6 Analysis Of Nucleic Acids Flashcards
*Q: Explain the term hybridisation, used for binding of a probe to nucleic acid.
A: single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA
Q: How does cell-cased DNA cloning (in vitro) occur? (6)
A:Cutting a target sequence and a replicon with the same restriction endonuclease so that the ends of the two DNA sequences are compatible -> Purify and mix -> join the DNA fragments by using DNA Ligase -> Transformation of recombinant DNA molecules into host cells.
Selective propagation of individual cell colonies on agar plate. This can be done by using selectable antibiotic resistance marker in the replicon so only cells which have taken up the replicon survive.
Expansion of cell culture and isolation of recombiant DNA
Q: What are the 2 types of DNA cloning?
A: cell-Based DNA Cloning (in vivo),
Restriction Endonucleases
Q: Define replicon. eg?
A: sequence capable of independent replication e.g. plasmid
Q: How can you clone DNA using restriction endonucleases? Produce?
A: Type II Restriction Endonucleases are enzymes (usually dimers) that cleave DNA at specific recognition sequences (usually 4-8bp palindromic sequences) -> They can produce blunt or sticky ends
Q: What is the result of a longer recognition site?
A: the less frequently it occurs in DNA
Q: How are DNA fragments separated?
A: Electrophoresis
DNA is negatively charged because of its phosphate backbone and it moves towards the anode when an electrical force is applied to the DNA solution.
When DNA is forced to travel through a porous gel matrix, small fragments travel further than large fragments
Q: What is a method of detecting specific sequences in our DNA? Relies on? How? Significance?
A: nucleic Acid Hybridisation
This method relies on having single stranded DNA to which a probe can bind.
The probe is made so that it can be visualised i.e. radioactive or fluorescent = Allows you to identify related target molecules in a mixture of unlabelled nucleic acids.
You design the probe to match the sequence that you are trying to find.
important and versatile in nucleic acid analysis
Q: What are hybridisation assays? What occurs?
A: Target DNA is immobilised on a solid support (nylon or nitrocellulose membrane) which readily binds single-stranded nucleic acids (e.g. denatured DNA or RNA).
This is then hybridised with a solution of (fluorescently or radioactively) labelled probe.
Q: What does hybridisation (blotting) of DNA fragments after separation do?
A: After resolution, DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridisation with a labelled probe detected by exposure to photographic film.
Q: How do you detect restriction fragment length polymorphism in human genomic DNA? (3) with? Used where? Process. (2)
A: by restriction digestion, gel electrophoresis and hybridisation with a gene-specific probe
Used in familial genetic analysis
When you bind a probe to some sample of DNA, the length of DNA that is recognised will be slightly different depending on the genotype of the individual.
When the fragments undergo electrophoresis, the different alleles can be visualised depending on fragment length.
Q: What is the use of in situ hybridisation? means? Process.
A: locate specific genes on chromosomes
In situ - IN THE ORIGINAL/NATURAL LOCATION
Can mix with several different probes to see whether they have the correct copy number and to check for the presence/absence of certain genes.
Q: How is denaturation of a probe DNA achieved? Where does the energy needed depend on? (4)
A: by heating until the hydrogen bonds between the bases holding the two strands together are disrupted.
energy needed to achieve this depends on:
Strand Length - longer strand = more hydrogen bonds to break
Base Composition - G-C has 3 hydrogen bonds whereas A-T has 2 so G-C are harder to break
Chemical Environment - eg. (Na+) STABILISE the DNA duplex by neutralising charge of phosphate backbone.
*Q: What is stringency? What does high or low mean?
A: how specifically the probe binds to the target DNA. Low stringency means that the probe can bind to target DNA with some degree of mismatch. High stringency means that it only binds to perfectly complementary target DNA.
At HIGH stringency, duplexes form only between strands with perfect one-to-one complementarity.
Lower stringency allows annealing between strands with some degree of mismatch between bases.
Q: What is the melting temperature (Tm)? Defined as? What is it for mammalian DNA? property?
A: measure of nucleic acid duplex stability
It is defined as - the midpoint temperature of transition from double stranded to single stranded forms of nucleic acids.
For mammalian DNA where you will have around a 40% G-C complex - Tm is 87oC
