1. 13-1 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Chapter 13: Genetic engineering and biotechnology

2. 13-2 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Restriction enzyme mapping Restriction endonucleases cut double-stranded DNA at defined sequences Each restriction enzyme cuts a particular palindromic sequence The enzymes have been isolated from bacteria which use them to inactivate foreign DNA Identical DNA molecules will be cut into fragments of the same length based on the position of the endonuclease recognition sites on the molecule (cont.)

3. 13-3 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.1b: Restriction endonucleases

4. 13-4 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Restriction enzyme mapping (cont.) Cutting identical molecules with different enzymes produces a different pattern of fragments The patterns will overlap—cutting with two enzymes together produces a greater number of smaller fragments which are equivalent in total length to either enzyme alone This allows the relative positions of the DNA recognition sequences to be mapped (cont.)

5. 13-5 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Restriction enzyme mapping (cont.) Fragments are separated by size using gel electrophoresis The electric current causes fragment migration through the gel, with small fragments moving faster than large fragments

6. 13-6 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.2: Electrophoretic separation of fragments

7. 13-7 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Recombinant DNA molecules Restriction enzymes cut at defined sites regardless of the origin of the molecule DNA from different sources can be joined to form a recombinant molecule as long as the same restriction enzyme was used to cut each molecule Some enzymes produce staggered cuts in which short single-stranded regions protrude The molecules adhere at these sites and are ligated together by DNA ligase

8. 13-8 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.3: Ligation of DNA fragments

9. 13-9 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint DNA vectors Production of multiple copies of the DNA fragment requires ligation into a self-replicating vector molecule –plasmids –bacteriophage –cosmids –YACs (yeast artificial chromosomes) and –BACs (bacterial artificial chromosomes) Replication of the recombinant vector occurs in the appropriate bacterial or yeast host (cont.)

10. 13-10 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.4: Cloning a human gene (top)

11. 13-11 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.4: Cloning a human gene (bottom)

12. 13-12 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint DNA vectors (cont.) Regardless of their size or origin vector molecules must have the following –an origin of replication –at least one unique restriction site for insertion of DNA fragment –a gene for an inducible character, such as antibiotic resistance, to ensure efficient replication in the host organism –a means of distinguishing between vector alone and recombinant vector molecules

13. 13-13 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.5a: Plasmid DNA vector

14. 13-14 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.5b: Selecting cells

15. 13-15 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.5c: Distinguishing cells

16. 13-16 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Genomic DNA libraries Entire genomes are fragmented and ligated into a vector Millions of resulting colonies or plaques are produced, each one of which contains a piece of the genome If the library is large enough each fragment of genome should be present at least once

17. 13-17 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.6: Constructing a human genomic library (top)

18. 13-18 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.6: Constructing a human genomic library (bottom)

19. 13-19 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint cDNA libraries Genomic DNA libraries contain all DNA sequences cDNA libraries contain only those coding sequences present in transcribed genes mRNA molecules are copied by reverse transcriptase into complementary cDNA cDNA molecules are ligated into vectors and a library constructed Each clone is derived from a gene being expressed at the time of the mRNA isolation

20. 13-20 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.7: Constructing a library of cDNA

21. 13-21 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Identifying cloned sequences Hybridisation –colonies or plaques grown on plates –recombinant DNA in the colonies is denatured –a replica of the plate is made on a membrane filter and the adherent cells lysed to reveal their DNA –a labelled, single-stranded probe to the gene of interest is hybridised to complementary sequences on the membrane –the original colony or plaque can be recovered from the plate and used in further analysis

22. 13-22 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Isolating genes by PCR Polymerase chain reaction (PCR) allows the amplification of specific sequences without the need for cells –amplification is selective and repeated, using heat-stable DNA polymerase and deoxynucleotide triphosphates –specificity is determined by the use of oligonucleotide primers to known sequences flanking the fragment of interest –each cycle of annealing and extension doubles the fragment copy number

23. 13-23 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.10: PCR (top)

24. 13-24 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.10: PCR (bottom)

25. 13-25 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint DNA (and RNA) blotting Called Southern blotting after its inventor Edwin Southern –DNA isolated and cut into different sized fragments –fragments separated physically by size using gel electrophoresis –separated fragments are denatured and transferred to a membrane filter –radiolabelled single-strand probe is bound to the fragment of interest, making it visible A similar technique is used to identify mRNA molecules

26. 13-26 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.12a: Sequence determination of a short DNA fragment

27. 13-27 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.12b: Sequence determination of a short DNA fragment

28. 13-28 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.12c: Sequence determination of a short DNA fragment

29. 13-29 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Nucleotide sequence analysis The base sequence of DNA can be determined in vitro by DNA synthesis and electrophoresis –each synthesis reaction contains normal deoxynucleoside triphosphates and a chain-terminating dideoxynucleoside triphosphate (ddNTP) –four reactions are employed, each containing a different ddNTP to stop the reaction –a series of fragments is generated with different lengths but each terminating in the same nucleotide (the ddNTP) –each reaction is labelled with a different colour and the sequence read as a series of fluorescent bands

30. 13-30 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.13: Southern (DNA) blotting

31. 13-31 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Analysing genetic variation Base changes in a gene result in restriction fragment length polymorphisms (RFLPs) The consistent presence of a particular RFLP in people with the disease being investigated is strong evidence of the mutation causing the disease—also permits localisation of the gene in which the mutation has occurred RFLPs can be distinguished by Southern hybridisation or by PCR

32. 13-32 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint DNA in forensic science Developed as a way of defining specific differences in DNA sequences between people –differences must be extensive and detailed enough to minimise risk of accidental identity –gene sequences are not used for this –microsatellites and minisatellites: regions of repeat- sequence DNA, where short sequences (2–5 nucleotides) may be repeated many times –VNTRs (variable number tandem repeats) are similar. They vary in number between individuals, so looking at several VNTRs at once provides a unique ‘fingerprint’ of sequence lengths for that person

33. 13-33 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.18: Find the murderer!

34. 13-34 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Mapping genes Classical gene linkage analysis has limitations, especially in mammals DNA sequence polymorphisms can be used as landmarks to detect recombination in offspring of heterozygous parents Association of linkage markers with disease alleles is important in the location and isolation of the disease gene The physical location on a chromosome of a gene can be found using a labelled probe from a cloned sequence (see Fig. 13.20)

35. 13-35 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.20: Mapping genes to chromosomes by FISH (fluorescence in situ hybridisation) (a) (b)

36. 13-36 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Biotechnology Recombinant protein production –gene products such as drugs, hormones and enzymes can be produced in large quantities in cell culture systems Modifying agricultural organisms –inserting genes for improved yield or pest resistance into plants –cloning domestic animals chosen for their superior qualities (cont.)

37. 13-37 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.23: Animal cloning (top)

38. 13-38 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.23: Animal cloning (bottom)

39. 13-39 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Biotechnology (cont.) Gene therapy –the introduction of a modified gene into the cells of a patient suffering a genetic disease to correct the abnormality –still experimental –problems associated with directing the vector to the target cells and maintaining expression Cell therapy –the use of multipotent stem cells which can be induced to differentiate in vitro –introduced into patient to replace absent or damaged cells

40. 13-40 Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint Fig. 13.24: Cell therapy