Nucleic Acid Detection Methods Flashcards
What is aragose gel electrophoresis?
A technique used to separate nucleic acid molecules
What is the mobility of nucleic acids effected by?
Their shape
Group from biggest to smallest: relaxed circle, supercoiled state and linear form?
Relaxed circle > linear form > supercoiled state
What is Southern blotting used for?
to identify, size and reveal an abundance of DNA
What is the Southern hybridisation technique?
Identifying a piece of DNA or gene with a probe
What is a probe?
a sequence of nucleotides with a complimentary base sequence
What are the steps of the Southern blotting technique? (5)
Separation of DNA fragments; blotting of DNA fragments’ immobilisation of DNA fragments; Hybridisation of DNA fragments; Detection of probe bound DNA
What do non-radioactive probes use?
biotin
What happens in a dot blot? (2)
DNA is dotted directly onto a membrane. The dotted DNA is hybridised with a probe and visualised.
What causes DNA to migrate in electrophoresis?
Its negative charge
What is the purpose of Southern blotting?
To identify a specific gene.
What is the process of DNA synthesis? (3)
At 95C dsDNA separates into 2 strands. When cooled the sequence will join again, primers will bind at complimentary sequences. DNA polymerase synthesises new DNA by extending the primers.
What is PCR?
Polymerase chain reaction is a primer extension reaction amplifying specific nucleic acids. It is used to make a substantial number of copies of a specific DNA region.
What are the 3 PCR cycle reactions?
(All repeats for 30-40 cycles) Denaturation 95C: Strands melt open into single strands, no enzyme activity. Renaturation/Annealing 55C: allows primers to bind to complimentary sequences. Synthesis 72C: ideal temp for enzymes, bases added to 3’ end.
What are the 3 limitations of PCR?
Prior knowledge of target DNA, very high risk of contamination and requires proofreading enzymes for accuracy.
