Chapter 6: Biotechnology - Its Tools & Techniques Flashcards
ingredients required for PCR
template DNA strand
free nucleotides
taq DNA polymerase enzyme
primers
buffer solution
what are primers
short, single stranded pieces of chemically synthesised DNA which flank the target region
define buffer solution
liquid into which all the all of the other ingredients are added: prevents sudden pH changes.
where is PCR done?
to do a PCR, mixture is put into a thermal cycler, so it can be heated and cooled in a controlled way. PCR uses variations in temperature to control the replication process
steps of PCR
- Denaturation
- PCR reaction mixture is heated to 95 degrees celsius
- DNA is denatured, i.e. separates to form two single strands - Annealing
- PCR reaction mixture is cooled down to 55 degrees celsius
- allows primers to anneal (=bind) to the template DNA - Extension
- PCR reaction mixture is heated to 72 degrees celsius
- DNA polymerase moves down the template, synthesising new DNA.
does the amount of DNA per PCR cycle quadruple?
In each cycle, the number of DNA molecules doubles.
what does gel electrophoresis do ?
separates ad visualises DNA fragments based on their size
what is DNA charge?
negative due to the phoshpate group
steps of gel electrophoresis
- prepare the set up:
- Load DNA samples and ladder:
- Run the gel
well definitnion
hole with a specific size
Setting up stage of gel electrophoresis
- prepare the set up:
- pour the liquid agarose gel into a mould
- before it sets, insert a well comb.
- once mould has set, place it into a gel box which is set up with a negative electrode on one end, and a positive electrode on the other end. wells are on the side with the negative electrode
- Immerse gel with buffer solution (can conduct electricity)
loading DNA samples and ladder stage of gel electrophoresis
- Load DNA samples and ladder:
- Each DNA sample is transferred into its own well, these are usually amplified using PCR before they are run through the gel, so there are enough to use.
- A DNA ladder is added into one well, this ladder is a mixture of known DNA fragments, each of which has a specific size.
running the gel phase of gel electrophoresis
- Run the gel:
- Turn on the power - a current runs through the gel
due to the negative charge of DNA from its phosphate groups, and the fact that opposites attract, DNA moves through gel.
- DNA migrates through the gel, towards to positive pole.
why can DNA be separated based on size
Gel acts as a matrix. smaller DNA fragments move faster than bigger longer ones do.
what does the electircal current in gel elcotrorei do
provides a force to move the DNA
what does the agarose gel do in gel elelctoroepfero
strucutres (tiny pores) filters DNA by size.
what does the DNA ladder allow us to do?
to infer how many base pairs are in each fragment in the wells. thus, how long each of them are.
visualising the DNA fragments phase of gel electrophoresis
- Visualising the DNA fragments:
- Add a dye to stain the DNA fragments, by either:
a) to liquid agarose, before its poured into the mould
b) to gel, after it has been run.
define band in gel eleoeroeikvl;
each of the lines in the gel. they contain large numbers of DNA fragments of the same size, which have ultimately travelled to the same position in the gel.
Applicaitons of gel electrophoresis.
DNA sequencing
DNA profiling
Recombinant DNA
Describe how a specific gene from extracted DNA could be amplified.
PCR can be used to amplify this specific region of DNA. Complementary primer strands to the start and end of the gene are added. This is followed by PCR cycling (heating and cooling) with polymerase enzymes to replicate the region, amplifying the gene.
