DNA Gels, Blots and Probes Flashcards
How can DNA fragments of different lengths be separated?
Gel electrophoresis.
Where is the digested DNA sample placed?
Into a well in a porous polysaccharide gel immersed in a buffer solution.
Where does the electrical current run?
Runs between the two electrodes placed in the buffer.
Where does the DNA move and why?
To the positive electrode because it is negatively charged.
What causes the fragments to separate along their lane according to their size?
The charge is uniformly distributed along the sugar phosphate backbone of the sugar-phosphate backbone of the molecule. The smaller fragments move faster and further through the gel.
What might a well in a gel usually contain?
A DNA ladder.
What can a DNA ladder be used for?
It displays a selection of known lengths of DNA that can be used as a reference scale.
How can DNA become denatured?
Heating it up to approximately 95 degrees C, braking the H bonds holding the complementary bases together
What is annealing or hybridising DNA?
The formation of a double-stranded molecule from single strands.
Why is the melting point of DNA with a high proportion of C:G pairs higher?
Because C:G pairs have more hydrogen bonds
Why are DNA blotting procedures used?
To make an accurate record of the final positions of the DNA fragments after electrophoresis.
What are blotting procedures used to do?
Used to draw the DNA fragments out of the gel and onto a nitrocellulose or nylon filter.
Why is the DNA denatured chemically with sodium hydroxide before the procedure?
To enhance the attachment of DNA to the filter.
What are DNA probes?
They are short single stranded sequences of DNA that have been labelled either radioactively or with a fluorescent tag.
What is specific about the design of a DNA probe?
Their sequence is designed to be complementary to a DNA sequence.
When a blot is incubated with a probe, what does the probe do?
The probe binds with any complementary sequences of DNA that are present in the sample.
Why is a blot washed?
To remove any unbound probe.
How are the bands where the probe has bound revealed?
By using photographic film or light of a specific wavelength.
What are single locus probes?
Those that have a complementary sequence at only one location within the genome being produced. So they will only hybridise at this one location.
What does a band on a single locus probe gel indicate?
The presence of the sequence in the fragment at that location in the gel.
What can single locus probes be used for?
Used for paternity tests and to screen for mutations within gene sequence.
What are multi-locusprobes?
Those that have a complementary sequence which occurs at many locations within the genome being probed.
Under what condition will the multi-locus probes hybridise at many locations and produce a pattern of bands known as a DNA profile or fingerprint.
When the genome is digested with a restriction enzyme.
What does DNA microarray technology allow?
A sample of DNA to be tested by many thousands of different probes at one time.
