DNA Technology and Genomics

1. DNA Technology and Genomics

2. What is biotechnology? • Manipulation of organisms or their components to make useful products • Which of these are “biotechnology”? • Use of microbes to make red wine or cheese • Selective breeding of livestock • Modify specific genes and move them between organisms

3. KWL Write either something you know or something you want to know about biotechnology on your sticky notes (with your initials) and place them on the board Want to Know • Know

4. How does DNA control the cell??

5. You Must Know • The terminology of biotechnology • The steps in gene cloning with special attention to the biotechnology tools that make cloning possible • The key ideas that make PCR possible • How gel electrophoresis can be used to separate DNA fragments or protein molecules

6. Key Questions • What is biotechnology (or DNA technology), and what are some practical applications that affect our lives? • How are restriction enzymes used to create recombinant DNA? • How are genes cloned? (hint: there are generally 5 steps) • What is PCR, what is it used for, and what are the steps of the process? • What is gel electrophoresis, what is it used for, and what are the steps of the process? • What are microarrays and what are they used for? • How is DNA technology used in diagnosis of disease, gene therapy, pharmaceuticals, forensics, environmental work, and agriculture? • What important safety and ethical questions does DNA technology raise?

7. Commonly Used Terms • Genetic engineering – the process of manipulating genes and genomes • Biotechnology – the process of manipulating organisms or their components for the purpose of making useful products • Recombinant DNA – DNA that has been artificially made, using DNA from different sources and often different species. Example: introducing a human gene (like the insulin gene) into an E. coli bacterium • Gene cloning – the process by which scientists can produce multiple copies of specific segments of DNA that they can then work with in the lab

8. What is biotechnology? • Manipulation of organisms or their components to make useful products • Modify specific genes and move them between organisms • We need to be able to cut out specific genes and reinsert them somewhere else!

9. Restriction Enzymes • Restriction enzymes – used to cut strands of DNA at specific locations called restriction sites; derived from bacteria • When DNA is cut by restriction enzymes, the result will be a set of restriction fragments which will have at least one single stranded end called a sticky end • Sticky ends can form H-bonds with complementary single-stranded pieces of DNA which are sealed with DNA ligase

10. What is biotechnology? • Manipulation of organisms or their components to make useful products • Modify specific genes and move them between organisms • Why would we want to modify, move, and clone genes? • Engineer bacteria to produce medicine or clean oil spills; investigate the function of a gene (super important in cancer research!); identify criminal at crime scene; etc

11. Cloning Genes Generally occurs in 5 steps • Isolate the vector (plasmid) and gene-source DNA (ex: human gene). The plasmid should include a gene for antibiotic resistance (for example, ampicillin resistance denoted ampR).

12. Cloning Genes • Insert the DNA (human gene) into the vector. • Digest plasmid and human DNA with same restriction enzyme (this creates compatible sticky ends on both the human DNA and the plasmid) • Mix the fragments of human DNA with the clipped plasmids; the sticky ends of a plasmid base-pair with the sticky ends of the human DNA fragment • Join the DNA molecules using DNA ligase

13. Cloning Genes • Introduce the cloning vector (recombinant plasmid) into the cells. Plasmids are taken up by bacteria by transformation

14. Cloning Genes • Clone the cells (and foreign genes) and select for cells that have been transformed. • Transformed bacteria are plated on solid nutrient medium containing the antibiotic for which the plasmid confers resistance (for example, ampicillin) so that only bacteria that have been transformed (those that have taken up the plasmid with ampR) can survive and reproduce. • A reporter gene, such as green fluorescent protein, may also be linked to the plasmid to help identify colonies of transformed bacteria. • Each reproducing bacterium forms a colony and in the process, the human genes are also cloned.

15. Cloning Genes • Identification of cell clones carrying the gene of interest (human gene). The gene itself or the protein it produces can be identified. • Nucleic acid hybridization • Nucleic acid probe

16. Cloning Genes • Identification of cell clones carrying the gene of interest (human gene). • Nucleic acid hybridization – the method for detecting the DNA of a gene directly using base pairing between the gene and a complementary sequence n another nucleic acid molecule • Nucleic acid probe – the short, single stranded molecule of DNA or RNA that is complementary to a portion of the gene; making the probe radioactive or fluorescent makes the gene easier to track

17. Check for Understanding Place the events involved in gene cloning in the correct order A. Add DNA ligase to bond sticky ends B. Isolate plasmid (vector) DNA and human DNA C. Plate cells onto medium with antibiotic D. Put plasmids into bacteria by transformation E. Mix DNAs (plasmid and human gene) F. Identify clone/colony containing gene of interest G. Cut both DNAs with same restriction enzyme

18. Check for Understanding The correct order is… B. Isolate plasmid (vector) DNA and human DNA G. Cut both DNAs with same restriction enzyme E. Mix DNAs (plasmid and human gene) A. Add DNA ligase to bond sticky ends D. Put plasmids into bacteria by transformation C. Plate cells onto medium with antibiotic F. Identify clone/colony containing gene of interest

19. Key Questions Working with your partner, discuss and write down answers to the following questions: • What is biotechnology (or DNA technology), and what are some practical applications that affect our lives? • How are restriction enzymes used to create recombinant DNA? • How are genes cloned? (hint: there are generally 5 steps)

20. E. Coli Insulin Factory • In this activity we will simulate the process of inserting a human gene (insulin) into a bacterial plasmid which can then be taken up by a bacteria, causing the bacteria to produce the human gene product

21. Key Questions • What is biotechnology (or DNA technology), and what are some practical applications that affect our lives? • How are restriction enzymes used to create recombinant DNA? • How are genes cloned? (hint: there are generally 5 steps) • What is PCR, what is it used for, and what are the steps of the process? • What is gel electrophoresis, what is it used for, and what are the steps of the process? • What are microarrays and what are they used for? • How is DNA technology used in diagnosis of disease, gene therapy, pharmaceuticals, forensics, environmental work, and agriculture? • What important safety and ethical questions does DNA technology raise?

22. PCR • PCR (or polymerasechainreaction) is a method used to greatly amplify a particular piece of DNA in vitro without the use of cells • PCR is used when the source of DNA is scanty or impure • crime scene • ancient artifacts

23. PCR • Starting material • double stranded DNA containing sequence that is “targeted” for copying • heat-resistant DNA polymerase • supply of all 4 nucleotides • supply of primers specific to target sequences

24. PCR • Heat solution briefly to separate DNA strands

25. PCR • Cool to allow specific primers to H-bond

26. PCR • DNA polymerase adds nucleotides to 3’ of each primer

27. Repeat – solution is again heated, cooled to allow primer binding, and DNA polymerase adds nucleotides to 3’ of each primer…

28. PCR

29. Key Questions Working with your partner, discuss and write down answers to the following question: • What is PCR, what is it used for, and what are the steps of the process?

30. 20.2 – DNA technology allows us to study the sequence, expression, and function of a gene • Genomics – the study of whole sets of genes and their interactions • Gel electrophoresis • Southern blotting • DNA microarray assays

31. Gel Electrophoresis • Gel electrophoresis – a lab technique that is used to separate macromolecules, primarily DNA and proteins, on the basis of their size and charge with the use of an electric current

32. Gel Electrophoresis • In separating DNA, the negative charges on the phosphates in the molecule cause DNA to move toward the positive pole of the electrophoresis set-up • The gel allows smaller molecules to move more easily than larger fragments of DNA, thus the fragments are separated by size

33. Gel Electrophoresis • Samples containing a mixture of DNA molecules of different sizes are placed in wells near one end of the gel; electrodes are attached to both ends and voltage is applied • The negatively charged DNA molecules migrate toward the positive electrode (the anode); longer molecules travel slower, shorter molecules travel more quickly and thus farther • When the current is turned off, the DNA molecules are arrayed in “bands” along a “lane” according to their size

34. Southern Blotting • Southern blotting – combines gel electrophoresis and nucleic acid hybridization to allow researchers to find a specific human gene; specific enough to differentiate between alleles • Ex: southern blotting can distinguish between a normal hemoglobin allele and a sickle-cell allele

35. Southern Blotting

36. DNA Microarray Assays • DNA microarray assays – used to detect and study the expression of thousands of genes at one time • ex – used to see differences in gene expression of a breast cancer tumor cell and a noncancerous breast cell

37. DNA Microarray Assays • Small amounts of single-stranded DNA fragments representing different genes are fixed to a glass slide in a tight grid which is termed a DNA chip

38. DNA Microarray Assays • All mRNA molecules from the cell being tested are isolated and converted to cDNA by reverse transcriptase, then tagged with a fluorescent dye • The cDNA bonds to the ssDNA (single-stranded DNA) on the chip, indicating which genes are “on” in the cell (actively producing mRNA); the dye alerts the researcher

40. DNA Microarray Assays 2 1 3

41. 20.3 – The practical applications of DNA technology affect our lives in many ways For the following applications, give at least one example of how the technology is being used. • Diagnosis of disease • Gene therapy • Pharmaceuticals • Forensic applications • Environmental cleanup • Agricultural applications Safety and Ethical Concerns • List at least 3 major safety measures and concerns and at least 3 major ethical questions that arise from use of DNA technology.