3.5 part 1
- 1. Part 1 3.5: Genetic Modification and Biotechnology
- 2. Understanding s Gel electrophoresis is used to separate proteins or fragments of DNA according to size. PCR can be used to amplify small amounts of DNA. DNA profiling involves comparison of DNA. Genetic modification is carried out by gene transfer between species. Clones are groups of genetically identical organisms derived from a single original parent cell. Many plant species and some animal species have natural methods of cloning. Animals can be cloned at the embryo stage by breaking up the embryo into more than one group of cells. Methods have been developed for cloning adult animals using differentiated cells.
- 3. Applications/Skill s Guidanc e A: Use of DNA profiling in paternity and forensic investigations. A: Gene transfer in bacteria using plasmids makes use of restriction endonucleases and DNA ligase. A: Assessment of the potential risks/benefits associated with greater genetic modification of crops. A: Production of cloned embryos produced by somatic cell nuclear transfer. S: Design of an experiment to assess one factor affecting the rooting of stem cuttings. S: Analysis of examples of DNA profiles. S: Analysis of data on risk to monarch butterflies of Bt crops. Students should be able to deduce whether or not a man could be the father of a child from the pattern of bands on a DNA profile. Dolly can be used as an example of somatic-cell transfer. A plant species should be chosen
- 4. Uses for PCR Using the technique called polymerase chain reaction (PCR), researchers are able to create vast quantities of DNA identical to trace samples. This process is also known as DNA amplification. Many procedures in DNA technology require substantial amounts of DNA to work with, for example; DNA sequencing DNA profiling/fingerprinting Gene cloning Making artificial genes Sometimes DNA samples can be hard to obtain: A crime scene (body tissue samples) A single viral particle (from an infection) Fragments of DNA from a long extinct animal
- 5. Steps in the PCR Process The laboratory process called the polymerase chain reaction or PCR involves the following steps 1- 3 each cycle: Separate Strands Separate the target DNA strands by heating at 98°C for 5 minutes Add Reaction Mix Add primers (short RNA strands that provide a starting sequence for DNA replication), nucleotides (A, T, G and C) and DNA polymerase enzyme. Incubate Cool to 60°C and incubate for a few minutes. During this time, primers attach to single-stranded DNA. DNA polymerase synthesises complementary strands. Repeat for about 25 cycles Repeat cycle of heating and cooling until enough copies of the target DNA have been produced.
- 6. Although only three cycles of replication are shown here, following cycles replicate DNA at an exponential rate and can make literally billions of copies in only a few hours. The process of PCR is detailed in the following slide sequence of steps 1-5. Polymerase Chain Reaction PCR cycle s No. of target DNA strands 1 2 2 4 3 8 4 16 5 32 6 64 7 128 8 256 9 512 10 1024 11 2048 12 4096 13 8192 14 16 384 15 32 768 16 65 536 17 131 072 18 262 144 19 524 288 20 1 048 576 21 2 097 152 22 4 194 304 23 8 388 608 24 16 777 216 25 33 554 432 Cycle 1 Cycle 2 Cycle 3 Original DNA Sample
- 7. Primer annealed A DNA sample called the target DNA is obtained DNA is denatured (DNA strands are separated) by heating the sample for 5 minutes at 98C Primers (short strands of mRNA) are annealed (bonded) to the DNA
- 8. Nucleotides Nucleotides After one cycle, there are now two copies of the original sample. The sample is cooled to 60°C. A thermally stable DNA polymerase enzyme binds to the primers on each side of the exposed DNA strand. This enzyme synthesises a complementary strand of DNA using free nucleotides. Media showcase animation McGraw-Hill PCR animation PCR song PCR song 2
- 9. Testing for GMOs using PCR PCR primers do not bind unless there is a complementary sequence of nucleotides ◦ A = T ◦ T = A ◦ G ≡ C ◦ G ≡ C One test for GM ingredients in food involves a primer that will only bind to the GM DNA. If GM DNA is present, the PCR process will amplify the DNA If no GM DNA is present, the PCR has no effect
- 10. Gel Electrophoresis Gel electrophoresis can be used to separate large molecules (including nucleic acids or proteins) on the basis of their size, electric charge, and other physical properties. DNA is split into fragments using restriction enzymes The DNA samples are placed in wells and covered with a buffer solution that gradually dissolves them into solution. Wells Buffer solution Cathode Anode Gel Plastic frame Buffer Sample
- 11. DNA has a negative charge because the phosphate groups are negatively charged. The DNA fragments in the gel move through the gel towards the positive terminal of the electric field. Smaller molecules move at a faster rate through the gel; longer fragments take longer to work through the small spaces in the gel. Groups of DNA fragments can be seen as bands on the gel – usually seen with the help of a dye. negative terminal positive terminal Small fragments Large fragments Tray: Contains the set gel. DNA solutions: Mixtures of different sizes of DNA fragments are loaded into each well. DNA markers: A mixture of DNA molecules with known molecular weights. They are used to estimate the sizes of the DNA fragments in the sample lanes. DNA fragments: The gel matrix acts as a sieve for the DNA molecules. Wells: Holes created in the gel with a comb. Gel electrophoresis demo lab
- 12. DNA profiling (DNA fingerprinting) is a technique for genetic analysis, which identifies the variations found in the DNA of every individual. The profile refers to the distinctive pattern of fragments which is used to identify an individual. DNA profiling does not determine a base sequence for a sample but merely sorts variations in base sequences. Only one in a billion (i.e. a thousand million) persons is likely to have an identical DNA profile, making it a useful tool for forensic investigations and paternity analysis
- 13. the presence of a particular gene, (such as cystic fibrosis) in a family. genetic relatedness of different organisms e.g. checking on pedigree in stock breeding programs. e.g. checking that captive populations of endangered species are not inbred. DNA profiling can be used for investigating:
- 14. DNA fingerprints from tissue samples can be used as evidence in the same way traditional fingerprinting is used. Which DNA fingerprint from the three suspects matches that of the tissue sample submitted as evidence? Why would the DNA from the victim be included in this test? The DNA from the victim must be excluded from the evidence. In a paternity test, the DNA from the mother must also be included to exclude her contribution to the banding patterns in the child’s profile. DNA profiling can be used for forensic purposes:
- 15. We expect 100% match as the cells left behind at the scene are the perpetrator’s cells
- 16. We expect 100% match as the cells left behind at the scene are the perpetrator’s cells The overlapping bands between victim and suspect indicate a close genetic relationship
- 17. No. Without a stronger match, the evidence is insufficient to convict the suspect. He should be released and a new suspect found. DNA evidence is being reviewed in many wrongful convictions.
- 18. Because the child inherits half its genetic material from each parent, any band that the child has not inherited from his mother, he must have inherited from his father. In this case, determine which man (1 or 2) is the biological father of the child.
- 19. Which male (1 or 2) is the father of the child? Explain.
- 20. Which suspect(1,2 or 3) was present at the crime scene? Explain.
- 21. “DNA is better at proving innocence than guilt.” Discuss this statement
- 22. TOK How do you think a child would feel if he/she were to find out from DNA profiling that her father was not her biological father? How would a man feel if he found out he was not his child’s father? What effect would such a result have on the relationships between siblings or between spouses? What kind of emotions might someone feel after spending 18 years in prison and then being freed thanks to DNA testing?