Genetic Fingerprinting Flashcards
What are DNA probes? Give the 2 most commonly used
Short, single-stranded lengths of DNA that have a label attached, which makes it easily identifiable.
The two most commonly used are:
1. Radioactively labelled (identified using x-ray film)
2. Fluorescently labelled (emit fluorescence under certain conditions)
How are DNA probes used to identify particular alleles of genes?
- DNA probe has complementary base sequence to the target DNA sequence of the allele
- Target DNA is treated to separate its 2 strands
- Separated DNA strands are mixed with the probe
- Probe binds with DNA hybridisation
- Site of binding is identified - via radioactivity/fluorescence
What needs to be known before a probe is made?
The base sequence in the targeted allele
What gets the probe to bind? Describe it
DNA hybridisation: after heating the DNA to separate its 2 strands its allowed to cool and the complementary bases on the probe will anneal to the strands
Describe the use of DNA hybridisation using a probe for genetic screening for mutations
1.sequence mutated allele- consult gene library
2. Design and produce DNA probe complementary to target gene
3. PCR of probe to make more copies
4. Add label to probe - radioactive/ flourescent
5. Obtain DNA from person to be screened
6. Heat to separate DNA strands
7.cool and mix with probes
8.if target gene is present, the probe will bind by complementary bases pairing
9.wash to remove unattached probes
10. Hybridised DNA will be labelled
11. Use x-ray or photography or UV light to make visible
Discuss the uses of Gene probes
- Genetic screening for mutations that cause genetic disorders
—> screen for recessive alleles in heterozygous individuals to find carriers
—> screen for oncogenes and mutated tumour suppressor genes
2.personalised medicine
—>health care based on genotype
—>drug dosage e.g. for painkillers
—>vitamin E reduces risk of CVD among some with diabetes
3.genetic counselling
—>implications for having children e.g. a history of sickle cell anaemia, cystic fibrosis etc
—>early diagnosis
—>life choices to reduce risk (in case of oncogenes)
What is genetic fingerprinting
- its based on non-coding repetitive bases in introns of all eukaryotic cells
- these are called variable number tandem repeats (VNTRs)
-the number and lengths of VNTRs is unique to each individual
-more closely related two individual are, the more similar the VNTRs will be.
How can you find out the number and lengths of segments of DNA?
Gel electrophoresis
What are the stages of genetic fingerprinting?
1,extraction
2. Digestion
3.separation
4.hybridisation
5.development
Describe the stage of extraction in genetic fingerprinting
Extract DNA from biological sample, e.g hair. Separate DNA from the rest of the cell. As DNA quantity will be relatively small, use PCR to amplify it to make copies
Describe digestion as a stage in genetic fingerprinting
DNA is cut into fragments using restriction endonucleases. The restriction endonucleases are chosen for their ability to cut close to but not within target DNA.
Describe separation as a stage of genetic fingerprinting
DNA fragments are separated according to size (lengths) by gel electrophoresis. DNA fragments are placed onto agar gel and a voltage is applied across it. The gel is then immersed in alkali to separate the double strands
Describe hybridisation as a stage of genetic fingerprinting
Radioactive/fluorescent probes are used to bind with VNTRs to label the fragments. They bind under specific conditions e.g. temp/ph. Different probes are used which all bind with different target DNA sequences.
Describe development as a stage of genetic fingerprinting
-A nylon membrane with radioactively labelled DNA fragments is placed onto an x-ray film.
-film is exposed via radiation or if using fluorescence positions are located visually.
- series of bands are revealed, which correspond to the position of the DNA fragments as separated during gel electrophoresis
-these bands are unique to every individual except identical twins
How do you interpret genetic fingerprinting?
- For paternity:
-> all bands of child not matching mother must match father
-> as child inherits half of the VNTRs of each parent - For crime scenes:
->all bands must match to confirm DNA is from the same person - For plant/animal breading:
->use most dissimilar DNA fingerprints as these are least related to avoid inbreeding
Describe how the technique of genetic fingerprinting is carried out and how it can be used to identify a person
Restriction endonuclease is used to cut the DNA.
Gel electrophoresis is used to separate the bands according to length
DNA is made single-stranded which is transferred to membrane via southern blotting
Probe is applied
Radioactivity/fluorescence is detected
VNTRs identified - each pattern is uniques to every individual.
