3 Molecular genetic techniques Flashcards
Explain how PCR works.
Oligonucleotides define boundaries of synthesis. DNA synthesised used DNA polymerase. 93 heat denaturation. 55 primer annealing. 72 primer extension. PCR assay determines product size on base level
Explain how oligonucleotide ligation assays work.
Allele specific primers designed which have 3’ end nucleotides pairing with changed nucleotide.
Which technique is used when the diagnosis is known but the genetic basis is not?
Genotyping assay: genotyping a panel of known mutations.
For example - CFTR tests.
What is MLPA and what is it used for?
Multiplex Ligation-dependant Probe Amplification.
Quantitive measurements - amount of PCR product is proportional to the amount of target DNA in the sample.
Why are some mutations difficult to analyse via PCR based methods?
Gene is too big.
Fragile X mutation in FMR1 gene is repetitive tract of CGG (southern blotting used instead).
GC rich regions are difficult to PCR.
Northern, Southern and Western blotting - which does what?
Northern - RNA.
Southern - DNA.
Western - protein.
When is DNA sequencing used in genetic diagnoses?
When mutation is unknown, and the position or type of mutation is important.
How does Sanger sequencing work?
Specific oligonucleotide primer added, DNA polymerase uses dNTPS’s to make new strands. ddNTP’s disrupt chain (no 3’ hydroxyl group needed for phosphodiester bond). Random termination. Fluorescent sequencing gel to determine each base.
What DNA is sequenced by clonal next generation) sequencing?
PCR products.
Long PCR products.
Panels known to be mutated for a phenotype.
Large exome panels.
Which technique is used for genes that cannot be analysed via PCR?
Southern blotting.
Which technique is used to distinguish between the normal genetic code and a point mutation?
Oligonucleotide ligation assay.
