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
What does dNTP and ddNTP stand for?
dNTP - deoxynucleotide
ddNTP - dideoxynucleotide
How does ddNTP lead to the termination of strand synthesis?
ddNTP lacks 3’ hydroxyl group»_space; phosphodiester bond not formed.
At what end of does the strand terminate when ddNTPs are incorporated?
3’ end.
Do the fragments move towards the positive or negative charge in gel electorphoresis?
Move towards the positive charge.
- ‘pulls’ them towards it
- smaller fragments travel further
What disorder is MEGF10 screened for?
Muscular dystrophies.
What is another name for clonal sequencing?
‘Next generation’ sequencing.
- non-Sanger method
- billions of DNA strands sequenced in parallel
Describe the stages of clonal sequencing. (6)
- Prepare DNA sample
- Attach DNA to surface
- Bridge amplification
- Fragments become double stranded
- Denature double strands
- Complete amplification»_space; millions of DNA clusters
What DNA is sequenced by clonal sequencing?
- PCR products
- Panels of genes known to be mutated
- Large ‘exome’ panels
What is ‘1000 genomes’?
Genomes of 1092 individuals from 14 populations.
-helps show variation and understand genetics contribution to disease
