two or more loci, that descend from single, ancestral chromosomes. Linkage disequilibrium is wholly a measurement of proximal genomic space
What is phase?
The allele inherited from a grandparent
What is essential for gene tracking for LOAD disease?
Must have a tightly linked marker
What are the steps for gene tracking of LOAD disease?
- Tell the parents of the consultand apart to identify who has the diseased gene
- Identify phase to identify which allele the diseased gene is on
- Genotype the affected parent and their parent who has been identified as diseased
- Identify which alleles the consultand has recieved and make prediciton
What samples can be taken for direct testing?
Blood DNA
Mouthwash and Buccal scrapes
Chorionic villus biopsy (fetal DNA)
What is direct dideoxy sequencing?
The gold standard for genetic testing which can tell which base is mutated and whether it is a deletion or insertion
What are the problems associated with direct dideoxy sequencing approach?
Genes are frequently large and mutations often occur in introns (splice donor sites)
What are the problems associated with loss of function mutations and direct dideoxy sequencing?
Spectrum of mutations are often extremely heterogeneous and complexity may be reduced by sequencing the RNA transcript
What are the advantages of direct dideoxy sequencing?
It looks at the actual sequence that codes for the protein and does not contain introns that have been spliced out.
How is RNA converted to cDNA?
Reverse Transcription PCR
Give some examples of diseases that have the same mutation or one of a limited number of mutations
SCA, Hutingdon’s, CF and alpha- and beta- thalassaemia
What is Restriction digest of PCR amplified DNA; check size on gel?
Only when the mutation creates or destroys a natural restriction site
What is Hybridize PCR-amplified DNA to allele specific oligonucleotides (ASO)?
General method for specific point mutations; allows use of large arrays
What is PCR using allele specific primers (ARMS test)?
General method for point mutations;primer design critical. Can be adopted to chip technology
What are Oligonucleotide Ligation Assays?
Uses the enzyme DNA ligase
Relies on the two primers forming perfect hybrids on the target
Point Mutations
What is PCR with primers located on either side of a translocation breakpoint?
Successful amplification shows presence of the specific deletion or specific rearrangement
Point Mutations
What is Check size of expanded repeat?
Dealt with when discussing Huntington disease
May also be used for Fragile X syndrome
What is Dideoxy Fingerprinting?
Similar to conventional sequencing but run just two lanes-for example C and T
Less expensive than conventional sequencing, it has high sensitivity but can be complicated to interpret
What are Gene Chips?
Quick, high throughput can define all changes.
New technology, that is expensive and used only for a limited number of genes
“CHIP” is arrayed with 20mer sequences that correspond to wild type and all known mutations within a particular gene
Simply PCR sample and allow to hybridise (PCR product is labelled with a fluorescent dye
Possible to array 300 000 oligonucleotides within a 1.28 X 1.28 cm array
What is a Southern Blot?
detects major deletions and rearrangements. It is laborious expensive and lots of DNA
What is Heteroduplex Gel Mobilty?
Simple-Cheap
Sequence <200bp, limited sensitivity, does not reveal position.
Relies on the fact that most mutations occur as hetroduplex. DNA subject to PCR heated and denatured. Then subject to nondenaturing polyacrylamide gel electrophoresis
What is Single Strand Conformation Polymorphism (SSCP)?
Similar advantages disadvantages as Hetroduplex analysis. PCR molecule will have its own migration properties dependent on its base composition.
What is Denaturing gradient Gel Elecrophoresis(DGGE)?
High sensitivity (note than SSCP and hetroduplex analysis do generate false positive and false negative results) Requires careful initial optimisation (primer design) and conditions and does not reveal position of mutation
What is Denaturing High Pressure Liquid Chromatography (dHPLC)?
Similar principle to DGGE but a lot faster, with higher throughput and is quantitative
The disadvantages are that it again does not reveal the position of the change and the equipment is expensive
What is Mismatch cleavage (Chemical or Enymatic)?
Both have high sensitivity and show position of the change
Problems chemical method toxic both experimentally difficult and often poor results
These methods rely on the formation of a hetroduplex which is recognised by chemicals or restriction enzymes-usually those that are sensitive to single stranded DNA in the hetroduplex.
Generated products can then be resolved on a gel or sequenced
