Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings PowerPoint Lectures for Biology, Seventh Edition Neil Campbell and Jane Reece Lectures by Chris Romero Chapter 20 DNA Technology and Genomics

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 In broad terms, biotechnology is the manipulation of organisms or their components to make useful products

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 – 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

LE 20-1 Bacterium Bacterial chromosome Plasmid Gene inserted into plasmid Cell containing gene of interest Gene of interest DNA of chromosome Recombinant DNA (plasmid) 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 Protein harvested Gene of interest Copies of gene Basic research on gene Basic research on protein Basic research and various applications 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

LE 20-2 Restriction site DNA Restriction enzyme cuts the sugar-phosphate backbones at each arrow. One possible combination 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 DNA ligase seals the strands. Recombinant DNA molecule Sticky end

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Cloning a human gene in a bacterial plasmid can be divided into five steps

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The Techniques of Gene Cloning Cloning a human gene in a bacterial plasmid can be divided into five steps: 1.Isolate a gene of interest, for example, the gene for human insulin 2.Insert the gene into a bacterial plasmid 3.Insert the plasmid into a vector, a cell that will carry the plasmid, such as a bacterium. To accomplish this, a bacterium must be made competent (be able to take up the plasmid) 4.Clone the gene. As the bacteria reproduce themselves by fission, the plasmid and the selected gene are also being cloned. Millions of copies of the gene are being produced. 5.Identify the bacteria that contain the selected gene and harvest it from the bacteria.

LE 20-3_1 Isolate plasmid DNA and human DNA. 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. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site amp R gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Recombinant DNA plasmids

LE 20-3_2 Isolate plasmid DNA and human DNA. 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. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site amp R gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria

LE 20-3_3 Isolate plasmid DNA and human DNA. 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. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site amp R gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments 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 non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Identifying Clones Carrying a Gene of Interest All the methods for detecting the DNA of a gene directly depend on base pairing between the gene and a complementary sequence on another nucleic acid molecule, a process called nucleic acid hybridization. – The complementary molecule, a short, single stranded nucleic acid is called the nucleic acid probe. If we know at least part of the nucleotide sequence of our gene, we can synthesize a probe complementary to it. – We trace the probe, which will hydrogen bond specifically to complementary single strands of the desired gene, by labeling it with a radioactive isotope or a fluorescent tag. – DNA probes can be used in this way to identify a person who carries an inherited genetic defect, such as sickle cell, Tay-Sachs, Huntington’s, and others.

LE 20-4 Master plate Filter Solution containing probe Filter lifted and flipped over Radioactive single-stranded DNA Probe DNA Gene of interest Single-stranded DNA from cell Film Hybridization on filter Master plate Colonies containing gene of interest A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter. The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter. The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography). After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Storing Cloned Genes in DNA Libraries See figure 20.5 in textbook: A genomic library is a set of thousands of DNA segments from a genome, each carried by a plasmid, phage, or other cloning vector. 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 (introns are removed ) because bacteria lack introns and have no way to edit them out for transcription. – In order to clone a human gene into a bacterium, scientists must insert a gene with NO introns by extracting fully processed mRNA from cells and then using the enzyme reverse transcriptase (obtained from retroviruses) to make DNA transcripts of this RNA.

LE 20-6 Bacterial clones Recombinant plasmids Recombinant phage DNA or Foreign genome cut up with restriction enzyme Phage clones Plasmid libraryPhage library

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Cloning and Expressing Eukaryotic Genes As an alternative to screening a DNA library, clones can sometimes be screened for a desired gene based on detection of its encoded protein After a gene has been cloned, its protein product can be produced in larger amounts for research Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter (YACs)

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 without using cells. – A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Concept 20.2: Restriction Fragment Analysis Restriction fragment analysis detects differences in the nucleotide sequences of DNA molecules – Such analysis can rapidly provide comparative information about DNA sequences 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 A technique called Southern blotting combines gel electrophoresis 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

LE 20-8 Cathode Power source Anode Mixture of DNA molecules of different sizes Gel Glass plates Longer molecules Shorter molecules

LE 20-9 Normal  -globin allele 175 bp201 bpLarge fragment Sickle-cell mutant  -globin allele 376 bpLarge fragment 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

LE DNA + restriction enzyme Restriction fragments  Normal  -globin allele  Sickle-cell allele  Heterozygote Preparation of restriction fragments.Gel electrophoresis.Blotting.  Nitrocellulose paper (blot) Gel Sponge Alkaline solution Paper towels Heavy weight Hybridization with radioactive probe.  Radioactively labeled probe for  -globin gene is added to solution in a plastic bag Paper blot Probe hydrogen- bonds to fragments containing normal or mutant  -globin Fragment from sickle-cell  -globin allele Fragment from normal  -globin allele Autoradiography.  Film over paper blot

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

LE DNA (template strand) 5 3 Primer 3 5 DNA polymerase DeoxyribonucleotidesDideoxyribonucleotides (fluorescently tagged) 3 5 DNA (template strand) Labeled strands 3 Direction of movement of strands LaserDetector

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

LE 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

LE 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. Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. Isolate mRNA. Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA microarray Size of an actual DNA microarray with all the genes of yeast (6,400 spots)

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Future Directions in Genomics Genomics is the study of entire genomes Proteomics is the systematic study of all proteins encoded by a genome Single nucleotide polymorphisms (SNPs) provide markers for studying human genetic variation

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Concept 20.5: 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 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

LE DNA RFLP marker Disease-causing allele Normal allele Restriction sites

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

LE Cloned gene Retrovirus capsid Bone marrow cell from patient Inject engineered cells into patient. 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. Viral DNA carrying the normal allele inserts into chromosome. Bone marrow

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Pharmaceutical Products Some pharmaceutical applications of DNA technology: – Large-scale production of human hormones and other proteins with therapeutic uses – Production of safer vaccines

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

LE Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V)

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Environmental Cleanup & Agricultural Productivity 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 DNA technology is being used to improve agricultural productivity and food quality

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE – Genetic Engineering in Plants Agrobacterium tumefaciens Ti plasmid Site where restriction enzyme cuts DNA with the gene of interest T DNA Recombinant Ti plasmid Plant with new trait The Ti plasmid is the most commonly used vector for introducing new genes into plant cells. Agricultural scientists have endowed a number of crop plants with genes for desirable traits

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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