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What does isolating DNA mean?
Extracting DNA from cells
What steps are involved in isolating DNA?
- The biological sample eg skin, blood, etc is collected
- The cell membrane is broken down by using heat, salt and/or detergents. This releases DNA and other cellular components into solution
- The solution is cooled on ice to precipitate out cell components other than DNA. These are removed by filtration or centrifuging.
- Ice cold alcohol is then used to precipitate the DNA into solution
What is Gel Electrophoresis?
This is the separation of different sizes of DNA through a gel using electricity (a charge)
How does Gel Electrophoresis work?
DNA is negatively charged. It is therefore attracted to a positive charge. By putting DNA into a 'gel' and putting a charge across it the DNA moves. By running DNA samples along side a DNA marker you can determine what size they are
What size of pieces move faster in Gel Electrophoresis?
What technique can be used in a lab to replicate DNA?
PCR - polymerase chain reaction
What ingredients do you need for PCR?
- Template DNA
- 2 Primers
- Taq DNA polymerase
- DNA ligase
What do the two primers do in PCR?
They are short sections of DNA which complement the section of DNA to be replicated. Nucleotides join on to these to make the new DNA chains. There may be many sets of the two primers.
Why is taq DNA polymerase used in PCR?
As it is able to withstand very high temperatures
What are the steps involved in PCR?
- The ingredients are put into microtubules in a PCR machine where the following occurs:
- The tubules are heated to ~95°C to break the hydrogen bonds between the two antiparallel DNA strands - the DNA is denatured.
- They are then cooled to ~50°C to allow the primers to anneal to seperate the strands of DNA
- Finally, at ~72°C taq DNA polymerase elongates the new strand from the starting point of the primer. DNA ligase seals any gaps in the sugar-phosphate backbone.
- This process is repeated over and over until there are many strands of the desired piece of DNA
What is the sequence of DNA?
The order of the nucleotides in DNA
What are the two differences between the ingredients used in PCR and DNA sequencing?
- There is only one type of primer in DNA sequencing as only one sid of the DNA is amplified
- There are normal nucleotides as well as modified nucleotides. Modified nucleotides are altered by either adding a fluorescent dye to them or removing the hydroxyl group on the third carbon atom
What does removing the hydroxyl group on the third carbon atom of a nucleotide prevent?
It prevents the sugar-phosphate bond in the backbone of DNA forming. When modified nucleotides are added the chain stops short.
How do the fluorescent dyes help to figure out the sequence of the DNA?
At the end of many reactions, where the nucleotides are incorporated randomly making different sized pieces of DNA, all the different sized pieces of DNA are sorted by running them through a column or gel. A sequencing machine reads the colours and tells you the sequence of the piece of DNA you are looking at.
What do restriction enzymes do to DNA?
They digest (cut) DNA by breaking the sugar-phosphate backbone
The number of times the DNA is cut can tell us how many _____ ______ there are and where they are on DNA?
Probes are made from a piece of DNA that _______ the sequence of DNA you are looking for?
How does a probe work?
The probe is tagged with a radioactive or fluorescent probe. The probe is then washed over chromosomes of the DNA. If it binds then it shows that the gene/DNA of interest is present
DNA can be replicated by clonin DNA in _____ which are replicated in bacteria or yeast?
How is DNA replicated using plasmids?
First, the DNA strand that you are interested in is cut from the chromosome using a specific enzyme. Then the vector (plasmid) is vut with the same enzyme. As both the DNA of interest and the plasmid have both been cut by the same enzyme it means that they can 'stick together' because the ends of the strands will pair with each other. The enzyme ligase helps them to join DNA by sealing gaps in the sugar-phosphate backbone. The bacteria/yeast replicate producing many copies of the desired DNA. This DNA can then be extractd from the plasmids for use or further research.