DNA Technology. Chapter 12 CHAPTER 12. DNA Technology. RPTSE Biology Fall 2015, Dr. Jong B. Lee 1. Recombinant DNA Technology. Jong B. Lee, Ph.D.

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1 CHAPTER 12 DNA Technology Chapter 12 DNA Technology PowerPoint Lectures for Essential Biology, Third Edition Neil Campbell, Jane Reece, and Eric Simon Essential Biology with Physiology, Second Edition Neil Campbell, Jane Reece, and Eric Simon Lectures by Chris C. Romero Copyright 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Dec. 04, 2015 Jong B. Lee, Ph.D. Recombinant DNA Technology Recombinant DNA technology was born with E. coli genetics : In 1946, Lederberg and Tatum demonstrated that two individual E. coli can exchange genes. In the 1970s, research on E. coli led to the development of recombinant DNA technology, a set of techniques for combining genes from different sources (even different species) into a single DNA molecule : A molecule carrying DNA from more than one source. It is called recombinant DNA. Conjugation ( 접합 ) Plasmid 의발견 A host that carries DNA acquired from a different species is called transgenic organisms or genetically modified organism (GMO). DNA technology has revolutionized biotechnology. cf ) Biotechnology ( 생명공학 ); the use of organisms to perform practical tasks, 이미오래전부터존재 RPTSE Biology Fall 2015, Dr. Jong B. Lee 1

2 From Humulin to Genetically Modified Foods By transferring the gene for a desired protein product into a bacterium, yeast, cultured mammalian cells that is easy to grow, proteins that are present naturally in only small amounts can be produced in large quantities Making Humulin In 1982, the world s first genetically engineered pharmaceutical product, Humulin, human insulin, was produced by genetically modified bacteria. By the biotechnology company Genentech DNA 재조합기술로생산및판매되는대표적물질 HGH (human growth hormone) :Growth hormone from other species did not work for human 1985, recombinant HGH was produced by transgenic E. coli EPO (erythropoietin): one of glycoproteins present in blood serum, Growth factor of RBC for treatment of anemia. Mammalian cells are also being used for recombinant proteins Vaccine : A harmless variant or derivative of a pathogen, a disease-causing organism (virus, bacteria) is used for vaccines. Vaccine for hepatitis B virus is produced using transgenic yeast Genetically Modified (GM) Foods Today, DNA technology is quickly replacing traditional plant-breeding programs: In the United States today, roughly one-half of the corn crop and over three-quarters of the soybean and cotton crops are genetically modified. Corn has been genetically modified to resist insect infestation ( 출몰 ): This corn has been damaged by the European corn borer. Growing insect-resistant plants reduces the need for chemical insecticides 그밖의 GM foods Golden rice has been genetically modified to contain beta-carotene: our bodies use beta-carotene to make vitamin A Modified strawberry produce bacterial proteins that act as a natural antifreeze. Modified potatoes produce harmless proteins derived from the cholera bacterium: these modified potatoes can serve as an edible vaccine to produce immunity against the disease cholera in developing nations RPTSE Biology Fall 2015, Dr. Jong B. Lee 2

3 Pharm Animals While transgenic plants are used today as commercial products, transgenic whole animals are currently only in the testing phase. All transgenic animals carrying a recombinant human gene, could then serve as a grazing pharmaceutical factory pharm animals The human blood protein from the sheep s milk is being tested as a treatment of cystic fibrosis. Recombinant DNA Techniques Bacteria are the workhorses of modern biotechnology. To work with genes in the laboratory, biologists often use bacterial plasmids. Plasmids are small, circular DNA molecules that are separate from the much larger bacterial chromosome Plasmids Plasmid, just like the main bacterial chromosome, is copied by the cell s replication machinery Plasmids are readily taken up by bacterial cells (= transformation) Plasmid Bacterial cell Plasmid Isolate plasmids. Bacterial clone Gene of interest Cut both DNAs with same enzyme. Gene of interest Recombinant DNA plasmids Recombinant bacteria Other genes Mix the DNAs and join them together. Bacteria take up recombinant plasmids. Clone the bacteria. DNA fragments from cell Isolate DNA. Cell containing the gene of interest DNA Transgenic organism or genetically modified organisms (GMO) Plasmid acts as a vector, DNA carrier that moves genes from one cell to another Any foreign DNA that has been inserted will be replicated right along with the rest of the plasmid Gene for pest resistance Some uses of genes Gene for toxic-cleanup bacteria The gene and protein Genes may be of interest are isolated inserted into from the bacteria. other organisms. Find the clone with the gene of interest. Some uses of proteins Protein for stone-washing Harvested jeans proteins may be used directly. Protein for dissolving clots RPTSE Biology Fall 2015, Dr. Jong B. Lee 3

4 A Closer Look: Cutting and Pasting DNA with Restriction Enzymes Recombinant DNA is produced by combining two ingredients: 1. A bacterial plasmid 2. The gene of interest To combine these ingredients, this splicing process can be accomplished using restriction enzymes. These enzymes cut DNA at specific nucleotide sequences : The places where DNA is cut are called restriction sites Restriction enzymes & Ligase These cuts produce pieces of DNA called restriction fragments That may have sticky ends that are important for joining DNA from different sources. This union is then made permanent by the pasting enzyme DNA ligase. A Closer Look: Obtaining the Gene of Interest How can a researcher obtain DNA that encodes a particular gene of interest? The shotgun approach is one way to synthesize a gene of interest: Millions of recombinant plasmids containing different segments of foreign DNA are produced. This collection of millions of recombinant plasmids is called a genomic library Once a genomic library is created, biologists must identify the bacterial clone containing the desired gene. A specific sequence of radioactive nucleotides (=Complementary DNA) matching those in the desired gene can be created. This type of labeled nucleic acid molecule is called a nucleic acid probe. 세포의 whole genomic DNA (genome) 를한종류의제한효소로절단한다 그결과생성된모든 DNA fragments 를 plasmid 에삽입하여 recombinant plasmid 를만든다 그많은 recombinant plasmids 을하나씩지니고있는 bacteria 를제작한다 (=shotgun cloning) 이러한 bacteria 의모음을 genomic library 라고부른다 RPTSE Biology Fall 2015, Dr. Jong B. Lee 4

5 이 radioactive probe 와 recombinant plasmids 와섞고반응하면 radioactive probe 가상보적인서열을지닌 recombinant plasmids 와결합하고그결합여부는방사능의탐지를통해서알수있다 This called autoradiograph. Using a reverse transcriptase is another method for synthesizing a gene of interest: An artificial DNA gene from a molecule of mrna 즉 complementary DNA (=cdna) 의제작방법이다 Genomic DNA library 와는달리조직세포마다 cdna library 는다르다. Why? A final approach is to synthesize a gene using a automated DNA synthesizer. DNA Profiling and Forensic Science DNA profiling has rapidly revolutionized the field of forensics ( 법의학 ). Forensics is the scientific analysis of evidence from crime scenes. DNA profiling : a procedure that analyzes a person's unique collection of DNA: To produce a DNA profile, scientists compare genetic markers, sequences in genome that vary from person to person. DNA profiling can be used to determine whether or not two samples of genetic material come from the same individual Murder and Paternity DNA profiling has become a criminology and has provided crucial evidence, 다양한예? The Innocent Project in New York Exonerate over 180 convicted criminals, including several on death row World Trade Center Attack, on : 신원확인으로이용 Settle a question of paternity: of historically interest: Thomas Jefferson & one of the children of his slave Sally Hemings RPTSE Biology Fall 2015, Dr. Jong B. Lee 5

6 Ancient DNA DNA profiling is very specific and powerful that the starting DNA material can even be a partially degraded state Cheddar Man, 9,000-year-old skeleton found in a cave near Cheddar DNA profiling The results showed that Cheddar Man was a direct ancestor (300 generations) of a present-day schoolteacher living only a half mile from the cave DNA profiling Techniques : The Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR) is a technique by which any segment of DNA can be copied quickly and precisely. Through PCR, scientists can obtain enough DNA from even minute amounts of blood or other tissue to allow DNA profiling. ( 소량으로존재하는 DNA 를쉽게증폭할수있는기술이다 ) PCR mixture: 1. DNA template, 2. Taq DNA polymerase (heat stable E.) 3. nucleotide monomers 4. synthetic single-stranded DNA (= primer) PCR Cycle 이들 mixture 가 automatic PCR machine 에서한 cycle 을돌때마다 DNA 의수는두배씩증폭한다. 30 cycle 증폭하면 (2-3 시간소요 ) 2 30 = 10 7 ( 약 1 천만배 ) 정도증폭된다. 최종분자수는 개정도. 1. Denaturing (parental DNA 의이중나선구조를열로서풀어줌 : 94 o C) 2. Annealing (parental DNA 의특정염기서열에 primer 가상보적으로결합할수있도록온도를낮춰줌 : o C) 3. Polymerization or Extension (Taq DNA polymerase 에의해서 parental DNA 를주형으로복사하여새로운 daughter DNA 를합성 : 72 o C 왜냐하면 Taq DNA pol. 의최적온도이기때문에 ) Figure RPTSE Biology Fall 2015, Dr. Jong B. Lee 6

7 Short Tandem Repeat (STR) Analysis DNA profiling relies on indirect methods to compare samples The genetic markers most often used in DNA profiling are inherited variations in the lengths of repetitive DNA segments, which are a series of short noncoding DNA segments that vary from person to person Such a variable region in the genome is a genetic marker, a chromosomal landmark whose inheritance can be studied Some of this DNA consists of short sequences repeated many times in a row (tandemly); such sequences are called short tandem repeats (STRs) For example, one person might have the sequence AGAT repeated 12 times at one place in the genome, the sequence GATA repeated 15 times at a second place. STR analysis is a method for producing and comparing DNA profiling that reflect the lengths of STR sequences at specific sites in the genome Most commonly, STR analysis compares the number of repeat of 13 specific four-nucleotide DNA sequences scattered throughout the genome Once a set of DNA fragments is prepared, The next step in STR analysis is to determine the lengths of these fragments. Gel electrophoresis ( 전기영동 ): a method for sorting macromolecules primarily on the basis of their electrical charge and length. Gel electrophoresis To create a DNA fingerprint, a scientist uses PCR to specifically amplify the STR regions. RPTSE Biology Fall 2015, Dr. Jong B. Lee 7

8 This photo shows Earl Washington just before his release in 2001, after 17 years in prison. Each band consists of DNA molecules of the same length Innocent Project The DNA fragments are visualized as bands on the gel. The bands of different DNA samples can then be compared The bands can be made visible by staining, by exposure onto photographic film or by measuring fluorescence Source of sample Semen on victim Earl Washington Kenneth Tinsley STR marker 1 17, 19 16, 18 17, 19 STR marker 2 STR marker 3 These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder. 13, 16 12, 12 14, 15 11, 12 13, 16 12, 12 One common application of gel electrophoresis is RFLP analysis, Genomics Genomics is the science of studying whole genomes. In which DNA molecules to be compared are exposed to a series of restriction enzymes. The resulting fragments are separated and made visible in a gel. 판정? Figure RPTSE Biology Fall 2015, Dr. Jong B. Lee 8

9 The Human Genome Project In 1990, an international consortium of government-funded researchers and Celera Genomics began the Human Genome Project The goal of the project was to sequence the human genome Completed in (Over 99% ) The human genome is very large: 2.9 billion base pairs, 21,000 genes from 24 different chromosomes Only a small amount of our total DNA is contained in genes that code for proteins, only 2% is responsible for coding sequence (for protein, trna, rrna) 98% is nonocoding sequences: Some of these noncoding DNA is made up of gene control sequences such as promoters, enhancers, and micrornas. Other regions include intron, repetitive DNA, and other noncoding DNA between genes and at the end of chromosome. Some non-coding DNA is important to our health. The potential benefit of having a complete map of the human genome Hundreds of disease-associated genes have already been identified. ex: Mapped a very small number of genes of Parkinson s disease to a specific gene 유전적이다 An altered version of the protein encoded by this gene has also been tied to Alzheimer s disease Sense of smell, song learning in rats Tracking the Anthrax ( 탄저병 ) Killer In October 2001, a Florida man died from inhalation anthrax, a disease caused by breathing spores of the bacterium Bacillus anthracis. 그후여러유사사건발생. Investigators analyzed the genome of the anthrax spores used in each attack. Investigators were able to establish that the spores from all of the cases were identical This suggested a single perpetrator of the crime This investigation is an example of the new field of comparative genomics ( 비교유전체학 : the comparison of whole genomes). RPTSE Biology Fall 2015, Dr. Jong B. Lee 9

10 Comparative Genomics 를이용한성과물 : A Florida dentist transmitted HIV to several patients in 1991 In 1993, after the Aum Shinrikyo cult released anthrax spores in Tokyo, genomic analysis showed why their attack did not kill anyone: They used a harmless veterinary vaccine strain Investigation of the West Nile virus outbreak in 1999 proved that a single natural strain of virus was infecting both birds and humans. In 2005, humans and chimpanzees share 96% of the genomes Genome-Mapping Techniques Sequencing human chromosomal DNA at once is simply not possible. Instead, chromosomes must be chopped up into smaller pieces using restriction enzymes Each piece is inserted into a vector and cloned The clone is then sequenced According to sequences, the fragments are aligned Reassemble into proper order In 1998, the private company Celera Genomics entered the race to map the human genome: It was able to produce a draft of the human genome within three years The Whole Genome Shotgun Method Major challenges Figuring out the functions of the genes and other sequences How they (genes) work together to direct the structure and functions of a living organism Clone each restriction fragment into plasmids Sequence of nucleotides Whose genome was sequenced? The answer is no one s or at least not any person s (compiled from a group of individuals). The genome sequenced by Celera consisted primarily of DNA sampled from the company s president This representative sequence will serve as a standard. RPTSE Biology Fall 2015, Dr. Jong B. Lee 10

11 The process of Science: Can genomics cure cancer? In 2003, FDA approved the drug gefitinib for the treatment of lung cancer Gefitinib targets a protein called EGFR (This protein is found on the outside of cells that line the lungs, it is also found in lung cancer tumors) Unfortunately, gefitinib is ineffective for many patients Why?? Question: Are genetic differences among lung cancer patients responsible for the differences in gefitinib s effectiveness? Hypothesis: mutations in the EGFR gene was causing different responses to gefitinib Prediction: the EGFR gene would have a different DNA sequence in the tumors of responsive patients compared to the tumors of unresponsive patients Experiment: sequencing of the EGFR gene Results: mutations in EGFR only from responsive patients Observation: A few patients responded quite dramatically to the drug Implication (Perspectives): custom-tailored therapy to the genetic makeup of each patient Proteomics Proteomics are a systematic studies of the full protein sets that genomics encode The number of proteins in humans far exceeds the number of genes (about 100,000 proteins) And since proteins, not genes, actually carry out the activities of the cell, scientists must study when and where proteins are produced and how they interact in order to understand the functioning of cells and organisms Human Gene Therapy Human gene therapy is a recombinant DNA procedure that seeks to treat disease by altering the genes of the afflicted person The mutant version of a gene is replaced or supplemented with a properly functioning one, permanently. Which cell type most fits for this purpose? : Bone marrow cells, which include the stem cells are prime candidates Figure RPTSE Biology Fall 2015, Dr. Jong B. Lee 11

12 Ex: Treating Severe Combined Immunodeficiency (SCID) SCID is a fatal inherited disease caused by a single defective gene (=adenosine deaminase), whose absence prevents the development of the immune system SCID patients quickly die unless treated with a bone marrow transplant (Patients: remain isolated within protective bubble ) The first trial of human gene therapy In 1990, NIH, USA, 4-year-old girl with SCID However, the SCID study was halted in 2002, after three patients developed leukemia-like symptoms : The retrovirus used as a vector activated an oncogene, creating cancerous blood cells Although the concept of gene therapy remains promising, very little evidence of safe and effective gene therapy has yet appeared The patient is still alive SAFETY AND ETHICAL ISSUES As soon as scientists realized the power of DNA technology, they began to worry about potential dangers The possibility of the creation of hazardous new pathogens What happen if the transfer of cancer-causing genes are transferred into infectious bacteria and viruses? To address this concerns, scientists developed a set of guidelines that in US and other countries have become formal government regulations Strict laboratory safety procedures have been designed to protect researchers from infection by engineered microbes In addition, strains of microorganisms to be used in recombinant DNA experiments are genetically crippled to ensure that they cannot survive outside laboratory Most concerns to GM foods rather than recombinant microbes in these days RPTSE Biology Fall 2015, Dr. Jong B. Lee 12

13 The Controversy Over Genetically Modified Foods GM strains account for a significant percentage of several agricultural crops in the United States In 1999, controversy over the safety of these foods prompted protests throughout Europe EU suspended the introduction of new GM crops and started considering the possibility of banning the import of all GM foods In US and other countries, the labeling of GM foods is now being debated but has not yet become law A major concern: transgenic plants might pass their new genes to close relatives in nearby wild areas lawn and crop grasses commonly exchange genes with wild relatives via pollen transfer. If domestic plants carrying genes for resistance to herbicides, diseases, or insect pests pollinated wild plants, the offspring might become superweeds that would be very difficult to control. However, researchers may be able to prevent the escape of such plant genes by engineering plants so that they cannot hybridize. Negotiators from 130 countries (including the United States) agreed on a Biosafety Protocol The protocol requires exporters to identify GM organisms present in bulk food shipments and allows importing countries to decide whether the shipments pose environmental or health risks RPTSE Biology Fall 2015, Dr. Jong B. Lee 13