7P: Molecular genetics I Flashcards
Genetic diagnosis of Fragile-X syndrome
- Cytogenetic examination: Cell culture on folate poor substrate
- PCR based examination
- Southern-blot based examination
Disadvantages of cytogenetic diagnosis
- Lot of work
- Expensive
- Takes long
- High rate of false positive and false negative results
Classification of molecular genetic methods (basic principles)
- Cut and paste
- Hybridization
- Amplification
- Visualization
Restriction endonucleases
Enzymes that cut DNA at specific (palindromic) recognition nucleotide sequences
Hybridization
Making a complimentary strand to a ssDNA or an RNA strand
- Eg. FISH, PCR, blotting techniques
Applications of Southern blot
- Detection of fragile X syndrome
- Identification of methylation sites
Possible error of PCR
If the DNA is not clean enough, contamination will be amplified as well
Needed for PCR
Primer, nucleotides, Taq polymerase, template, DNA
Advantage of capillary electrophoresis
In the separation of DNA sequences (e.g. PCR products) of very small size (1bp) difference
Fragile X syndrome gene
FRAXA gene
CGG-repeats in FRAXA gene causing disease
55-230 -> Premutation (healthy)
230-1000 -> Full mutation
PCR-RFLP method
1) The DNA region is amplified with specific primers in PCR
2) Digestion of DNA with restriction enzymes (for PCR-RFLP)
3) Gel electrophoresis
4) Staining with DNA-binding dyes, e.g. Ethidium bromide
5) Evaluation in UV light
Clinical significance of VNTR
- Triplet repeat disease (expansion of triplet in mutant allele)
- Wild type allele - few triplet repeat, but in mutant the number of repeats are multiplied
Multiplex PCR
Allows simultaneous amplification of multiple target regions within single reaction using different primer pairs
Largest gene of human genome
Dystrophin gene
