AP Biology Ch. 20

1. AP Biology Ch. 20 Slides for Test

2. DNA Cloning • Plasmids -used to insert foreign DNA • Recombinant plasmid is inserted into a bacteria • Reproduction in bacteria cell results in cloning of the plasmid including foreign gene • Useful for making copies of a gene and producing a protein product

3. Making recombinant DNA • Use bacterial restriction enzymes • Cuts DNA at specific restriction sites • Cuts covalent bonds between sugar phosphate backbone • Making restriction fragments • Most useful restriction enzymes cut DNA in a staggered way forming “sticky ends” • DNA ligase seals the bonds between restriction fragments. • Cloning vector is original plasmid carrying foreign gene into the host cell.

4. Storing Cloned Genes in DNA Libraries • A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome. • A genomic library that is made using bacteriophages is stored as a collection of phage clones. • A bacterial artificial chromosome (BAC)is a large plasmid that can carry a large insert with many genes.

5. Storing Cloned Genes in DNA Libraries • Complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell. • cDNA library represents only part of the genome--only the subset of genes transcribed into mRNA in th original cells

6. Fig. 20-6-3 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA

7. Fig. 20-6-4 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase

8. Fig. 20-6-5 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase cDNA

9. Screening a Library for Clones Carrying a Gene of Interest • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene • This process is called nucleic acid hybridization

10. A probe can be synthesized that is complementary to the gene of interest • For example, if the desired gene is – Then we would synthesize this probe … … 5 G G C T A A C T T A G C 3 C C G A T T G A A T C G 5 3

11. The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest • Once identified, the clone carrying the gene of interest can be cultured

12. Fig. 20-7 • TECHNIQUE Radioactivelylabeled probemolecules ProbeDNA Gene ofinterest Multiwell platesholding library clones Single-strandedDNA from cell Film Nylon membrane Nylonmembrane Location ofDNA with thecomplementarysequence

13. Detecting DNA sequence by hybridization with a nucleic acid probe • Results • The location of the black spot on the photographic film identifies the clone containing the gene of interest. • By using probes with different nucleotide sequences, researchers can screen the collection of bacterial clones for different genes.

14. Expressing Cloned Eukaryotic Genes • After gene has been cloned, its protein products can be produced in larger amounts for research. • Cloned genes can be expressed as protein in either bacterial or eukaryotic cells.

15. Problems with expressing eukaryotic genes in bacterial host cells • Scientists use an expression vector, a cloning vector that contains a highly active prokaryotic promoter • This allows the bacteria to recognize the promoter and proceed to express the foreign gene. • This allows synthesis of many eukaryotic proteins in bacteria cells. • Another problem is the presence of introns in eukaryotic genes. • Bacteria cells do not have RNA splicing machinery. • This can be overcome by using cDNA which includes only the exons.

16. Eukaryotic cloning and Expression Systems • The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems • YACs behave normally in mitosis and can carry more DNA than a plasmid • Eukaryotic hosts can provide the post-translational modifications that many proteins require

17. Amplifying DNA using Polymerase Chain reaction (PCR) • PCR can produce many copies of a specific target segment of DNA • Three-step cycle-heating--cooling--and replication • Brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

18. Fig. 20-8 3 5 TECHNIQUE Targetsequence 3 5 Genomic DNA 1 5 3 Denaturation 5 3 2 Annealing Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides Cycle 2yields 4 molecules Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

19. Fig. 20-8a 5 3 TECHNIQUE Targetsequence Genomic DNA 3 5

20. Fig. 20-8b 1 5 3 Denaturation 3 5 2 Annealing Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides

21. Fig. 20-8c Cycle 2yields 4 molecules

22. Fig. 20-8d Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

23. Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene • DNA cloning allows researchers to • Compare genes and alleles between individuals • Locate gene expression in a body • Determine the role of a gene in an organism • Several techniques are used to analyze the DNA of genes

24. Gel Electrophoresis and Southern Blotting • Gel electrophoresis uses a gel as a molecular sieve to separate nucleic acids by size • A current is applied to the gel that causes the charged molecules to move • DNA is negatively charged and it moves towards a positive pole. • Molecules are sorted into “bands” by their size

25. Fig. 20-9a TECHNIQUE Powersource Mixture ofDNA mol-ecules ofdifferentsizes Anode Cathode – + Gel 1 Powersource – + Longermolecules 2 Shortermolecules

26. Fig. 20-9b RESULTS

27. In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis • Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene • The procedure is also used to prepare pure samples of individual fragments

28. Fig. 20-10 Normal -globin allele Normalallele Sickle-cellallele 175 bp Large fragment 201 bp DdeI DdeI DdeI DdeI Largefragment Sickle-cell mutant -globin allele 376 bp 201 bp175 bp Large fragment 376 bp DdeI DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

29. A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization • Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

30. Fig. 20-11a TECHNIQUE Heavyweight Restrictionfragments I II III DNA + restriction enzyme Nitrocellulosemembrane (blot) Gel Sponge I Normal-globinallele II Sickle-cellallele III Heterozygote Papertowels Alkalinesolution 2 3 1 Preparation of restriction fragments Gel electrophoresis DNA transfer (blotting)

31. Fig. 20-11b Radioactively labeledprobe for -globin gene Probe base-pairswith fragments I II III I II III Fragment fromsickle-cell-globin allele Film overblot Fragment fromnormal -globin allele Nitrocellulose blot 5 4 Hybridization with radioactive probe Probe detection

32. Studying the Expression of Interacting Groups of Genes • Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays • DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions

33. Fig. 20-15 TECHNIQUE Tissue sample 1 Isolate mRNA. 2 Make cDNA by reversetranscription, usingfluorescently labelednucleotides. mRNA molecules Labeled cDNA molecules(single strands) DNA fragmentsrepresentingspecific genes 3 Apply the cDNA mixture to amicroarray, a different gene ineach spot. The cDNA hybridizeswith any complementary DNA onthe microarray. DNA microarray DNA microarraywith 2,400human genes 4 Rinse off excess cDNA; scanmicroarray for fluorescence.Each fluorescent spot represents agene expressed in the tissue sample.

34. Concept 20.3: Cloning organisms may lead to production of stem cells for research and other applications • Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell

35. Cloning Plants: Single-Cell Cultures • One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism • A totipotent cell is one that can generate a complete new organism

36. Fig. 20-16 EXPERIMENT RESULTS Transversesection ofcarrot root 2-mgfragments A singlesomaticcarrot celldevelopedinto a maturecarrot plant. Fragments werecultured in nu-trient medium;stirring causedsingle cells toshear off intothe liquid. Singlecellsfree insuspensionbegan todivide. Embryonicplant developedfrom a culturedsingle cell. Plantlet wascultured onagar medium. Later it wasplantedin soil.

37. Cloning Animals: Nuclear Transplantation • In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell • Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg • However, the older the donor nucleus, the lower the percentage of normally developing tadpoles

38. Fig. 20-17 Frog egg cell Frog embryo Frog tadpole EXPERIMENT UV Fully differ- entiated (intestinal) cell Less differ-entiated cell Donornucleustrans-planted Donor nucleus trans- planted Enucleated egg cell Egg with donor nucleus activated to begin development RESULTS Most develop into tadpoles Most stop developing before tadpole stage

39. Reproductive Cloning of Mammals • In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell • Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus

40. Fig. 20-18 TECHNIQUE Mammarycell donor Egg celldonor 2 1 Egg cellfrom ovary Nucleusremoved Cells fused 3 Culturedmammary cells 3 Nucleus frommammary cell Grown inculture 4 Early embryo Implantedin uterusof a thirdsheep 5 Surrogatemother Embryonicdevelopment 6 Lamb (“Dolly”)genetically identical tomammary cell donor RESULTS

41. Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs • CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”

42. Fig. 20-19

43. Problems Associated with Animal Cloning • In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth • Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development

44. Stem Cells of Animals • A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types • Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem cells; these are able to differentiate into all cell types • The adult body also has stem cells, which replace nonreproducing specialized cells

45. Fig. 20-20 Embryonic stem cells Adult stem cells From bone marrowin this example Early human embryoat blastocyst stage(mammalian equiva-lent of blastula) Cells generatingall embryoniccell types Cells generatingsome cell types Culturedstem cells Differentcultureconditions Differenttypes ofdifferentiatedcells Blood cells Nerve cells Liver cells

46. The aim of stem cell research is to supply cells for the repair of damaged or diseased organs

47. Concept 20.4: The practical applications of DNA technology affect our lives in many ways • Many fields benefit from DNA technology and genetic engineering One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases

48. Human Gene Therapy • Gene therapy is the alteration of an afflicted individual’s genes • Gene therapy holds great potential for treating disorders traceable to a single defective gene • Vectors are used for delivery of genes into specific types of cells, for example bone marrow • Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

49. Fig. 20-22 Clonedgene Insert RNA version of normal alleleinto retrovirus. 1 Viral RNA Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured. 2 Retroviruscapsid Viral DNA carrying the normalallele inserts into chromosome. 3 Bonemarrowcell frompatient Bonemarrow Inject engineeredcells into patient. 4

50. Pharmaceutical Products • Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases