15. Molecular Techniques and Diagnosis - Analysis Of DNA at Nucleotide, Gene and Chromosome Level Flashcards
How does southern hybridisation using an allele-specific probe work?
The normal-specific oligo matched the allele perfectly but a diseased oligo won’t match or bind as tightly to the probe.
How does PCR using allele-specific probes work?
If the probe and genomic sequence match, there will be a PCR product. If they don’t match, there won’t be a PCR product.
What does western hybridisation study?
Proteins.
What does northern hybridisation analyse?
RNA.
How does reverse transcriptase PCR work?
RNA degrades quickly so isn’t nice to work with. Therefore reverse transcriptase creates an mRNA strand from mature mRNA and cDNA is made. RNAase breaks it down to leave a single strand of DNA for PCR. This allows it to be seen when a gene is switched on.
What are the purposes of using microarray technology?
For looking at 1000s of genes simultaneously, investigating chromosome deletions or duplications and to investigate conditional gene expression.
How does DNA hybridisation microarray technology work?
RNA is isolated form diseased cells and normal cells. They both undergo reverse transcriptase labelling. The disease for has red fluorescent probes and the healthy has green fluorescent probes. The two are combined and where it is brown shows where both are being expressed. It shoes the difference in gene expression between healthy and diseased cells.
How can array comparative genome hybridisation be used?
Cells from a patient and normal control are DNA is extracted. Each are labelled with different fluorochromes. Equal quantities are mixed together. The green:red ratio for each cell is worked out and aligned to a database of clones.
Who was DNA fingerprinting invented by and when?
Professor Sir Alex Jeffreys in 1984.
When is DNA profiling used?
In forensics and in paternity disputes.
How can chromosomes be analysed?
Staining then for karyotyping o rushing FISH.
What does FISH stand for?
Fluorescent in situ hybridisation.
What is FISH used to investigate?
Genes in situ, chromosome structure, chromosome number and chromosome behaviour.
What are ethical consideration for DNA sequencing?
Who would be interested in the genome information (family, police, insurance companies, potential spouse), can the knowledge prevent illness later, does it open up areas for discrimination and who owns the DNA sequence?
