Learning Objectives. What is genomics?

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1 What is genomics? Learning Objectives Sequencing basics Very basic mechanics of genetic engineering Understanding the relationship between cdna and mrna What is reverse transcriptase? Restriction endonucleases Recognition sites Sticky ends Importance in genetic engineering What is PCR and why is it important? Very general overview of DNA fingerprinting Banding patterns are the fingerprint How is genetic engineering used to generate vaccines? How is genetic engineering used in agriculture? What s the difference between genetic engineering and genetic modification? How are animals cloned? Why do they have shortened life spans? What are stem cells? Embryonic vs. Adult Therapeutic cloning How does gene therapy work?

2 Genomics is a field that compares the entire DNA content of different organisms Genomics The genome is the full complement of genetic information of an organism (i.e., all of its genes and other DNA) DNA sequencing is a process that allows scientists to read each nucleotide in a strand of DNA Genomics is about comparisons

3 DNA Sequencing In DNA sequencing, a fragment of DNA is amplified so that there are thousands of copies These fragments are sequenced by synthesis using DNA polymerase Chemical tags that form complementary base pairs just like the nucleotide bases are incorporated during synthesis Incorporation of a chemical tag causes synthesis to halt

4 Electrophoresis The fragments of DNA are then separated according to size by gel electrophoresis In an electrical current, DNA will migrate toward a positive electrode (Why?) In gel electrophoresis, an electrical current is run through an agarose gel, and DNA fragments migrate through the porous gel faster or slower based on size The fragments become arrayed in order and the sequence of nucleotides can be read one nucleotide at a time Which fragments will migrate through a gel faster?

5 The Human Genome The publication of the sequence of the entire human genome occurred on June 26, 2000 the human genome contains more than 3 billion base pairs it is estimated that there are 20,000 to 25,000 proteinencoding genes, only 1% of the genome The race to sequence the human genome was a tie between Craig Venter s company and the publicly funded Human Genome Project Celera pioneered whole genome shotgun sequencing 1

6 Table 13.1 Some Eukaryotic Genomes

7 Figure 13.2 The human genome

8 Where Are the Genes? The number of genes is much less than expected based on unique mrnas found in human cells in eukaryotic DNA, protein-encoding segments called exons are interspersed among noncoding introns alternative splicing of exons accounts for the fact that about 25,000 genes can encode four times as many proteins Some chromosomes are packed with genes; others contain relatively few genes

9 Types of Non-Coding DNA There are four major types of non-coding DNA, which account for 99% of the DNA in humans noncoding DNA within genes these are introns structural DNA tightly coiled DNA that remains untranscribed repeated/duplicated sequences simple sequence repeats (SSRs) and duplicated sequences that are scattered about chromosomes transposable elements bits of DNA that jump from one location on a chromosome to another

10 Animation: Simple Transposition Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at

11 A Scientific Revolution Genetic engineering is moving genes from one organism to another the first stage in a genetic engineering experiment is to chop up the source DNA and obtain a copy of the gene you want to transfer restriction enzymes, or restriction endonucleases, bind to specific short sequences on the DNA and make a specific cut the sequence is symmetrical palindromic the cut generates DNA fragments that are sticky because the incision made by the restriction enzyme is usually made to the side of the sequence, not in the center of it

12 Restriction Enzymes DNA from another source that is cut with the same restriction enzyme will have the same sticky ends these ends can be joined together by the enzyme ligase Restriction enzymes are the basic tools of genetic engineering

13 Figure 13.5 How restriction enzymes produce DNA fragments with sticky ends

14 Animation: Restriction Endonucleases Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at

15 Animation: Early Genetic Engineering Experiment Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at

16 cdna The need for complementary DNA (cdna) Recall that eukaryotic genes contain both exons and introns before the gene is translated and expressed, the introns are cut out from the primary RNA transcript enzymes stitch together the remaining exon fragments to form mrna, which is eventually translated in the cytoplasm Bacteria lack the enzymes to remove introns It is desirable for genetic engineers to transfer an intron-free copy of the gene into bacteria

17 Reverse Transcriptase Forming cdna to obtain DNA without introns, genetic engineers isolate first the processed mrna corresponding to a particular gene the enzyme reverse transcriptase can then produce a DNA version of this mrna, called complementary DNA (cdna) How does this fit into the Central Dogma?

18 Figure 13.6 cdna: Producing an intron-free version of a eukaryotic gene for genetic engineering

19 Animation: cdna Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at

20 DNA Fingerprinting DNA fingerprinting is a revolutionary technique used in forensic evidence the process uses probes on DNA samples that have been cut with the same restriction endonucleases the probes are complementary to unique DNA sequences found in non-coding regions of human DNA that are highly variable among individuals Why might they be variable? the resulting DNA fingerprints consist of autoradiographs, rows of parallel bands on X-ray film each band represents the position of a DNA fragment that complementarily binds to a probe How would it bind?

21 DNA Evidence DNA fingerprints have been used in courts of law since 1987 while an individual DNA fingerprint is not 100% accurate, it is as reliable as traditional fingerprinting used in evidence when multiple probes are used any source of DNA (i.e., a hair, a speck of blood, or semen) can be used in DNA fingerprinting to convict or to clear a suspect however, laboratory analyses of DNA must be carried out properly to ensure accuracy

22 Figure 13.7 DNA Fingerprints that led to conviction

23 Animation: DNA Fingerprinting Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at 2

24 PCR Polymerase chain reaction (PCR) is a method which produces millions of copies of DNA from a single copy a double-stranded DNA fragment is heated to separate the strands each of the strands is copied by DNA polymerase the cycle is repeated many times, each time doubling the number of DNA copies PCR fun facts: PCR was invented by surfer, scientist, and frequent LSD user Kary Mullis in 1983 PCR revolutionized molecular biology and made $300 million for Dr. Mullis company. Dr. Mullis received a $10,000 bonus

25 Genetic Engineering and Medicine Much of the promise of genetic engineering lies treating illness Many genetic defects occur because our cells fail to make critical proteins through genetic engineering, the genes encoding insulin have been introduced in bacteria, which can cheaply produce large quantities of insulin Other proteins, such as factor VIII to promote clotting, are now safely produced by bacteria, which eliminates the risks associated with donated blood

26 Engineering Vaccines Genetic engineering is also used to create subunit vaccines against viruses, such as herpes and hepatitis engineers splice genes from the coat of the virus into a fragment of cowpox (vaccinia) virus genome, which is used as a vector to carry the viral coat genes the recombinant virus is injected into humans and cause the production of antibodies against the virus, which is essentially harmless to humans this method of using one virus as a vector to introduce fragments of the DNA of a disease-causing virus produces what are called piggyback vaccines

27 Figure 13.9 Constructing a subunit, or piggyback, vaccine for the herpes simplex virus 3

28 Genetic Engineering and Agriculture Engineering crops to be resistant to insect pests reduces the need to add insecticides to the environment for example, genes from the soil bacterium Bacillus thuringiensis (Bt), which produces a protein that is toxic when eaten by crop pests, have been inserted into the chromosomes of tomatoes because the plants can synthesize Bt protein, they are toxic to pests, such as the tomato hornworm 4

29 Herbicide resistance has also been genetically engineered glyphosate is a powerful herbicide that kills most actively growing plants and is used to control weeds using a gene gun, engineers inserted into crop plants an isolated gene from a bacterium that is resistant to glyphosate the glyphosate can now be widely applied to fields and orchards where it retards weed growth but not crop growth Roundup-Ready

30 Figure Shooting genes into cells

31 Golden Rice The real promise of genetically modified (GM) plants is to produce crops with desirable traits that directly benefit the consumer for example, to combat iron and vitamin A micronutrient deficiencies among the world s rice eaters, genetic engineers created GM golden rice this transgenic rice contains genes from a bean, a fungus, wild rice, and a daffodil to increase its nutritional value

32 Figure Transgenic golden rice Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Beans Aspergillus fungus Wild rice Daffodil Ferritin gene is transferred into rice from beans. Phytase gene is transferred into rice from a fungus. Metallothion in gene is transfer red into rice from wild rice. Enzymes for beta-carotene synthesis are transferred into rice from daffodils. Fe Pt Rice chromosome S A 1 A 2 A 3 A 4 Ferritin protein increases iron content of rice. Phytate, which inhibits iron reabsorption, is destroyed by the phytase enzyme. Metallothionin protein supplies extra sulfur to increase iron uptake. Beta-carotene, a precursor to vitamin A, is synthesized. Golden Rice Humanitarian Board:

33 GM Crops Genetically modified (GM) crops are commonly cultivated in the United States some of the benefits of GM crops include increased soil preservation and reduced pesticide usage Is eating GM food dangerous? does adding genes introduce novel proteins that maybe potentially harmful when consumed? could introduced proteins become allergens? all introduced proteins are extensively tested and the risk of eating GM crops seems to be slight

34 Table 13.3 Genetically Modified Crops Genetically engineered crops Non-GMO crops that have been genetically modified

35 GM Crops and the Environment Are GM crops harmful to the environment? possibility of unintentional harm to other organisms weeds might be important sources of food and shelter for non-pest insects potential for new resistance pests might be likely to become resistant to the engineered proteins farmers are required to plant some non-gm crop alongside the GM crop in order to slow the selection pressure for resistance gene flow into neighboring plants modified genes may spread to non-gm species due to interbreeding

36 A New Life for an Old Crop GE tobacco currently provides the best hope for an Ebola cure. Transgenic tobacco grows antibodies used to treat two American workers. Charles Arntzen

37 Reproductive Cloning Hans Spemann proposed in 1938 that cloning might be possible by removing the nucleus from an egg cell and replacing it with a nucleus from another cell Attempts at cloning were made many years later by several researchers Some success was obtained but only with a donor nucleus from an early embryo, not an adult nucleus

38 Starving the Cells Keith Campbell, a geneticist, proposed that, in order for a successful nuclear transplant to take place, both the egg and the donated nucleus need to be in the same stage of the cell cycle by first starving the cells so that they paused at the G 1 checkpoint, researchers succeeded in cloning farm animals from advanced embryos

39 Dolly the Sheep Campbell s colleague Wilmut attempted to transfer a nucleus from an adult cell into an enucleated egg Wilmut used an adult sheep s mammary gland as the nuclear donor both the donor mammary cells and the enucleated eggs were first starved and a brief electrical shock allowed the contents to fuse together the resulting embryo developed into a blastula and was implanted into a surrogate mother Dolly the cloned lamb was born on July 5, 1996

40 Figure Wilmut s animal cloning experiment Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Mammary cell is extracted and grown in nutrientdeficient solution that arrests the cell cycle. Nucleus containing source DNA Egg cell is extracted. Nucleus is removed from egg cell with a micropipette. Mammary cell is inserted inside covering of egg cell. Electric shock opens cell membranes and triggers cell division. Preparation Cell fusion Cell division Embryo Embryo begins to develop in vitro. Embryo is implanted into surrogate mother. After a five-month pregnancy, a lamb genetically identical to the sheep from which the mammary cell was extracted is born. Development Implantation Birth of clone Growth to adulthood Courtesy The Roslin Institute, The University of Edinburgh

41 Cloned Animals A wide variety of farm animals have been cloned since Dolly the sheep Cloning procedures have become increasingly efficient However, most cloned animals do not live a normal lifespan 6 Clone Troopers also have a shortened lifespan. Science!

42 Epigenetics and Cloning What may be wrong with cloned embryos is that as mammalian eggs and sperm mature, their DNA is conditioned by the parent by a type of reprogramming called epigenetics chemical changes are made to the DNA that alter when particular genes are expressed genes can be locked in the off position by methylation, in which a CH 3 group is added to some of the cytosine nucleotides

43 Stem Cells Embryonic stem cells are special cells that form early in development embryonic stems are called totipotent, meaning that each cell has the ability to form any body tissue, even an adult animal Adult stem cells are pluripotent because they can differentiate into many different cell types, but not all

44 Stem Cells Tissues As development proceeds, some of the embryonic stem cells begin to become committed to forming a certain type of tissue only every major tissue is formed from its own tissue-specific adult stem cell Because they can develop into any tissue, embryonic stem cells offer the exciting possibility of restoring damaged tissues 7

45 Figure Using embryonic stem cells to restore damaged tissue

46 Therapeutic Cloning Experiments using stem cells to repair damaged tissues have been carried out in mice without functioning immune systems for stem cell therapy to work in humans, the problem of immune rejection needs to be addressed Therapeutic cloning (also called somatic cell nuclear transfer) addresses the issue of immune acceptance in therapeutic cloning, the nucleus from a patient is inserted into an enucleated egg cell and the egg develops in vitro the embryonic stem cells are removed and used to replace the patient s damaged or lost tissue

47 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 The nucleus from a skin cell of a diabetic patient is removed. Nucleus of skin cell Enucleated egg cell 2 The nucleus is removed from a human egg cell. Diabetic patient 7 The stem cells develop into healthy pancreatic islet cells that are injected into or transplanted into the patient. 3 The skin cell nucleus is inserted into the enucleated human egg cell. Early embryo 4 Cell cleavage occurs as the embryo begins to develop in vitro. Healthy pancreatic islet cells Stem cells 5 The embryo reaches the blastocyst stage. Therapeutic cloning Reproductive cloning Blastocyst 6 Embryonic stem cells are extracted and grown in culture. 6a The blastocyst, if kept intact, could be implanted into the uterus of a surrogate mother. Fig a The resulting baby would be a clone of the diabetic patient.

48 Therapeutic Cloning: Bioethics Many people regard therapeutic cloning to be ethically unacceptable Reprogramming adult cells to be embryonic stem cells might be an alternative approach

49 Gene Therapy Gene transfer therapy involves introducing healthy genes into cells that lack them early work with a cold virus vector, called adenovirus, was unsuccessful in humans because of immune attack a smaller vector, adenoassociated virus (AAV), does not elicit a strong immune response and seems promising

50 Figure Using gene therapy to cure a retinal degenerative disease in dogs