Molecular Genetics - techniques and applications Flashcards
Which common technologies are based on DNA:DNA hybridisation? (2)
- PCR
- Ligation assay
What are the main features of PCR?
- Synthesis of large amounts of DNA by copying
- Primers define boundaries
- DNA polymerase = enzyme
Describe the basic process of PCR.
Heat denaturation (94)
» primer annealing (55)
» primer extension (72*)
» REPEAT
In what direction are nucleotides added in PCR?
5’ to 3’.
How are PCR products separated for analysis?
Gel electrophoresis.
What are the main functions of PCR?
- Determine presence / absence of product
- Determine product size
What type of PCR determines if a product is present / absent?
Allele-specific PCR.
-must know disease-causing point mutation and normal allele
What is a common mutation in CF?
F508 deletion.
- 3bp from CTFR
What does cystic fibrosis testing on newborns involve?
Immunoreactive trypsin protocol.
What mutation is the commonest cause of inherited deafness?
Connexin 26 (GJB2). -PCR size analysis can detect base deletion
What is ligation?
Joining of 2 DNA strand by phosphate ester linkage.
What happens in an oligonucleotide ligation assay if there is a point mutation at the ligation site?
No ligation product forms.
-ligation fails for both primers due to mis-match
What is the cystic fibrosis genotyping assay?
Quantitative in vitro test to genotype a panel of mutations in CFTR.
-used for carrier screening
What are the results of the cystic fibrosis genotyping assay not used for?
- Foetal diagnosis
- Stand-alone diagnosis
What does MLPA analysis stand for?
Multiplex Ligation-dependent Probe Amplification analysis.
What is MLPA analysis?
Multiplex PCR for detecting chromosomal DNA copy number changes in targets.
How does MLPA analysis work?
2 Probes contain forward and reverse sequences.
- forward is fluorescently labelled
- probes are hybridised against target DNA
- ligation / amplification only occurs if target DNA is present
Is MLPA analysis a qualitative or quantitative test?
Quantitative test.
-PCR product is proportional to amount of target DNA present
When is PCR unsuitable?
- Gene too big
- GC-rich regions
Why is fragile X syndrome with a mutation in FMR1 gene unsuitable for PCR?
Repetitive CGG sequence.
-GC-rich regions hard for PCR
What method can be used if PCR is unsuitable?
Southern blotting.
What are the different blotting methods? (3)
Southern - DNA
Northern - RNA
Western - protein
What is Southern blotting used for?
Detection of a specific DNA sequence in DNA samples.
How is DNA sequenced?
-DNA produced by PCR >>use primers to anneal strands >> strand synthesis using DNA polymerase and dNTPS >> small amount of ddNTPS >> strands of different lengths produced
