22 terms

Manipulating genomes

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The polymerase chain reaction (PCR)
Used to select fragments of DNA and amplify it to produce millions of copies
Steps in PCR
1) Reaction mixture that contains the DNA sample, free nucleotides, primers, and DNA polymerase.
2) The DNA mixture is heated to 95degrees Celsius to break the hydrogen bonds between the two strands of DNA.
3) the mixture is cooled to between 50 and 65 degrees Celsius so that the primers can bind to the strands. (ANNEALING).
4) the reaction mixture is heated to 72 degrees Celsius, so DNA polymerase can work and line up free DNA nucleotides along each template strand. Complementary base pairing occurs forming complementary strands.
How many new copies of fragment DNA are formed in one cycle of PCR?
2
When the cycle starts again how many strands are used as templates?
All 4; two original and two new.
Why is DNA polymerase used
It does not denature at high temperatures and so many cycles of PCR can be carried out without having to use new enzymes.
What is a primer
Short pieces of DNA that are complementary to the bases at the start of a fragment you want
How much does the DNA amplify after each cycle of PCR
It doubles
What is the function of electrophoresis?
To separate out fragments of DNA, RNA and proteins depending on their size.
What is the first step needed before electrophoresis?
To pour agarose gel into a gel tray and leave it to solidify. A row of wells is created at one ends of the gel, this end needs to be closest to the negative electrode on the gel box. Buffer solution then needs to be added to the reservoirs of the gel boys so that the surface becomes covered in buffer solution.
Why is loading dye added to the fragmented DNA samples?
So that they sink to the bottom of the wells and are easier to see.
How are the DNA fragments placed into the wells?
Using a clean micropipette each time, add a set volume of the DNA sample to each well, by making sure the top of the micropipette is in the buffer solution Just above the opening of the wells.
How is electrophoresis carried out?
-connect the gel box to the power supply and set it to the reared voltage so that a current passes through the gel.
- negatively charged DNA fragments move towards the anode (positive electrode). Smaller fragments move faster and so will move further along the gel.
- remove gem tray from gel box and remove excess buffer solution
- strain the DNA fragments so that the different strands are visible and the size can be measured in bases.
What is done with proteins before they undergo electrophoresi?
They are mixed with a chemical that denatures them so that they all have the same charge (as some proteins are positively charges and some are negatively charged.)
What are some of the uses of the electrophoresis of proteins?
To identity protein present in urine or blood samples which can help in diagnosis of disease.
How much does the DNA amplify after each cycle of PCR
It doubles
What is the function of electrophoresis?
To separate out fragments of DNA, RNA and proteins depending on their size.
What is the first step needed before electrophoresis?
To pour agarose gel into a gel tray and leave it to solidify. A row of wells is created at one ends of the gel, this end needs to be closest to the negative electrode on the gel box. Buffer solution then needs to be added to the reservoirs of the gel boys so that the surface becomes covered in buffer solution.
Why is loading dye added to the fragmented DNA samples?
So that they sink to the bottom of the wells and are easier to see.
How are the DNA fragments placed into the wells?
Using a clean micropipette each time, add a set volume of the DNA sample to each well, by making sure the top of the micropipette is in the buffer solution Just above the opening of the wells.
How is electrophoresis carried out?
-connect the gel box to the power supply and set it to the reared voltage so that a current passes through the gel.
- negatively charged DNA fragments move towards the anode (positive electrode). Smaller fragments move faster and so will move further along the gel.
- remove gem tray from gel box and remove excess buffer solution
- strain the DNA fragments so that the different strands are visible and the size can be measured in bases.
What is done with proteins before they undergo electrophoresi?
They are mixed with a chemical that denatures them so that they all have the same charge (as some proteins are positively charges and some are negatively charged.)
What are some of the uses of the electrophoresis of proteins?
To identity protein present in urine or blood samples which can help in diagnosis of disease.
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