DNA profiling and techniques Flashcards
What are the three techniques used to study genes?
PCR
Gel electrophoresis
Cutting out DNA using restriction enzymes
What does PCR do?
amplify millions of copies of a DNA fragment
What is placed in the reaction mixture for PCR?
excess nucleotide bases, the DNA sample, primers and DNA polymerase
What is a primer?
short sequence of bases that are complementary to the bases at the start of the fragment you want
What is the first stage of PCR?
DNA mixture is heated to 95*C to break hydrogen bonds between the two DNA strands
What happens after hydrogen bonds in DNA break during PCR?
temperature is decreased to 55*C and primers bind to the ends of the DNA strands
What is it called when the primers bind to the end of DNA strand?
annealing
What happens after primers have bound to DNA in PCR?
reaction mixture is heated to 72*C so that DNA polymerase can work and add bases to the primer building up the complementary strand.
What does gel electrophoresis do?
separates out DNA fragments, RNA fragments or proteins dependant on their size
What is the gel tray made of?
solidified agarose gel
Where does the gel tray go?
in the gel box with the wells closest to the negative electrode (cathode)
How is pH maintained in the gel plate/box?
cover in a buffer solution
How can you provide a reference for fragment sizing in gel electrophoresis?
add DNA fragments of known length into first and last well so that you can then compare it to the rest
What happens when an electrical current is passed through the gel?
DNA fragments move towards the anode as they are negatively charged
How are DNA fragments separated on the gel plate?
smaller fragments move faster and travel further through the gel so they separate due to size
What is done after the current is turned off in gel electrophoresis in order to expose the bases?
the gel is placed in an alkaline buffer solution to denature the DNA fragments, exposing the bases
What is Southern blotting?
the technique by which DNA strands are passed onto a nylon membrane in the same positions as they were on the gel
How are DNA strands cut into small fragments?
use of restriction endonucleases
What is the name given to short non coding pieces of DNA that are repeated many times?
satellite DNA
What is a minisatellite?
a region of 20-50 base pairs that are repeated several hundred times, also called variable number tandem repeats
What is a microsatellite?
2-4 base pairs repeated 5-15 times, also called short tandem repeats
How are the fragments on the nylon membrane tagged?
use of a radioactive or fluorescent DNA probes that bind to the DNA fragments and then can be viewed under UV light or X-ray
What can DNA profiling be used for?
forensic science and medical diagnosis
How can DNA profiling find people at risk of certain diseases?
certain non coding microsatellites are associated with increased risk of disease, these regions can be identified in DNA profiling
Why use non coding regions when profiling a human?
genome is very similar, coding sequences would not provide a unique profile and non coding regions contain the short tandem repeats
