Overview: Understanding and Manipulating Genomes

1. Overview: Understanding and Manipulating Genomes • Sequencing of the human genome was largely completed by 2003 • DNA sequencing has depended on advances in technology, starting with making recombinant DNA • In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule • Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes

2. DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products

3. Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment • To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called gene cloning

4. DNA Cloning and Its Applications: A Preview • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids • Cloned genes are useful for making copies of a particular gene and producing a gene product

5. LE 20-2 Cell containing gene of interest Bacterium Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome Plasmid put into bacterial cell Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

6. Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites • A restriction enzyme usually makes many cuts, yielding restriction fragments • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary “sticky ends” of other fragments • DNA ligase is an enzyme that seals the bonds between restriction fragments

7. LE 20-3 Restriction site 5¢ 3¢ DNA 3¢ 5¢ Restriction enzyme cuts the sugar-phosphate backbones at each arrow. Sticky end DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme One possible combination DNA ligase seals the strands. Recombinant DNA molecule

8. Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there

9. Producing Clones of Cells • Cloning a human gene in a bacterial plasmid can be divided into six steps: 1. Vector and gene-source DNA are isolated 2. DNA is inserted into the vector 3. Human DNA fragments are mixed with cut plasmids, and base-pairing takes place 4. Recombinant plasmids are mixed with bacteria 5. The bacteria are plated and incubated 6. Cell clones with the right gene are identified Animation: Cloning a Gene

10. LE 20-4_1 Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids

11. LE 20-4_2 Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria

12. LE 20-4_3 lacZ gene (lactose breakdown) Bacterial cell Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone

13. A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell • A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells

14. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA • A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

15. LE 20-7 5¢ 3¢ Target sequence Genomic DNA 3¢ 5¢ 5¢ 3¢ Denaturation: Heat briefly to separate DNA strands 3¢ 5¢ Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Cycle 1 yields 2 molecules Primers Extension: DNA polymerase adds nucleotides to the 3¢ end of each primer New nucleo- tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

16. Concept 20.2: Restriction fragment analysis detects DNA differences that affect restriction sites • Restriction fragment analysis detects differences in the nucleotide sequences of DNA molecules • Such analysis can rapidly provide comparative information about DNA sequences

17. Gel Electrophoresis and Southern Blotting • One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis • This technique uses a gel as a molecular sieve to separate nuclei acids or proteins by size Video: Biotechnology Lab

18. LE 20-8 Mixture of DNA molecules of differ- ent sizes Longer molecules Cathode Shorter molecules Power source Gel Glass plates Anode

19. 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

20. LE 20-9 Normal b-globin allele 175 bp 201 bp Large fragment Ddel Ddel Ddel Ddel Sickle-cell mutant b-globin allele 376 bp Large fragment Ddel Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of -globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles

21. Restriction Fragment Length Differences as Genetic Markers • Restriction fragment length polymorphisms (RFLPs, or Rif-lips) are differences in DNA sequences on homologous chromosomes that result in restriction fragments of different lengths • A RFLP can serve as a genetic marker for a particular location (locus) in the genome • RFLPs are detected by Southern blotting

22. Concept 20.3: Entire genomes can be mapped at the DNA level • The most ambitious mapping project to date has been the sequencing of the human genome • Officially begun as the Human Genome Project in 1990, the sequencing was largely completed by 2003 • Scientists have also sequenced genomes of other organisms, providing insights of general biological significance

23. Genetic (Linkage) Mapping: Relative Ordering of Markers • The first stage in mapping a large genome is constructing a linkage map of several thousand genetic markers throughout each chromosome • The order of markers and relative distances between them are based on recombination frequencies

24. LE 20-11 Chromosome bands Cytogenetic map Genes located by FISH Genetic (linkage) mapping Genetic markers Physical mapping Overlapping fragments DNA sequencing

25. Physical Mapping: Ordering DNA Fragments • A physical map is constructed by cutting a DNA molecule into many short fragments and arranging them in order by identifying overlaps • Physical mapping gives the actual distance in base pairs between markers

26. DNA Sequencing • Relatively short DNA fragments can be sequenced by the dideoxy chain-termination method • Inclusion of special dideoxyribonucleotides in the reaction mix ensures that fragments of various lengths will be synthesized

27. LE 20-12 DNA (template strand) Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) 3¢ 5¢ 5¢ DNA polymerase 3¢ DNA (template strand) Labeled strands 3¢ 5¢ 3¢ Direction of movement of strands Laser Detector

28. Linkage mapping, physical mapping, and DNA sequencing represent the overarching strategy of the Human Genome Project • An alternative approach to sequencing genomes starts with sequencing random DNA fragments • Computer programs then assemble overlapping short sequences into one continuous sequence

29. LE 20-13 Cut the DNA from many copies of an entire chromosome into overlapping frag-ments short enough for sequencing Clone the fragments in plasmid or phage vectors Sequence each fragment Order the sequences into one overall sequence with computer software

30. Concept 20.4: Genome sequences provide clues to important biological questions • In genomics, scientists study whole sets of genes and their interactions • Genomics is yielding new insights into genome organization, regulation of gene expression, growth and development, and evolution

31. Identifying Protein-Coding Genes in DNA Sequences • Computer analysis of genome sequences helps identify sequences likely to encode proteins • The human genome contains about 25,000 genes, but the number of human proteins is much larger • Comparison of sequences of “new” genes with those of known genes in other species may help identify new genes

33. Determining Gene Function • One way to determine function is to disable the gene and observe the consequences • Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function • When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype • In nonmammalian organisms, a simpler and faster method, RNA interference (RNAi), has been used to silence expression of selected genes

34. Studying 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

35. LE 20-14 Tissue sample Isolate mRNA. mRNA molecules Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organism’s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Labeled cDNA molecules (single strands) DNA microarray Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. Size of an actual DNA microarray with all the genes of yeast (6,400 spots)

36. Comparing Genomes of Different Species • Comparative studies of genomes from related and widely divergent species provide information in many fields of biology • The more similar the nucleotide sequences between two species, the more closely related these species are in their evolutionary history • Comparative genome studies confirm the relevance of research on simpler organisms to understanding human biology

37. Diagnosis of Diseases • Scientists can diagnose many human genetic disorders by using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the disease-causing mutation • Even when a disease gene has not been cloned, presence of an abnormal allele can be diagnosed if a closely linked RFLP marker has been found

38. LE 20-15 RFLP marker DNA Disease-causing allele Restriction sites Normal allele

39. 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 cells • Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

40. LE 20-16 Cloned gene Insert RNA version of normal allele into retrovirus. Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Retrovirus capsid Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Bone marrow Inject engineered cells into patient.

41. Pharmaceutical Products • Some pharmaceutical applications of DNA technology: • Large-scale production of human hormones and other proteins with therapeutic uses • Production of safer vaccines

42. Forensic Evidence • DNA “fingerprints” obtained by analysis of tissue or body fluids can provide evidence in criminal and paternity cases • A DNA fingerprint is a specific pattern of bands of RFLP markers on a gel • The probability that two people who are not identical twins have the same DNA fingerprint is very small • Exact probability depends on the number of markers and their frequency in the population

43. LE 20-17 Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V)

44. Environmental Cleanup • Genetic engineering can be used to modify the metabolism of microorganisms • Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials

45. Agricultural Applications • DNA technology is being used to improve agricultural productivity and food quality

46. Animal Husbandry and “Pharm” Animals • Transgenic organisms are made by introducing genes from one species into the genome of another organism • Transgenic animals may be created to exploit the attributes of new genes (such as genes for faster growth or larger muscles) • Other transgenic organisms are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use

47. Genetic Engineering in Plants • Agricultural scientists have endowed a number of crop plants with genes for desirable traits

48. Safety and Ethical Questions Raised by DNA Technology • Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures • Most public concern about possible hazards centers on genetically modified (GM) organisms used as food