Manipulating Genomes Flashcards
What are the three techniques used to study genes
- Polymerase chain reaction (PCR)
- Gel electrophoresis
- Cutting out DNA fragments using restriction enzymes
What can the polymerase chain reaction be used for
To select a fragment of DNA and amplify it to produce millions of copies in a few hours
What are the three stages of PCR
Separation, annealing of primers, synthesis of DNA
What happens during the separation stage of pcr
A reaction mixture containing DNA sample, free nucleotides, primers and DNA polymerase is set up. It is heated to 95 degrees Celsius to break the hydrogen bonds between DNA strands.
What happens to DNA polymerase during the separation stage of PCR
It does not denature even at this high temperature so many cycles of PCR can be carried out with the same enzymes
What happens during the annealing of primers stage in PCR
The mixture is cooled to 55 degrees Celsius so that the primers can anneal (bind) to the strands.
What happens during the DNA synthesis stage of PCR
The reaction mixture is heated to 72 degrees Celsius so that DNA polymerase can work. It lines up free DNA nucleotides alongside each template strand. Complementary strands can then be formed.
How many new copies of the fragment of DNA are formed in one cycle of PCR
2
What are primers
Short pieces of DNA that are complementary to the bases at the start of the fragment you want
What is DNA polymerase
An enzyme that creates new DNA strands
What is electrophoresis
A procedure that uses an electrical current to separate out DNA fragments, RNA fragments or proteins dependent on their size.
What is the set up that allows electrophoresis to occur
- Performed using agarose gel in a gel tray.
- This is then placed into a gel box or tank.
- At the negative end (cathode), the gel has wells. Using a micropipette, the fragmented DNA samples with loading dye should be added to the wells.
- There should be a buffer solution added to the reservoirs at the side of the gel box so the surface of the gel becomes covered.
How to carry out electrophoresis
- Put the lid on the gel box and connect the leads from gel box to the power supply.
- Turn on the power supply and set to required voltage (100 V).
- DNA fragments are negatively charged and therefore will move through the gel towards the positive electrode at the far end of the gel
- Let the gel run for around 30 mins (or until dye is about 2cm from end of gel) and turn off power supply
Which DNA fragments move the quickest in electrophoresis
Small DNA fragments will travel faster and further as they will separate according to size
What to do once electrophoresis has finished
Remove the gel tray from the gel box and tip off any excess buffer solution.
Wearing gloves stain the DNA fragments by covering the surface of the gel with a staining solution then rinsing the gel with water. The bands will now be visible
What must you do before carrying out electrophoresis on proteins and why
They may be positively or negatively charged so they are mixed with a chemical that denature the protein so they all have the same charge.
What is the use of electrophoresis in proteins
To identify the proteins present in urine or blood samples - help diagnose disease
What are restriction enzymes
Enzymes that recognise specific palindromic or recognition sequences and cut the DNA at these places
What are palindromic or recognition sequences
A palindromic sequence is a nucleic acid sequence in a double-stranded DNA or RNA molecule whereby reading in a certain direction on one strand is identical to the sequence in the same direction on the complementary strand.
Why do different restriction enzymes cut at different recognition sequences
Because the shape of the recognition sequence is complementary to an enzymes active site
What can occur if recognition sequences are present at either side of the DNA fragment you want
You can use restriction enzymes to separate it from the rest of the DNA
What happens to the DNA sample with specific restriction enzymes
It is incubated and the restriction enzymes cuts the DNA fragment out via a hydrolysis reaction
What is sometimes left after using restriction enzymes to cut DNA fragments and what can it be used for
Sticky ends (small tails of unpaired bases at each end of the fragment. They can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with complementary sequences
Why is electrophoresis used
To produce DNA profiles as:
The number of times a persons non-coding sections are are repeated differs from person to person, so the length of nucleotides differ too
The number of times a sequence is repeated at different specific places in a persons genome to create a DNA profile
The probability of two individuals having the same DNA profile is very low