Chapter 15 Studying and Manipulating Genes

1. Chapter 15Studying and Manipulating Genes

2. 15.1 Personal DNA Testing • About 99% of your DNA is the same as everyone else’s • A nucleotide difference found in at least 1% of a population is called a single-nucleotide polymorphism (SNP) • SNPs account for many differences in the way humans look, and in the way our bodies work

3. Personal DNA Testing • About 4.5 million SNPs in human DNA have been identified • Using commercial SNP chips, individuals can now be tested for some of the SNPs they carry • Personal genetic testing may soon allow physicians to customize treatments based on individual genetic makeup

4. Personal DNA Testing: SNP Chips

6. But how do you MAKE one of these?

9. CFU START

10. 15.2 Cloning DNA • Researchers cut up DNA from different sources, then paste the resulting fragments together • Cloning vectors can carry foreign DNA into host cells

11. Cutting and Pasting DNA • Restriction enzymes • Bacterial enzymes that cut DNA • Recombinant DNA • Composed of DNA from two or more organisms

12. DNA ligase (paste) mix restriction enzyme (cut) A restriction enzyme recognizes a specific base sequence (orange boxes) in DNA from any source. When the DNA fragments from the two sources are mixed together, matching sticky ends base-pair with each other. DNA ligase joins the base-paired DNA fragments. Molecules of recombinant DNA are the result. 1 3 4 The enzyme cuts DNA from two sources into fragments. This enzyme leaves sticky ends. 2 Making Recombinant DNA Stepped Art

13. DNA Cloning • Making recombinant DNA is the first step in DNA cloning • DNA cut is inserted into cloning vectors(plasmids) cut with the same enzyme • Cloning vectors with foreign DNA are placed in host cells clones

14. Kpn l Sph l Pst l Bam Hl Eco RI Sal l Acc l Xho l Xba l Bst XI Sac l lac P lacZ Kanamycin pDrive Cloning Vector 3.85 kb Ampicillin Not l Figure 15-3 p236

15. Figure 15-3a p236

16. A A restriction enzyme cuts a specific base sequence in chromosomal DNA and in a plasmid cloning vector. chromosomal DNA fragments chromosomal DNA B A fragment of chromosomal DNA and the plasmid base-pair at their sticky ends. DNA ligase joins the two pieces of DNA. + recombinant plasmid plasmid cloning vector cut plasmid C The recombinant plasmid is inserted into a host cell. When the cell multiplies, it makes multiple copies of the plasmids. DNA Cloning Stepped Art

17. cDNA Cloning mRNA mRNA cDNA DNA cDNA EcoRI recognition site

18. 15.3 Isolating Genes • DNA libraries and the polymerase chain reaction (PCR) help researchers isolate particular DNA fragments

19. DNA Libraries • Genome • The entire set of genetic material of an organism • DNA librariesare sets of cells containing various cloned DNA fragments • Genomic libraries (all DNA in a genome) • cDNA libraries (all active genes in a cell)

20. Probes • Probe • A fragment of DNA labeled with a tracer • Used to find a specific clone carrying DNA of interest in a library of many clones • Nucleic acid hybridization • Base pairing between DNA from different sources • A probe hybridizes with the targeted gene

21. A Individual bacterial cells from a DNA library are spread over the surface of a solid growth medium. The cells divide repeatedly and form colonies—clusters of millions of genetically identical descendant cells. B A piece of special paper pressed onto the surface of the growth medium will bind some cells from each colony. C The paper is soaked in a solution that ruptures the cells and releases their DNA. The DNA clings to the paper in spots mirroring the distribution of colonies. D A probe is added to the liquid bathing the paper. The probe hybridizes (base-pairs) with the spots of DNA that contain complementary base sequences. E The bound probe makes a spot. Here, one radioactive spot darkens x-ray film. The position of the spot is compared to the positions of the original bacterial colonies. Cells from the colony that made the spot are cultured, and the DNA they contain is harvested. Figure 15-5 p238

22. PCR • Polymerase chain reaction (PCR) • A cycled reaction that uses a heat-tolerant form of DNA polymerase (Taq polymerase) to produce billions of copies of a DNA fragment

23. PCR Techniques • DNA to be copied is mixed with DNA polymerase, nucleotides and primers that base-pair with certain DNA sequences • Cycles of high and low temperatures break and reform hydrogen bonds between DNA strands, doubling the amount of DNA in each cycle

24. Two Rounds of PCR

25. 15.4 DNA Sequencing • DNA sequencing reveals the order of nucleotide bases in a fragment of DNA

26. DNA Sequencing • DNA is synthesized with normal nucleotides and dideoxynucleotides tagged with different colors • When a tagged base is added, DNA synthesis stops; fragments of all lengths are made • Electrophoresis separates the fragments of DNA, each ending with a tagged base, by length • The order of colored bases is the sequence of DNA

27. DNA template 2 3 4 5 Figure 15-7a p240

28. pigment: green blue black red base: adenine cytosine guanine thymine dideoxynucleotides 1 Figure 15-7 p240

29. The Human Genome Project • Automated DNA sequencing and PCR allowed human genome projects to sequence the 3 billion bases in the human genome • 28,976 genes have been identified, but not all of their products or functions are known

30. Sequencing the Human Genome

31. A Human DNA Sequence

32. 15.5 Genomics • Comparing the sequence of our genome with that of other species is giving us insights into how the human body works • Unique sequences of genomic DNA can be used to distinguish an individual from all others

33. Genomics • The study of genomes (genomics) is a broad field that encompasses whole-genome comparisons, structural analysis of gene products, and surveys of small-scale variations in sequence

34. Comparing Genomes • All genomes are related to some extent – comparing genomes provides evidence of genetic relationships • Comparing genomes among species also shows that changes in chromosome structure do not occur randomly • Comparing the coding regions of genomes also offers medical benefits • Example:APOA5 mutations and triglycerides

35. Genomic DNA Alignment

36. DNA Profiling: SNPs • Identifying an individual by his or her unique array of DNA sequences is called DNA profiling • One type of DNA profiling involves SNP-chips with microscopic spots of DNA stamped on them • An individual’s genomic DNA hybridizes only with DNA spots that have a matching SNP sequence • Probes reveal where the genomic DNA has hybridized

37. SNP-Chip Analysis

38. DNA Profiling: STRs • Another method of DNA profiling involves analysis of short tandem repeats, sections of DNA in which a series of 4 or 5 nucleotides is repeated several times in a row. • Types and numbers of STRs vary greatly among individuals • Unless two people are identical twins, the chance that they have identical short tandem repeats in even three regions of DNA is 1 in a quintillion (1018)

39. Analyzing STRs • PCR is used to amplify DNA from regions of several chromosomes that have STRs • Electrophoresis is used to separate the fragments and create a unique DNA fingerprint • DNA fingerprints have many applications, including legal cases, forensics, and population studies

40. An STR Profile

41. 15.6 Genetic Engineering • Genetic engineering is a laboratory process by which deliberate changes are introduced into an individual’s genome

42. Some GMOs • The most common GMOs are bacteria and yeast • Some E. coli have been modified to produce a fluorescent protein from jellyfish – used to study gene expression • Some bacteria have been modified to produce medically important proteins such as human insulin and chymotrypsin • Other GMO-produced enzymes improve the taste and clarity of beer and fruit juice, slow bread staling, or modify fats

43. E. coli with Jellyfish Genes

44. 15.7 Designer Plants • Genetically engineered crop plants are widespread in the United States

45. Introducing New Genes into Plants • Foreign or modified genes can be introduced into plant cells by way of electric or chemical shocks, or by blasting them with DNA-coated micropellets • Genes can also be introduced biologically using the Ti plasmid, a plasmid of the bacterium Agrobacterium tumefaciens which contains tumor-inducing (Ti) genes

46. A A Ti plasmid carrying a foreign gene is inserted into an Agrobacterium tumefaciens bacterium. Figure 15-12a p245

47. B The bacterium infects a plant cell and transfers the Ti plasmid into it. The plasmid DNA becomes integrated into one of the cell’s chromosomes. Figure 15-12b p245

48. C The plant cell divides, and its descendants form an embryo. Several embryos are sprouting from this mass of cells. Figure 15-12c p245

49. D Each embryo develops into a transgenic plant that expresses the foreign gene. The glowing tobacco plant is expressing a gene from fireflies. Figure 15-12d p245

50. Genetically Engineered Plants • Crop plants are genetically modified to produce more food at lower cost • Resistance to disease or herbicides • Increased yield • Plants that make pesticides (Bt protein) • Drought resistance • Genetic modifications can make food plants more nutritious • Rice plants that make β-carotene