19 December 2019

1. 19 December 2019 Today’s Title: CW: DNA manipulation – separating and probing Learning Question:

2. Aims from specification (f) outline how DNA fragments can be separated by size using electrophoresis (HSW3); (g) describe how DNA probes can be used to identify fragments containing specific sequences;

3. DNA probes • A DNA probe is a short length of single stranded DNA that has a complementary base sequence to the gene you want to extract • The probe is “labelled” • E.g. with nucleotides containing an isotope of phosphorous, 32P, which emits beta radiation • When the probe is mixed with DNA fragments it forms hydrogen bonds with stretches of DNA complementary to its own base sequence (annealing)

4. Using DNA probes • Probes can be used to locate specific sequences • Identify the same gene on a variety of different genomes • Locate a specific desired gene • Identify the presence or absence of an allele for a genetic disease

5. Question • Why is it important that DNA probes are relatively short molecules? • What does the term annealing mean? • What type of bond is responsible for annealing?

6. Gel Electrophoresis • Electrophoresis separates different fragments of DNA according to their sizes • Tank set up containing agarose gel • Direct current is passed continuously through the gel • DNA fragments carry a small negative electric charge • DNA fragments are pulled through the gel towards the anode • The smaller the fragments the faster they move through the agarose matrix.

7. Gel Electrophoresis • When the current is turned off • DNA fragments will have ended up in different places • These can be transferred onto absorbent paper or by a technique called southern blotting

8. Using electrophoresis

9. A radioactive probe is added to bind to the invisible bands of DNA, so they can blacken an X-ray film Gel Electrophoresis

10. After electrophoresis and labelling of DNA samples, you can compare the DNA from different individuals. This is DNA profiling Gel Electrophoresis

11. Past paper questions

12. Polymerase Chain Reaction (PCR)

13. Polymerase Chain reaction - Stage 1 • The reactants are mixed together in a PCR vial. • The mixture contains: • the DNA which is to be amplified • the enzyme DNA polymerase • small primer sequences of DNA and • a good supply of the four nucleotide bases A,T,C and G. • The vial is placed in a PCR machine.

14. Polymerase Chain reaction - Stage 2 • The reaction mixture is heated to 90-95oC for about thirty seconds. • At this temperature the DNA strands separate as the hydrogen bonds holding them together break down.

15. Polymerase Chain reaction - Stage 3 • The mixture is cooled down to 55-60oC. At this temperature the primers bind (or anneal) to the single DNA strands. • The primers are short sequences of nucleotide bases which must join to the beginning of the separated DNA strands for the full copying process to start.

16. Polymerase Chain reaction - Stage 4 • In the final step the mixture is heated up again to 75oC for at least a minute. • This is the optimum temperature for the DNA polymerase enzyme. • The enzyme adds bases to the primers segments to build up complementary strands of DNA identical to the original molecule.

17. PCR • These last three steps can be repeated around thirty times to give around 1 billion copies of the original DNA. • The whole process takes only about 3 hours – and much of that is the time taken heating and cooling the reaction mixture in the PCR machine

18. Summary of PCR • Denaturing of double-stranded DNA molecules to make single stranded • High temperature 95oC • Annealing primers to the ends of the single-stranded DNA molecules • 55-60oC • Building complete new DNA strands using DNA polymerase • 72oC

19. Summary of PCR

20. Summary of PCR

21. Summary of PCR

22. Questions • Describe and explain how to produce multiple copies of a DNA fragment using PCR • Describe and explain how electrophoresis works

23. Answers 1 • DNA is mixed with free nucleotides, primers and DNA polymerase • The mixture is heated to 95oC to break the hydrogen bonds • The mixture is then cooled to between 50-65oC to allow the primers to bind/anneal to the DNA • The primers bind/anneal to the DNA because they have a sequence that is complementary to the sequence at the start of the DNA fragment • The mixture is then heated to 72oC and DNA polymerase lines up free nucleotides along each template strand, producing new strands of DNA • The cycle would be repeated over and over to produce lots of copies

24. Answers 2 • Fluorescent tag is added to all the DNA fragments in the mixture so they can be viewed under UV light • The DNA mixture is placed into a well in a slab of gel ad covered in a buffer solution that conducts electricity • An electrical current is passed through the gel and the DNA fragments move towards the positive electrode because DNA fragments are negatively charged. • The DNA fragments separate according to size because the small fragments move faster and travel through the gel • The DNA fragments are viewed as bands under UV light