Genetic techniques Flashcards
What is allele specific mutation detection?
- Distinguishes between 2 alleles that may only differ by a single nucleotide
- Distinguish between a known disease-causing point mutation and the normal allele
- Reaction won’t take place when 3’ end nucleotide not perfectly base-paired=distinguishes 2 alleles
Describe oligonucleotide ligation assay
1) Designed so their 3’ end nucleotides base-pair with the changed nucleotide that distinguishes the normal and mutant alleles
2) Primer binds to allele allowing shared primer to bind=ligation
3) ligation will not take place at 3’ end when nucleotide is not perfectly base-paired- point mutation
- CF mutations
What happens in Multiplex Ligation-dependent Probe Amplification analysis? (MLPA)
1) 2 adjacent probes contain the forward & reverse primer sequences for PCR
2) Forward primer fluorescently labelled- amplicons can be visualised in gel electrophoresis
3) Length of ‘stuffer’ sequence can be varied probes hybridised against target DNA & ligated
4) If ligation occurs then a functional PCR strand is created so amplification only happens if the target DNA is present in the sample
- Multiple probe pairs can be pooled & amplified with the same primer pair
In MLPA what is the amount of PCR product proportional to?
Amount of target DNA present in the sample-suitable for quantitative measurements
What difficulties in mutation analysis can occur? What other technique can be used instead?
- Template unsuitable for PCR
- Gene too big for PCR
- Fragile X mutation in FMR1 is repetitive tract CGG sequence
- GC-rich regions difficult to PCR
- Can use southern blotting
What happens in Chain Termination Chemistry/Sanga?
1) Starting material is a single DNA fragment (produced by PCR)
2) Anneal specific oligonucleotide primer and synthesise a new strand that is complementary to the template
3) Strand synthesis catalysed by DNA polymerase, requires dATP, dCTP, dGTP, dTTP
4) Small amount of ddTTP also is incorporated into the reaction mix
5) DNA polymerase doesn’t discriminate between dNTPs & ddNTPs so ddNTP is incorporated into the growing DNA strand
6) ddNTP at the end of each fragment of different lengths
7) Sequences then added to fluorescent sequencing gel
What are the 2 types of DNA sequencing?
- Traditional sequencing
- Clonal sequencing (next generation)
What DNA is sequenced by clonal sequencing?
- PCR products
- Long PCR product covering a part/whole gene
- Panels selected genes known to be mutated for a particular phenotype
- Very large exome panels based on all coding genes in the human genome
What is the role of Connexin 26?
- Most common cause of inherited deafness
- Accurate size analysis
- Protein found on the GJB2 gene
What genetic testing can be carried out if we do/don’t know the identity of a mutation?
- Do= simple, cheap analytics
- Don’t= Discovery method (sanger DNA sequencing), clonal (next generation sequencing)
What technologies can be used based on DNA:DNA hybridisation?
- Ligation assay
- PCR
What is the function of PCR?
- Determine presence/absence of product (allele specific)
- Determine product size by gel electrophoresis (direct sequencing, ligation assay)
What is the function of MLPA analysis?
Multiplex PCR method for detecting chromosomal DNA copy number changes in multiple targets
What is the Illumina method in next generation sequencing?
1) Start with dsDNA which is fragmented
2) Adapters added to fragments
3) Fragments bound to a slide to provide strong fluorescent signal
4) DNA ‘bends’ to find a complementary primer on slide surface-amplification
5) Forms dense clusters of DNA in channels on the slide
6) One type of strand discarded
7) Sequencing primers, polymerase, nucleotides added
8) Laser used to activate fluorescence and colour is ‘read’
What is the function of clonal sequencing of gene panels?
Capture genomic (mutated) material of interest for sequencing of selected genes from PCR products
