Chapter 15 DNA Technology and Genomics

1. Chapter 15DNA Technology and Genomics

2. Biotechnology • Studies of DNA sequences reveal the organization of genes and the relationship between genes and their products • Recombinant DNA technologyallows researchers to splice together DNA from different organisms in the laboratory • Molecular modification (genetic engineering) alters an organism’s DNA to produce new genes with new traits • Biotechnologyincludes all commercial or industrial uses of cells or organisms

3. 15.1 DNA CLONING LEARNING OBJECTIVES: • Explain how a typical restriction enzyme cuts DNA molecules and give examples of the ways in which these enzymes are used in recombinant DNA technology • Distinguish among a genomic DNA library, a chromosome library, and a complementary DNA (cDNA) library; explain why one would clone the same eukaryotic gene from both a genomic DNA library and a cDNA library • Explain how researchers use a DNA probe • Describe how PCR amplifies DNA in vitro

4. Recombinant DNA Technology • Recombinant DNA technology began with genetic studies of viruses that infect bacteria (bacteriophages) • Restriction enzymes from bacteria are used to cut DNA molecules in specific places – a vector molecule transports the DNA fragment into a cell • Bacteriophages and plasmids are two examples of vectors

5. Recombinant DNA Technology (cont.) • A plasmid with foreign DNA spliced into it (recombinant plasmid) is introduced into bacteria by transformation • Once a plasmid enters a cell, it is replicated and distributed to daughter cells during cell division, producing many identical copies – the foreign DNA is cloned

6. Restriction Enzymes • Restriction enzymes enable scientists to cut DNA from chromosomes into shorter fragments in a controlled way • Eachrestrictionenzyme cuts DNA at a specific DNA sequence (restriction site), such as 5′-AAGCTT-3′ • Many restriction enzymes used for recombinant DNA studies cut palindromic sequences – the base sequence reads the same as its complement, in the opposite direction – such as 3′-TTCGAA-5′

7. Sticky Ends • Cutting both strands in a staggered fashion produces fragments with identical, complementary, single-stranded ends called sticky ends: 5′-A AGCTT -3′ 3′-TTCGA A-5′ • Sticky ends pair by hydrogen bonding with the complementary, single-stranded ends of other DNA molecules that have been cut with the same enzyme

8. DNA Ligase • Once the sticky ends of two molecules have been joined, they are treated with DNA ligase, an enzyme that covalently links the two DNA fragments to form a stable recombinant DNA molecule

9. Cutting DNA with a Restriction Enzyme

10. Plus HindIII restriction enzyme Sticky ends Fig. 15-1, p. 325

11. Recombinant DNA • The DNA to be cloned and plasmid (vector) DNA are cut with the same restriction enzyme • The two DNA samples are mixed, and complementary bases of the sticky ends are bonded • The result is recombinant DNA

12. Making Recombinant DNA

13. DNA of interest from another organism Plasmid from a bacterium 1 Clonable DNA fragment 2 Recombinant DNA 3 Fig. 15-2, p. 325

14. DNA of interest from another organism Plasmid from a bacterium 1 Clonable DNA fragment 2 Recombinant DNA 3 Stepped Art Fig. 15-2, p. 325

15. Plasmids • Plasmids used in recombinant DNA technology include features helpful in isolating and analyzing cloned DNA: • One or more restriction sites • Genes that let researchers select cells transformed by recombinant plasmids

16. A Plasmid Vector

17. Aat I Xba I Ampicillin resistance Yeast origin of replication Hpa I E. coli origin of replication Tetracycline resistance Cla I Pvu II URA-3 Sal I Sma I Bam HI (a) This plasmid vector has many useful features. Researchers constructed it from DNA fragments they had isolated from plasmids, E. coli genes, and yeast genes. The two origins of replication, one for E. coli and one for yeast, Saccharomyces cerevisiae , let it replicate independently in either type of cell. Letters on the outer circle designate sites for restriction enzymes that cut the plasmid only at that position. Resistance genes for the antibiotics ampicillin and tetracycline and the yeast URA-3 gene are also shown. The URA-3 gene is useful when transforming yeast cells lacking an enzyme required for uracil synthesis. Cells that take up the plasmid grow on a uracil-deficient medium. Fig. 15-3a, p. 326

18. Plasmid in Bacterium

19. Bacterial chromosome Bacterium Plasmid (b) The relative sizes of a plasmid and the main DNA of a bacterium. Fig. 15-3b, p. 326

20. DNA Libraries • The total DNA in a cell is its genome • A genomic DNA libraryis a collection of thousands of DNA fragments that represent all of the DNA in the genome • A chromosome library contains all the DNA fragments in that specific chromosome • A human genomic DNA library is stored in a collection of recombinant bacteria, each with a different fragment of DNA

21. Producing a Genomic DNA Library or Chromosome Library

22. Sites of cleavage Fragment 1 Fragment 2 Fragment 3 Fragment 4 = Human DNA 1 Produce recombinant DNA 2 Gene for resistance to antibiotic R R R R 3 Transformation Bacterium without plasmid 4 Bacteria without plasmid fail to grow. Bacteria with plasmid live and multiply to form a colony. Plate with antibiotic-containing medium Fig. 15-4, p. 327

23. Locating a Sequence of Interest • To identify a plasmid containing a sequence of interest, each plasmid is cloned until there are millions of copies • A sample of bacterial culture is spread on agar plates so cells are widely separated – each cell divides many times, forming a colonyof genetically identical clones • The next task is to determine which colony out of thousands contains the fragment of interest

24. DNA Probes • A segment of DNA that is homologous (identical) to part of the sequence of interest (DNA probe)can be used to detect the specific DNA sequence • The DNA probe is a segment of single-stranded DNA that can hybridize(attach by base pairing) to complementary base sequences in target DNA • DNA that is complementary to that particular probe is detected

25. A cDNA Library • It is possible to clone intact genes and avoid introns by using DNA copies of mature mRNA to construct complementary DNA (cDNA) • Researchers use the enzyme reverse transcriptase to synthesize single-stranded cDNA, then DNA polymerase to make the cDNA double-stranded • A cDNA library is formed using mRNA from a single cell type as the starting material

26. Formation of cDNA

27. 1 Exon Intron Exon Intron Exon DNA in a eukaryotic chromosome Transcription Pre-mRNA RNA processing (remove introns) Mature mRNA 2 Reverse transcriptase Mature mRNA mRNA cDNA copy of mRNA 3 Degraded RNA cDNA 4 DNA polymerase 5 Double-stranded cDNA Fig. 15-6, p. 329

28. The Polymerase Chain Reaction • The polymerase chain reaction (PCR)can be used to amplify a tiny sample of DNA without cloning in a cell • PCR uses a heat-resistant DNA polymerase (Taq polymerase), nucleotides and primers to replicate a DNA sequence in vitro • Cycles of denaturing (heating) and replication double the number of cloned molecules with each cycle

29. The Polymerase Chain Reaction

30. Using PCR • PCR enables researchers to amplify and analyze tiny DNA samples from a variety of sources, ranging from crime scenes to archaeological remains • Example: Investigators have used PCR to analyze mitochondrial DNA obtained from the bones of Neandertals

31. KEY CONCEPTS 15.1 • Scientists use DNA technology to produce many copies of specific genes (gene cloning)

32. 15.2 DNA ANALYSIS LEARNING OBJECTIVES: • Distinguish among DNA, RNA, and protein blotting

33. Gel Electrophoresis • Gel electrophoresis is used to separate mixtures of certain macromolecules: proteins, polypeptides, or DNA fragments • Nucleic acids migrate through the gel toward the positive pole of the electric field because they are negatively charged due to their phosphate groups • DNA fragments are separated by size – small molecules move farther than large molecules

34. Gel Electrophoresis

35. 1 Standards of known sizes placed in well – Direction of movement Mixtures placed in well Gel Buffer solution + Fig. 15-8a, p. 332

36. 2 Anode Longer molecules Shorter molecules Cathode Fig. 15-8b, p. 332

37. 3 Fig. 15-8c, p. 332

38. Southern Blot • DNA separated by gel electrophoresis is denatured and transferred to a membrane, which picks up DNA like a blotter picks up ink – this Southern blot is a replica of the gel • The blot is incubated with a DNA probe, which hybridizes with any complementary DNA fragments – the probe is detected by autoradiography or chemical luminescence

39. Similar Blotting Techniques • When RNA molecules separated by electrophoresis are transferred to a membrane and detected using a nucleic acid probe, the result is called a Northern blot • When the blot consists of proteins or polypeptides separated by gel electrophoresis, it is called aWestern blot

40. Southern Blotting Technique

41. 1 2 DNA DNA fragments Buffer solution Agarose gel Weight 5 4 3 Absorbent paper Nitro- cellulose filter Longer DNA fragments Gel Wick Shorter DNA fragments Buffer 6 7 Radioactive probe solution Fig. 15-9, p. 333

42. Restriction Fragment Length Polymorphisms (RFLPs) • Random DNA mutations and recombination result in individuals with different lengths of fragments produced by a given restriction enzyme • These restriction fragment length polymorphisms (RFLPs) can be used to determine how closely related different members of a population are • A genetic polymorphism exists if individuals of two or more discrete types, or “morphs,” are found in a population

43. RFLP Analysis • RFLP analysis has been used for paternity testing and analyzing evidence found at crime scenes • RFLP analysis helped map the exact location of gene mutations, such as the mutation that causes cystic fibrosis • Today, RFLP analysis is rapidly being replaced by newer methods, such as automated DNA sequencing

44. Rapid DNA Sequencing • Automated DNA-sequencingmachines connected to powerful computers let scientists sequence huge amounts of DNA quickly and reliably

45. Automated DNA-Sequencing Results Fig. 15-11, p. 335

46. Sequencing Entire Genomes • Advances in sequencing technology have made it possible for researchers to study the nucleotide sequences of entire genomes in a wide variety of organisms • The Human Genome Project, which sequencedthe 3 billion base pairs of the human genome, was completed in 2001 • DNA sequence information is stored in large computer databases, many of which are accessed through the Internet

47. KEY CONCEPTS 15.2 • Biologists study DNA using gel electrophoresis, DNA blotting, automated sequencing, and other methods

48. 15.3 GENOMICS LEARNING OBJECTIVES: • Describe three important areas of research in genomics • Explain what a DNA microarray does • Define pharmacogenetics and proteomics

49. Genomics • Genomicsis the study of the entire DNA sequence of an organism’s genome to identify all the genes, determine their RNA or protein products, and how the genes are regulated • Structural genomics: mapping and sequencing • Functional genomics: functions of genes and nongene sequences • Comparative genomics: comparing genomes of different species (evolution) • Metagenomics: analyzing communities of microorganisms instead of individual organisms

50. RNA Interference (RNAi) • RNA interference (RNAi) can be used to quickly determine the function of a specific gene by inactivating the gene • A short stretch of RNA complementary to part of the DNA sequence being examined is put into cells to silence the gene • Biologists observe any changes in phenotype to help determine the function of the missing protein