Advanced Molecular Techniques Flashcards
How would you analyse DNA at nucleotide level
- DNA sequencing
- PCR
How would you analyse use DNA at the gene level ?
- southern hybridisation
- Reverse transcriptase - PCR
- microarray
- DNA fingerprinting
How would you analyse DNA at the chromosome level ?
- Karytotyping
- FISH
What are a few ethical considerations we need to consider when DNA sequencing ?
- does it open up areas for discrimanition in the work place
- would your health insurance be more expensive
- can the knowledge help prevent illness later in life ?
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What is allele specific PCR ?
Allele specific PCR is an application of the PCR that permits the direct detection of any point mutation in human DNA.
- the primers were designed to have a complementary nucleotide at their 3’ end to the DNA sample.
What are allele specific probes ?
Short piece of synthetic DNA complementary to the sequence of a variable target DNA used in hybridisation
Outline th eprocess of reverse transcriptase PCR
1) take a sample of mRNA from cell
2) using reverse transcriptase , create cDNA which is a complementary form of mRNA.
3) Now break the double stranded piece of RNA + DNA
4) using PCR, now you can amplify cDNA.
What is the use of microarray technology?
- scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously
- OR can be used to detect genomic copy number changes ( gains or losses in DNA eg insertions or deletions)
Outline an example of how scientists would use microarray technology to compare conditional gene expression in cancer and normal cells
1) extract mRNA from each the cancer and normal cells. Isolate this RNA
2) Using reverse transcriptase , create cDNA
3) label each cDNA from cancer and normal cells with a fluorescent probe ( red or green )
4) combine the targets and hybridise to microarray.
5) Now analyse, for example if in both cells the same genes are expressed then there would be the same amount of red/green for that dot. However , if there is a gene expressed in a cancer cell but not a normal cell there would be more red fluorescent in that dot than green.
MICROARRAY TECHNOLOGY : explain how you would use array comparative genome hybridisation to compare cells from a cancer patient and normal control
1) extract NORMAL DNA from each pateitn( cancer and control )
2) CUT dna into smaller pieces
3) label each groups of DNA with two different Fluorochromes ( usually red and green )
4) mix in equal quantities , hybridise to microarray
5) using computers , measure the relative intensities and work out the red:green ratio for each cell
6) Higher red:green ratio indicates a duplication in DNA , whereas a lower red : green ratio indicates a deletion in DNA
What is another term used to describe DNA fingerprinting?
DNA profiling
What are minisatellites
- tract of repetitive DNA which are repeated around 5-50 times in DNA
- they do not contribute to the functions of genes
- each individual has a unique pattern of minisatellites ( apart of identical twins)
Outline the procedure of DNA profiling ( the first method)
1) first you obtain a sample of cells ( eg skin or hair )
2) DNA is extracted and purified
3) restrictive enzymes cut DNA at specific locations
4) place them on gel electrophoresis and sort them into their sizes
5) southern hybridisation occurs ( the probes are small fragments of Minisatellite DNA tagged with radioactive phosphorus - these probes only attach to pieces of DNA that they are complementary to- attach to the minisatellites in the genome ). These minisatellites that the probes have attached to were visualised by exposing the nylon membrane to X-ray film. DARK BANDS exposed the DNA FINGERPRINT
Outline the procedure of DNA profiling - modern day technique
1) extract DNA from biological samples eg blood , saliva , hair
2) DNA profiling ( modern day technique ) does not use restriction enzymes to cut the DNA. Instead it uses PCR to amplify the Microsatellite sequences ( short tandem repeats)
3) run the fragments on gel electrophoresis to separate the fragments according to size
4) Fragments with fluroscent tags glow when fragments are passed beneath a laser. The output is displayed as a series of coloured peaks highlighting the colour and length of each STR sequence.
What is karyotyping
- lab proexedure that allows your doctor to examine your set of chromosomes- it examines these dividing cells
- the pairs of chromosomes are arranged by their size and appearance - it helps doctors determine if any chromosomes are missing or damaged
