DNA technology 2 Flashcards
DNA probe
Short, single-stranded section of DNA that has some sort of label attached that makes it easily identifiable.
What are the two most commonly used probes?
- Radioactively labelled probes -> nucleotides with isotope 32P. Identified using a photographic plate that’s exposed by radioactivity.
- Fluorescently labelled probes
How are DNA probes used to identify particular genes?
- A DNA probe is made that has bases that are complimentary to the portion of DNA sequence that makes up part of the gene whose position we want to find
- DNA that’s being tested is treated to separate its 2 strands
- Separated DNA strands are mixed with probe, which binds to complimentary bases on one of strands. DNA hybridisation
- Site at which the probe binds can be identified by the radioactivity of fluorescence that the probe emits
Sanger method
Uses modified nucleotides that can’t attach to the next base in the sequence when they are being joined together. Act as terminators, ending the synthesis of a DNA strand.
What do the test tubes in the first step of the Sanger method have to contain
- many single-stranded fragments of DNA to be sequenced -> acts as a template for complementary strand
- A mixture of nucleotides
- A small quantity of 1/4 terminator nucleotides
- A primer to start process of DNA synthesis which is radioactively labelled/ fluorescent dye
- DNA polymerase to catalyse DNA synthesis
How are fragments identified?
All fragments of new DNA in any of the test tubes will end with a nucleotide that has the same base. Primers attached to the other end of the DNA section are labelled.
How are DNA fragments of different lengths separated?
Gel electrophoresis
- DNA fragments are placed on to an agar gel and a voltage is applied across it.
- The resistance of the gel means that the larger the fragments, the more slowly they move.
- The smaller fragments move further than the larger ones over a fixed period.
- A sheet of photographic film is then placed over the agar gel for several hours
- Radioactivity from each DNA fragment exposes the film and shows where it’s situated on the gel.
Why is restriction mapping necessary?
Larger genes and whole genomes must be cut into smaller fragments by restriction endonucleases and each fragment sequenced. The sequenced fragments need to be put back together to make up the original gene/ genome.
Restriction mapping
Involves cutting DNA with a series of different restriction endonucleases. The fragments produced are separated by gel electrophoresis. The distance between the recognition sites can be determined by the patterns of fragments that are produced.
What are the differences in automated DNA sequencing and restriction mapping?
- In computerised systems, instead of radioactively labelling DNA primer, 4 types of terminators are labelled with fluorescent dye.
- Each type takes up a different colour.
- The DNA synthesis takes place in a single test tube and is speeded up by using PCR cycles.
- Electrophoresis is carried out in a single narrow capillary gel.
- Results are scanned by lasers and interpreted by computer software, giving DNA sequence in a fraction of the time taken by conventional methods.
- Further automation includes use of a PCR machine to produce the DNA fragments required in techniques
Why is genetic screening done?
Important to screen individuals who may carry a mutant allele. Such individuals often have a family history of disease. Potential parents at risk can obtain advice from a genetic counsellor on implications of having children.
How are genetic disorders tested for simultaneously?
Possible to fix many different DNA probes in an array on glass side. By adding sample of DNA any complementary DNA sequence in donor will bind to one or more probes.
Summarise the genetic screening process
- Order of nucleotides on mutated gene determined by DNA sequencing
- Fragment of DNA with complimentary bases to mutated portion of gene is produced
- DNA probe is formed by radioactively labelling DNA fragment
- PCR techniques are used to produce multiple copies of DNA probe
- Probe is added to single-stranded DNA fragments from person being screened
- If donor has mutated gene, some donor DNA fragments will have a nucleotide sequence that’s complimentary to probe and probe will bind to its complementary bases on donor DNA
- These DNA fragments will now be labelled with probe and can be distinguished from rest of DNA fragments using x-ray film
- If complementary fragments are present, DNA probe will be taken up and X-ray film will be exposed
What is genetic counselling?
Advice and information are given to enable people to make personal decisions about themselves or their offspring
What does genetic fingerprinting rely on?
Genome of any organism contains many repetitive sequences of introns.
