1. Chapter 20 DNA Technology

2. DNA Cloning  Gene cloning allows scientists to work with small sections of DNA (single genes) in isolation. –Exactly what does the gene code for?  Much of a DNA molecule is noncoding, and scientists are mostly interested in the genes.  Cloning makes identical copies of the same gene (or genes)

3. Figure 20.1 An overview of how bacterial plasmids are used to clone genes

4. Bacterial Plasmids  Plasmids are small, circular DNA molecules in bacteria.  By inserting genes into plasmids, scientists can combine eukaryotic and prokaryotic DNA. (Recombinant DNA)  Bacterial cells continually replicate the foreign gene along with their DNA.  Cloning using plasmids can be used to: –Identify a particular protein a gene makes (ie: for study) –Produce large amounts of a particular protein/gene (ie: for use in medicine)

5. Restriction Enzymes  Also used to make recombinan t DNA.  Specifically cut DNA molecules at precise base locations. (restriction)

6. Making Recombinant DNA (Fig 20.3)

7. …Still Making Recombinant DNA

8. …Almost Recombinant

9. DNA Technology Files Restriction Enzyme and Cloning Movies Restriction Enzyme and Cloning Movies

10. Why Use Bacteria as vectors? 1.Plasmids are easy to use to manipulate which genes are expressed in clones. 2. Bacteria replicate very quickly and allow you to produce a large number of a desired gene.

11. Identifying Clones  Not all of the reproduced bacteria are clones carrying the desired gene.  Two ways to identify which are clones: –Look for the gene –Look for the protein the gene codes for

12. Nucleic Acid Hybridization (find gene)  If you know the sequence of the cloned gene you are looking for, you can make a nucleic acid probe with a complementary sequence.  The probe is radioactively labeled and allowed to base pair with the denatured (separated strands) DNA.  The probes H-bond with their complement (cloned gene), thus identifying the cloned cells.  Identified cells are cultured to produce more.

13. Figure 20.4 Using a nucleic acid probe to identify a cloned gene

14. Expressing Euk. Proteins in Bacteria  It is more difficult to get the bacteria to translate the proteins because of differences in promotor sequences b/t prokaryotes and eukaryotes.  Expression vectors are plasmids that contain the promotor sequence just before the restriction site.  This allows the insertion of a eukaryotic gene right next to the prokaryotic promotor.

15. Expressing Euk. Proteins in Bacteria  Bacteria also lack the enzymes needed to remove introns from DNA.  Therefore, cDNA (no introns) is inserted into plasmids to allow expression of the eukaryotic gene.  Reverse transcriptase is the enzyme used to make cDNA from a fully processed mRNA strand.

16. Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene

17. Another Solution: Use Yeast (eukaryotic)  Why? –They grow quickly like bacteria –They are eukaryotes (similar enzymes, metabolic mechanisms, protein mods) –They have plasmids (rare for eukaryotes) –Can replicate artificial chromosomes as well as DNA in plasmids

18. Genomic Libraries  Plasmids and phages used to store copies of specific genes.

19. Polymerase Chain Reaction (PCR)

20. PCR  Faster and more specific method for amplifying short DNA sequences  After DNA is denatured (split), primers start new complementary strands with each strand producing more molecules of the sequence.  In vitro = doesn’t require living cells –In test tube: denatured DNA, free nucleotides, DNA primers (specific to gene desired), “special” DNA polymerase (can withstand high heat w/o denaturing)

21. PCR

22. Analyzing DNA  Gel electrophoresis separates molecules based on size, charge, density, etc.  Linear DNA – mainly separated by fragment length (size)  Molecules of DNA are separated into bands of molecules of the same length.

23. Gel Electrophoresis

24. Restriction Fragment Analysis

25. Southern Blotting

26. 1.Produce restriction fragments of DNA (rest. enzyme used) 2.Separate fragments (gel electrophoresis) 3.Blotting  Transfer DNA to nitrocellulose paper via cap. action 4.Hybridize with radioactive probes (know seq.) 5.Autoradiography to identify which have probes.

27. RFLPs (rif-lips)  Polymorphisms that result from differences in noncoding regions of DNA.  Restriction enzymes cut DNA into different fragments in each variant.  RFLP markers allowed scientists to more accurately map the human genome.  Genetic studies do not have to rely on phenotypic (appearance/proteins) differences to guide them anymore.

28. In Situ (on a slide) Hybridization  Radioactively (or fluorescently) labeled probes base pair with complementary denatured DNA on a microscope slide.  Autoradiography and staining identify the location of the bound probe.

29. Human Genome Project  Attempt to map the genes on every human chromosome as well as noncoding information.  Three stages –Genetic Mapping (linkage) –Physical Mapping –Gene (DNA) Sequencing  Genomes of species that give insight to human codes are also being done (fruit fly, E coli, yeast)

30. Genetic Mapping (Stage 1)  Linkage maps based on recombination frequencies created.  Linkage maps portray gene sequences as you physically move along a chromosome.  Genetic markers along the chromosome allow researchers to use them as reference points while studying other genes.

31. Physical Mapping (Stage 2)  Determines the actual distance between the markers along a chromosome (# of bases)  Utilizes chromosome walking to identify the distance between. –Use a series of probes to identify the DNA sequence of various restriction fragments, and ultimately the entire length of DNA sample.

32. Chromosome Walking

33. DNA Sequencing (Stage 3)  As of 1998, 3% of the human genome had been sequenced using automation. (Sanger Method)  Once the sequences of all the genes are known, scientists can begin to study all of their functions, and manipulate their products in many ways.

34. Applied Genetics  Diagnosis of Genetic Disorders –Sequence individuals before birth to know if their DNA contains abnormalities  Human Gene Therapy –Replace missing or fix damaged genes in affected individuals

35. Gene Therapy

36. Pharmaceuticals  Hormone production (ie: Human Growth)  Protein supplements –HIV treatment: “decoy” receptor protein used to inhibit HIV virus’ ability to enter cell  Vaccines –Proteins that stimulate immune response can be used instead of traditional vaccines  Antisense Nucleic Acids –Block translation of certain proteins

37. Other Uses of DNA Tech  DNA Fingerprinting for forensic cases  Environmental cleanup  Agriculture –Animal Husbandry –Genetic Engineering of Plants

38. The Future of Genetics  The future of science lies in genetics.  The question is not whether or not we can do the things discussed in this chapter, but whether or not we should. This is a question you will ultimately have to help answer.