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Chapter 12 DNA Technology PowerPoint® Lectures for Campbell Essential Biology, Fourth Edition – Eric Simon, Jane Reece, and Jean Dickey Campbell Essential Biology with Physiology, Third Edition – Eric Simon, Jane Reece, and Jean Dickey Lectures by Chris C. Romero, updated by Edward J. Zalisko © 2010 Pearson Education, Inc. 1. Recombinant DNA technology 1.Application 2.Techniques 2. DNA profiling and Forensic Science 1.Techniques 3. Genomics and proteomics 1.Human genome project 2.Comparing genomes 3.Genome mapping techniques 4.Proteomics 4. Human Gene Therapy © 2010 Pearson Education, Inc. DNA Technology • • • Modern laboratory techniques for studying and manipulating genetic material. Scientists can modify specific genes and move them between organisms like bacteria, plants, and animals. Biotechnology today means the use of DNA technology, methods for: Studying and manipulating genetic material, Modifying specific genes, Moving genes between organisms Recombinant DNA Technology • When scientists combine pieces of DNA from two different sources (different species) to form a single DNA molecule. • Recombinant DNA technology is widely used in genetic engineering, the direct manipulation of genes for practical purposes © 2010 Pearson Education, Inc. Cutting and Pasting DNA with Restriction Enzymes • Recombinant DNA is produced by combining two ingredients a bacterial plasmid and a gene of interest • To combine these ingredients, a piece of DNA must be spliced into a plasmid. • This splicing process can be accomplished using restriction enzymes which cut DNA at specific nucleotide sequences – these cuts produce pieces of DNA called restriction fragments with “sticky ends” important for joining DNA from different sources • DNA ligase connects the DNA pieces into continuous strands by forming bonds between adjacent nucleotides. Recombinant DNA Techniques Bacteria represent the workhorses of modern biotechnology. To manipulate genes in the laboratory, bacterial plasmids - small, circular DNA molecules that are separate from the much larger bacterial chromosome are used - are ideal for gene cloning, the production of multiple identical copies of a gene-carrying piece of DNA • Can easily incorporate foreign DNA • Are readily taken up by bacterial cells • Can act as vectors, a DNA carriers Plasmids that move genes from one cell to Bacterial chromosome another Remnant of bacterium © 2010 Pearson Education, Inc. Colorized TEM Plasmids: Cutting and pasting DNA Recognition sequence for a restriction enzyme DNA A restriction enzyme cuts the DNA into fragments. Restriction enzyme A DNA fragment is added from another source. Fragments stick together by base pairing. DNA ligase joins the fragments into strands. Recombinant DNA molecule © 2010 Pearson Education, Inc. DNA ligase Using recombinant DNA technology to produce useful products Cut both DNAs with same enzyme. Gene of Other interest genes Mix the DNAs and Gene of interest join them together. Bacterial cell DNA fragments from cell Isolate DNA. Cell containing the gene of interest Isolate plasmids. Recombinant DNA plasmids Bacteria take up recombinant plasmids. Plasmid DNA Bacterial clone Recombinant bacteria Clone the bacteria. Find the clone with the gene of interest. Some uses of genes Gene for pest resistance Inserted into plant Some uses of proteins Protein for dissolving clots in heart attack therapy Gene for toxic-cleanup bacteria © 2010 Pearson Education, Inc. Genes may be inserted into other organisms. The gene and protein of interest are isolated from the bacteria. Harvested proteins may be used directly. Protein for “stone-washing” jeans Applications of DNA Technology : From Humulin to Foods to “Pharm” Animals DNA technology is used to produce medically valuable molecules, including: • • • Humulin is human insulin produced by genetically modified bacteria Human growth hormone (HGH) The hormone EPO (Erythropoietin), which stimulates production of red blood cells Vaccines, harmless variant of a disease - causing microbe (bacteria or virus) that is used to prevent an infectious disease. © 2010 Pearson Education, Inc. Applications of DNA Technology: Genetically Modified Foods • DNA technology is improving productivity of agriculture and quickly replacing traditional plant-breeding programs • The modified organisms have acquired one or more genes artificially. • If the new gene is from another organism (another species) the recombinant organism is called a transgenic organisms. • 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 © 2010 Pearson Education, Inc. Applications of DNA Technology: GM Foods II “Golden rice” has been genetically modified to contain beta-carotene. – Our bodies use beta-carotene to make vitamin A. © 2010 Pearson Education, Inc. Applications of DNA Technology: “Pharm” Animals • While transgenic plants are used today as commercial products, transgenic whole animals are currently only in the testing phase. • These transgenic sheep carry a gene for a human blood protein. – This protein may help in the treatment of cystic fibrosis. • While transgenic animals are currently used to produce potentially useful proteins, none are yet found in our food supply. • It is possible that DNA technology will eventually replace traditional animal breeding. © 2010 Pearson Education, Inc. Transgenic animals raised for the purposes of producting pharmaceuticals are called pharm animals DNA Profiling and Forensic Science • • • Forensics the scientific analysis of evidence from crime scenes, has been revolutionized by DNA technology. DNA profiling is used to determine whether two DNA samples come from the same individual. To produce a DNA profile, scientists compare genetic markers, sequences in the genome that vary from person to person Investigating Murder, Paternity, and Ancient DNA DNA profiling (DNA fingerprinting) can be used to establish innocence or guilt of a criminal suspect, identity victims, determine paternity, identify illegally exported animals products, trace the evolutionary history of organisms and contribute to research. © 2010 Pearson Education, Inc. Overview of DNA profiling 1. Collect cells 2. Extract DNA 3. Cut the DNA in fragments using the same restriction enzyme 1 DNA isolated Crime scene 2 DNA amplified 4. Separate the fragments using gel electrophoresis 3 DNA compared © 2010 Pearson Education, Inc. Suspect 1 Suspect 2 Profiling Techniques The Polymerase Chain Reaction (PCR)-specific segment of DNA is targeted and copied. Scientists can obtain enough DNA in blood or tissues to allow profiling. Short Tandem Repeat (STR) Analysis- used to compare genomes in two samples to prove they came from the same person. This is used by a series of short sequences (Repetitive DNA) that is repeated many times one after another. Gel Electrophoresis - Comparing length of DNA fragments by sorting macromolecules. © 2010 Pearson Education, Inc. Profiling Techniques: The Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR): • Is a technique to copy quickly and precisely any segment of DNA and • Can generate enough DNA, from even minute amounts of blood or other tissue, to allow DNA profiling • a single DNA molecule can be replicated in a test tube to make 30 million identical copies in a few hours © 2010 Pearson Education, Inc. Polymerase Chain Reaction: DNA Replication in a Test Tube Exponential Increase in the Number of DNA Molecules each Cycle © 2010 Pearson Education, Inc. Profiling Techniques: Short Tandem Repeat (STR) Analysis How do you test if two samples of DNA come from the same person? •Repetitive DNA: Compares dozen of short segment of repetitive sequences, present in multiple copies, that makes up much of the DNA that lies between genes in humans •Short tandem repeats (STRs) are short sequences of DNA that are repeated many times, tandemly (one after another), in the genome Used as a method of DNA profiling by comparing the lengths of STR sequences at certain sites in the genome © 2010 Pearson Education, Inc. Short tandem repeat (STR) sites STR site 1 AGAT STR site 2 GATA Crime scene DNA Different numbers of short tandem repeats Same number of short tandem repeats Suspect’s DNA AGAT GATA STR sites contain tandem repeats of 4-nucleotide sequences. The number of repetitions at each site vary from individual to individual. Here, both the samples have same number of repeats (7) at first site but different numbers in the second site © 2010 Pearson Education, Inc. Gel Electrophoresis STR analysis compares the lengths of DNA fragments • Uses gel electrophoresis, a method for sorting macromolecules usually proteins or nucleic acids—primarily by their electrical charge and Size • The DNA fragments are visualized as “bands” on the gel. • The differences in the locations of the bands reflect the different lengths of the DNA fragments. Mixture of DNA fragments of different sizes Band of longest (slowest) fragments Power source Gel © 2010 Pearson Education, Inc. Band of shortest Completed gel (fastest) fragments Visualizing STR fragment patterns • Difference in the locations of the band reflect the different length of DNA fragments • Provide evidence that crime scene DNA did not come from the suspect © 2010 Pearson Education, Inc. Amplified crime scene DNA Amplified suspect’s DNA Longer fragments Shorter fragments Genomics and Proteomics Genomics the study of complete sets of genes. Bacteria were the first targets of genomics. As of 2009, the genomes of nearly 1000 species have been published, including Baker’s yeast, Mice, Fruit flies, Rice and Sorghum Genome - Mapping Techniques The whole- genome shotgun method involves sequencing DNA fragments from an entire genome and then assembling the sequences. Proteomics The systematic study of the full set of proteins found in organisms. © 2010 Pearson Education, Inc. The Human Genome Project • In 1990 an international consortium of government-funded the Human Genome Project. The goal was to sequence the human genome so scientists could have roadmap for finding genes • Completed in 2004: – Over 99% of the genome had been determined to 99.999% accuracy – 3.2 billion nucleotide pairs were identified, about 21,000 genes were found and about 98% of the human DNA was identified as noncoding • The Human Genome Project can help map the genes for specific diseases such as: – Alzheimer’s disease – Parkinson’s disease © 2010 Pearson Education, Inc. Genome-Mapping Techniques Genomes are most often sequenced using whole-genome shotgun method in which: – The entire genome is chopped into fragments using restriction enzymes – The fragments are cloned and sequenced – Computers running specialized mapping software reassemble the millions of overlapping short sequences into a single continuous sequence for every chromosome—an entire genome • Begun in 2006, the Human Variome Project: – Seeks to collect information on all of the genetic variations that affect human health © 2010 Pearson Education, Inc. Chromosome Chop up with restriction enzyme DNA fragments Sequence fragments Align fragments Reassemble full sequence Human Gene Therapy What is it? Human Gene Therapy a recombinant DNA procedure intended to treat disease by altering an afflicted person's genes. How? Some cases, a mutant version of a gene is replaced or supplemented with the normal allele. Or replaced long enough to fix the medical problem. © 2010 Pearson Education, Inc. HUMAN GENE THERAPY • Human gene therapy: – Is a recombinant DNA procedure Normal human gene isolated and cloned – Seeks to treat disease by altering the genes of the afflicted person – Often replaces or supplements the mutant version of a gene with a properly functioning one © 2010 Pearson Education, Inc. Normal human gene inserted into virus Harmless virus (vector) Virus containing normal human gene Bone marrow Healthy person Virus injected into patient with abnormal gene Bone of person with disease Ethics of DNA Technology Controversy over Genetically Modified Food • The debate about genetically modified crops centers on whether they might harm humans or damage the environment by transferring genes through cross-pollination with other species. Ethical Questions raised by DNA Technology • Some ethical questions are how far should we take technology? • Some controversies are whether or not it is morally right to know your DNA when you are born and have a DNA profile, and how private that would be. • Also whether or not parents should be able to give their child who has dwarfism a growth hormone to make them grow. • Scientist are still weighing out the positive and negative effects of DNA technology. © 2010 Pearson Education, Inc. The diagram here summarizes _____. A) how a gene is cloned B) how a human could be cloned C) human gene therapy D) how a vaccine is made E) how viruses can cause disease Answer: C Skill: Knowledge/Comprehension © 2010 Pearson Education, Inc. Please read the following scenario then answer the following question(s). Molecular biologists have perfected DNA fingerprinting so that it is possible to use the technique to provide evidence to solve crimes and even identify a child's parents. Recently, a U.S. immigrant asked the U.S. Citizenship and Immigration Services for permission to have her young daughter who was living with grandparents in their homeland join her. Her request was denied because there was an apparent mix-up with the child's birth certificate and it could not be used as proof of maternity. Proof is required in cases such as this. The mother requested DNA fingerprinting to make her case. Samples of DNA were taken from the mother (Mom) and daughter (D1) as well as from another daughter (D2) and a son (S) living in the United States with her. Tandem repeat analysis was run on the four samples, and the results are shown here. © 2010 Pearson Education, Inc. 1) The results indicate that ______. A) she is not the mother of the son B) she is the mother of daughter D1 C) she is not the mother of daughter D1 D) daughter D1 and daughter D2 are identical twins E) the mother could not be the mother of both daughter D1 and daughter D2 © 2010 Pearson Education, Inc.