Common techniques of manipulating genomes

  • Created by: amyquince
  • Created on: 09-06-19 17:45

TECHNIQUES USED TO STUDY GENES

- POLYMERASE CHAIN REACTION (PCR)

- GEL ELECTROPHORESIS

- cutting out DNA fragments using RESTRICTION ENZYMES

- DNA PROFILING

- DNA SEQUENCING

- GNETIC ENGINEERING

- GENE THERAPY

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POLYMERASE CHAIN REACTION

- can be used to amplify millions of copies of DNA

1. a reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase.

primers - are sohrt pieces of DNA that complementary to the bases at the start of the fragment you want.

DNA polymerase is an enzymes that creates new DNA strands

2. DNA mixture is heated to 95 degrees to break down hydrogen bonds between the two strands of DNA. this is important as it means many cycles of PCR can be carried out without having to use a new enzyme everytime

3. mixture cooled to 50-60 so primer can bind to strands

4. reaction mixture is heated to 72 so DNA polymerase can work

5. DNA polymerase lines up free DNA nucleotides alongside each template strand, complementary base pairing means new complementary strands are formed. 

6. 2 new copies of fragments DNA are formed are one cycle of PCR is complete

7. cycle starts again but mixture kept at 95 this time all 4 strands used as templates (2 old 2 new)

8. each PCR cycle doubles the amount of DNA ( 1= 2x2=4, 2 =4x2=8, 3=8x2=16 and so on)

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ELECTROPHORESIS

1. performed using agarose gel that has been poured into gel tray and left to solidify. a row of wells is created at one end of the gel

2. put gel tray into gel box, make sure end of gel tray with wells is closest to negative electrode on gel box

3. add buffer solution to reservoirs at the sides of the gel box so surface of gel becomes covered in buffer solution

4.  take fragmented DNA samples and using a micropipette add same volume of loading dye to each - loading dye helps the samples to sink to the bottom of wells to make them seen easier

5. add set vol of DNA sample to first well. make sure tip of micropipette is in the buffer solution just abovethe opening of well. make sure dont pierce bottom of it

6. repeat process and add same volume of each of you other DNA samples to other wells in the gel. use clean micropipette tip each time

7. record which DNA sample you have added to each well.

8. put lid on gel box and connect leads from gel box to power supply

9. turn on power supply and set it to required voltage that causes electric current to be passed through the gel

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ELECTROPHORESIS

10. DNA fragments negatively charged so they'll move through gel towards pos electrode, small DNA fragments move faster and further so DNA separates according to size.

11. let gel run for about 30 min then turn off power.

12. remove gel tray and tip off any excess buffer solution, wearing gloves, stain DNA fragments by covering surface of gel with staining solution then rinsing gel with water. Bands of different DNA fragments now appear.

13. Size of DNA fragments is measured in bases eg. ATCC=4 bases or base pairs, 1000 bases is one kilobase (1kb)

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RESTRICTION ENZYMES

1. some sections of DNA have palindromic sequences of nucleotides. these sequences consist of antiparallel base pairs that read the same in oposite directions

2. Restriction enzymes are enzymes that recognise specific palindromic sequences and cut (digest) the DNA at these places

3. different restriction enzymes cut at different specific recognition sequences, because the shape of the recognition sequence is complimentary to the active site of the enzyme.

4. if recognition sequences are present at either side of the DNA fragment you want, you can use restriction enzymes to separate from the rest of the DNA

5. sample incubated with specific restriction enzyme which cuts DNA fragment out via a hydrolysis reaction

6. sometimes the cut leaves sticky ends - small tails of unpaired bases at each end of the fragment. they can be used to bind the DNA fragment to another piece of DNA with sticky ends by complementary base pairing.

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