Manipulating DNA: tools and techniques

1. Manipulating DNA: tools and techniques Chapter 12 • Key Knowledge: • tools and techniques: gel electrophoresis; DNA profiling; DNA sequencing; DNA recombination; DNA amplification; gene cloning, gene transformation; gene delivery systems;

2. Mitochondrial DNA (mtDNA) • Inherited maternally • Behaves like prokaryotic DNA • Haploid • Circular • More than one copy in the mitochondria • Useful in family studies • mtDNA D-Loop extremely variable, accumulates many different mutations because it is not transcribed

4. Mitochondrial DNA (mtDNA) • see Pages 419 – 421 of textbook “Murders at Ekaterinberg”.

5. Tools for Genetic Engineering

6. Restriction Enzymes The molecular biology revolution started with the discovery of restriction enzymes (restriction endonucleases) Enzymes cleave DNA at specific sites These enzymes are significant in two ways • Allow a form of physical mapping that was previously impossible • Allow the creation of recombinant DNA molecules (from two different sources)

7. Restriction Enzymes • Restriction enzymes cut at specific sites • Some form ‘blunt ends’ (straight cut) others form ‘sticky ends’ (staggered cut). e.g. EcoRI

9. Gel Electrophoresis • A technique used to separate DNA fragments by size • The gel (agarose or polyacrylamide) is subjected to an electrical field • The DNA, which is negatively-charged, migrates towards the positive pole • The larger the DNA fragment, the slower it will move through the gel matrix • DNA is visualised using fluorescent dyes.

10. Dye added to the DNA

11. Buffer solution added to the tank

12. DNA samples loaded into wells

13. Electrical current applied to the chamber

14. DNA is stained using ethidium bromide

15. Probes • A probe is used in order to find a specific sequence of DNA within a relatively large sample. • Probes are usually labelled with a marker (e.g.radioactive, fluorescent, etc.) and are complementary to the target sequence. • See Figure 12.9 in your textbook (pg 424).

17. Ligase • The enzyme Ligase is required to catalyse the joining of pieces of dsDNA at their sugar-phosphate backbone.

19. Transporting DNA into cells • DNA can be transported into cells through the use of vectors. • Vectors are cellular agents that have the capacity to carry DNA and transport it into target cells. • Bacteria are commonly used as vectors. • Bacteria have a small circular piece of DNA called a plasmid.

20. Steps to Transporting DNA into a Target Cell • DNA of the plasmid is cut using a restriction enzyme (the same restriction enzyme that the original DNA is cut with). • The plasmid and the foreign DNA are mixed and their ‘sticky ends’ pair • DNA ligase makes the joins permanent. • The plasmids that contain the recombinant DNA plasmid are then selected.

21. Steps to Transporting DNA into a Target Cell

22. Gene Isolation • It is easy to isolate the total DNA from human somatic cells • 46 chromosomes • 6 x 109 base pairs of DNA • 20,00 – 25,000 genes • Sequences of non-coding DNA

23. Gene Isolation • Harder to isolate one gene or part of a gene

24. Gene Isolation • Locate particular DNA fragments following separation by electrophoresis using a probe with a complementary base sequence

25. Gene Isolation • Synthesis DNA from nucleotides: Use a DNA synthesiser to artificially manufacture a specific DNA sequence with lengths greater than 50 bases to be used as primers or probes

27. Gene Isolation • Make a copy of DNA using a mRNA template: mRNA is isolated from specific cells and the enzyme Reverse Transcriptase is used to a make a complementary ssDNA strand. This is called copy DNA (cDNA). • DNA Polymerase may be used later to convert the cDNA into dsDNA. • This method only works for coding DNA: genes that produce mRNA

29. Gene Cloning Making multiple copies of a gene • The gene is copied and placed into a bacterial plasmid • The plasmid is inserted into the bacterial host cell • Inside bacterial host cell the plasmid and the gene make twenty copies of itself • The bacterial cell copies itself every twenty minutes by binary fission • Within several hours there are millions of copies of the bacterial host cell and the inserted gene. • In some case the gene is switched on and the product (protein) is harvested.

30. Polymerase Chain Reaction (PCR) • Polymerase chain reaction enables large amounts of DNA to be produced from very small samples. • There is a repeating cycle of: • Separationof double DNA strands • Annealing of primers to the sequence to be amplified. • Extension of DNA using the original DNA strand as a template.

31. The Reaction PCR tube THERMOCYCLER

32. Separation (heat to 95oC) Lower temperature to 56oC - Anneal with primers Increase temperature to 72oC - DNA polymerase + dNTPs = extension of DNA

34. Gene Transformation • First identified by Griffith in 1928 • Defined as the take up of naked DNA by cells • Occurs naturally in bacteria, yeast and some plants • May be induced • Mix bacteria with CaCl2 solution • Place in ice (0°C) • Then place at 37°C • Cells are now competent

37. Gene Delivery Systems • Gene Delivery is a process of inserting foreign DNA into host cells • There are many different processes • Viral • Non-viral • It is the key to Gene Therapy

38. Gene Delivery Systems – Viral Viral vectors • They are good at targeting and entering cells • Some can be engineered to target specific types of cells • They can be modified, so they do not replicate and destroy the cell

39. Gene Delivery Systems – Non Viral • Plasmids • microinjection • gene gun • impalefection • hydrostatic pressure • electroporation • continuous infusion • sonication • lipofection