Please enable JavaScript.
Coggle requires JavaScript to display documents.
Topic 8: The control of gene expression, DNA profiling
DNA profiling is a…
-
DNA profiling
DNA profiling is a forensic technique used to identify individuals by characteristics of their DNA. It can also be used to determine genetic relationships between organisms. One example is PCR.Polymerase chain reaction known as PCR is used to amplify DNA by making millions of copies of a given DNA sample. It occurs as following:
- A reaction mixture is set up by mixing the DNA sample, primers, free nucleotides and DNA polymerase which is the enzyme involved in creating new DNA strands.
- The mixture is then heated to 95 degrees to break the hydrogen bonds and to separate the two strands.
- The mixture is then cooled to a temperature between 50-65 degrees depending on the type of primers used, so that they can bind to the strands.
- Temperature is increased to about 70 degrees as this is the temperature DNA polymerase works at. The DNA polymerase is called Taq polymerase and is from bacteria that lives in hot springs.
- DNA polymerase creates a copy of the sample by complementary base pairing using the free nucleotides.
- This cycle is repeated around 30 times and gives rise to an amount of DNA sufficient to create a DNA profile.
Genetic technologies
In-vivo and in-vitro cloning
- In-vitro - this gene cloning can be done with PCR. This is fast, automated and reliable once conditions are established. This does not require living cells and can have problems such as contamination and errors*.
- In-vivo - gene cloning that can be done using recombinant plasmids in bacteria. This is accurate and useful as the gene is placed in cells where it can be expressed. The disadvantage thought is it is very time consuming and requires monitoring of cell growth.
DNA probes
A DNA probe is a short, single stranded DNA molecule that is designed to be complimentary to a sequence to be detected. DNA probes are made in smaller quantities and then amplified using PCR. The DNA labelling of the fragments either uses radioactive isotopes or a fluorescent dye which glows under certain wavelengths of
light.
DNA probes can be used in order to detect heritable conditions of health risks.
- The sequence of nucleotides on the mutated gene is determined by DNA sequencing. Genetic libraries now store of the DNA sequences of many of the genes responsible for common genetic diseases.
- Fragment of DNA with complimentary bases to the mutant allele of the gene is produced.
- DNA probe is formed by fluorescently labelling the DNA fragment.
- PCR techniques are used to produce multiple copies of the DNA probe.
- Probe is added to single-stranded DNA fragments from the person being screened.
- If the donor has the mutated gene, some donor DNA fragments will have a base sequence that is complimentary to the probe and the probe will bind to its complimentary bases on the donor DNA.
- These DNA fragments will now be labelled with the probe and can be distinguished from the rest of the DNA fragments.
- If complimentary fragments are present, the DNA probe will be taken up and the dye will fluoresce - this is detected by a special microscope. If complimentary fragments are not present, the DNA probe will not fluoresce.
Genetic fingerprinting
Genetic fingerprinting is a technique that can detect differences in people DNA. It uses variable number tandem repeats (VNTRs) which are short repeating sequences or bases. The probability of two individuals having identical VNTRs is extremely low therefore VNTR analysis can be used in genetic fingerprinting.Genetic fingerprinting can be used in the fields of forensic science, medical diagnosis as well as animal and plant breeding.
- Extraction DNA is extracted from the sample.
- Digestion Restriction endo-nucleases cut the DNA into fragments.
- Separation fragments are separated using gel electrophoresis.
- Separation DNA fragments are transferred from the gel to nylon membrane.
- Hybridization DNA probes are added to label the fragments . These radioactive probes attach to specific fragments.
- Development membrane with radioactively labelled DNA fragments is placed onto an X-ray Film.
- Development of the X-ray film reveals dark bands where the radioactive DNA probes have attached.
Gel electrophoresis is a process used to separate the DNA fragments and proteins according to their size using an electric current.
- Restriction enzymes cleave DNA into smaller segments of various sizes.
- DNA segments are loaded into wells in a porous gel. The gel floats in a buffer solution within a chamber between two electrodes.
3, When an electric current is passed through the chamber DNA fragments move toward the positively-charged cathode.
- Smaller DNA segments move faster and farther than larger DNA segments.