Manipulating genomes

common techniques

Polymerase chain reactions (PCR), using restriction enzymes and gel electrophoresis

PCR method

Set up a reaction mixture containing the DNA sample, DNA polymerase, primers and free nucleotides. Heat the reaction mixture to 95 degrees to break the hydrogen bonds between complementary nucleotides on the DNA strand. DNA polymerase doesn’t denature at this temp so can be reused for multiple cycles. Then cool the mixture to around 50-65 degrees so the primers can anneal to the DNA strands. Then heat the reaction mixture back up to 72 degreese so the DNA polymerase is at its optimum temperature. The DNA polymerase lines up free DNA nucleotides alongside each template strand causing complementary base pairing to occur forming two new DNA strands. Two new copies of the DNA fragment have been formed as one PCR cycle is complete. A new cycle can now start again using all four DNA strands as templates this time.

primers

Short pieces of DNA that are complementary to the bases at the start of a DNA fragment

PCR is used to

Amplify the amount of a DNA fragment you have by producing millions of copies of it

palindromic sequences

sequences that consist of the same order of bases when read backwards on the opposite strand

restriction enzyme

recognise specific palindromic sequences known as recognition sequences and cut the DNA at these places. The restriction sequences all have different shapes complementary to specific restriction enzymes active sites.

Use of restriction enzymes process

Done if recognition sequences are present either side of the DNA fragment wanted. First incubate the DNA sample with the specific restriction enzyme which cuts the DNA fragment via a hydrolysis reaction. Sometimes these cuts leave sticky ends which can then be used to anneale the DNA fragment to another piece of DNA with complemntary sticky ends

sticky ends

small tails of unpaired bases at each end of the fragment

electrophoresis

A process which uses an electrical current to separate out DNA and RNA fragments as well as proteins depending on their size

electrophoresis process

Perform elctrophoresis using an agrose gel that has been poured into a gel tray and left to solidify.Create a row of wells on one end of the gel tray. Place the gel tray into a gel box and with the wells closest to the negative electrode. Then add buffer solution to the resoviours at the side of the gel box so the gel’s surface gets covered in buffer solution.Using a micropippete add the same volume of loading dye to each DNA sample helping the samples sink to the bottom of the well. Then using a diffrent micropippete add a set volume of one DNA sample to each well. Ensure you don’t pierce the bottom of the wells by placing the micropippetes in the buffer solution just above the opening of the wells. Put the lid on the gel box and connect the leads from the gel box to a power supply. Turn on the power supply to 100V so an electrical current is passed through the gel. DNA fragments are negatively charged so will move through the gel to the posotive electrode. Small DNA fragments will move the fastes so will get furhter through the gel so the fragments will be separated by size. Let the gel run for about 30mins the turn the power supply of and remove the gel tray from the gel box and tip out excess buffer solution. Then wearing gloves stain the gel using staining solution so the bands of DNA fragments can be visible.

eoectropherisis for protein

Proteins can be positively or negativley charged so have to be denatured using chemicals. Can be used to identify proteins in blood and urine when diagnosing diseases