DNA probes Flashcards
(22 cards)
2 methods of identifying specific alleles of genes
DNA probes
DNA hybridisation
DNA probe
A short, single-stranded DNA molecule that has bases complementary with a target DNA base sequence
Stages of testing for a base sequence of an allele using DNA probes
Obtain sample of DNA by restriction endonuclease + amplify by PCR
Electrophoresis of sample + bound to nylon membrane
Make DNA probes with a complementary base sequence to target gene using gene machine
DNA hybridisation of probes
Detection
How to initially make DNA probes
Use a gene machine to make a specific base sequence + PCR to amplify it
Use fluorescent/ radioactive nucleotides so probe can be detected
Make probe single stranded by treatment with alkali
How can Radioactive nucleotides (used to make DNA probes) be detected after hybridisation?
The radiation emitted can be detected by blackening an X ray film laid over the nylon membrane (autoradiography)
How can fluorescent nucleotides (used to make DNA probes) be detected after hybridisation?
These glow when subjected to UV light, allowing the hybridised probe to be detected via a photo.
Why is it preferred to use fluorescent nucleotides than radioactive nucleotides to make a DNA probe?
This method is safer than using radioactive material
Also gives a much quicker result than waiting for X ray film to be exposed
Method to obtain DNA sample that will be tested for a specific base sequence of a gene
A DNA sample is extracted from cells
Amplified using PCR if necessary
Broken down into smaller fragments by restriction endonucleases, which cut at specific base sequences
Electrophoresis of DNA sample
The separation of DNA fragments by length by passing an electric current over a gel they are placed inside
Steps of electrophoresis
Place samples of DNA fragments into wells in a gel
Pass electric current through gel so DNA travels toward the positively charged electrode in the gel
Why does the DNA move toward the positively charged electrode during electrophoresis
DNA has negatively charged phosphate groups in the backbone so is attracted to positive electrode at the other end of the gel, and therefore repelled by negative electrode on the side of the gel where the wells are
What feature of DNA are the samples separated by in electrophoresis?
The length of the DNA fragment:
Shorter fragments with a smaller base sequence travel further compared to longer fragments with more base sequences
Negatively charged phosphate group to be attracted to positively charged electrode
What are the samples of DNA fragments transferred to after electrophoresis?
A nylon membrane
How can samples of DNA be transferred to a nylon membrane?
Alkaline transfer solution to make DNA fragments single stranded (denaturation)
Fix to nylon membrane using UV light
DNA hybridisation
When single stranded DNA probes are added to single stranded DNA on the nylon membrane and bind to target complementary base sequence by hydrogen bonds
What happens to the nylon membrane after DNA hybridisation
Unbound probes are washed off
So only hybridised DNA probes remain on the membrane
Detection of specific base sequences after DNA hybridisation
Only hybridised DNA probes are made
Which contain fluorescent or radioactive nucleotides so we can see if the target base sequence of the gene is present by the naked eye: autoradiography or fluorescence
Why do we use DNA probes?
To screen patients to see if they have alleles associated with:
Heritable conditions
Drug responses
Health risks
How can information about patient’s alleles (that were screened for health risks etc) be used?
In genetic counselling
Personalised medicine
Genetic counselling
Discusses the implications of patients alleles they were screened for in a sensitive and appropriate manner
Personalised medicine
Treatment for a patient is customised for an individual patient
Eg to check their genes for a possible response to a drug/medicine to check if its effective
Such as, the medicine only works due to a specific protein, translated by a specific gene
Advantages of personalised medicine
Faster patient recovery
More cost-effective - reduces use of drugs that aren’t going to work
Safer: avoids having to endure side effects of drug that later proves ineffective for that patient.
