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.
What is the role of thermostable DNA polymerase in PCR?
allows the denaturation without replicating DNA polymerase.
Why are primers required in PCR?
DNA polymerase cannot synthesise DNA without them.
What is RT-PCR?
A method of finding DNA from mRNA.
What is pyrosequencing?
A sequencing method based on synthesis, dNTPs are added one at a time. If a particular dNTP is incorporated into a growth chain Pi is released and seen as a flash of light.
What is the purpose of dideoxyneucleotides in Sagner DNA sequencing?
To terminate the DNA chain.
