Teresa Audesirk Gerald Audesirk Bruce E Byers Biology
Teresa Audesirk • Gerald Audesirk • Bruce E. Byers Biology: Life on Earth Eighth Edition Lecture for Chapter 13 Biotechnology Copyright © 2008 Pearson Prentice Hall, Inc.
Chapter 13 Outline • • • 13. 1 The World of Biotechnology, p. 252 13. 2 DNA Recombination in Nature, p. 252 13. 3 Biotechnology in Forensics, p. 254 13. 4 Biotechnology in Agriculture, p. 258 13. 5 Biotechnology and the Human Genome, p. 261 • 13. 6 Biotechnology in Medicine, p. 262 • 13. 7 Biotechnology and Ethics, p. 265
Section 13. 1 Outline • 13. 1 The World of Biotechnology – Traditional Applications of Biotechnology – Genetic Engineering – Recombinant DNA
Traditional Applications • Biotechnology is applied biology – Modern focus on genetic engineering, recombinant DNA technology, and analysis of biomolecules
Traditional Applications • Traditional (historical) applications of biotechnology date back to over 10, 000 years ago – Use of yeast to produce beer and wine in Egypt and Near East – Selective breeding of plants – Selective breeding of animals
Genetic Engineering • Genetic engineering refers to the modification of genetic material to achieve specific goals
Genetic Engineering • Major goals of genetic engineering – Learn more about cellular processes, including inheritance and gene expression – Provide better understanding and treatment of diseases, particularly genetic disorders – Generate economic and social benefits through production of valuable biomolecules and improved plants and animals for agriculture
Recombinant DNA • Genetic engineering utilizes recombinant DNA technology – Splicing together of genes or portions of genes from different organisms
Recombinant DNA • Recombinant DNA can be transferred to plants and animals – Modified animals are called transgenic or genetically modified organisms (GMOs) – Most modern biotechnology includes manipulation of DNA
Section 13. 2 Outline • 13. 2 DNA Recombination in Nature – Sexual Reproduction Recombines DNA – Transformation: Acquisition of DNA from the Environment – Viral Transfer: Movement of DNA Between Organism via Viruses
Recombination in Nature • Many natural processes recombine DNA
Sexual Reproduction • Due to crossing over during meiosis, each chromosome in a gamete contains a mixture of alleles from the two parental chromosomes – Thus, eggs and sperm contain recombinant DNA
Transformation • Bacteria can naturally take up DNA from the environment (transformation) and integrate the new genes into the genome (recombination)
Transformation • Small circular DNA molecules (plasmids) carry supplementary genes – Plasmid genes may allow bacteria to grow in novel environments – Plasmid genes may enhance virulence of bacteria in establishing an infection – Plasmid genes may confer resistance to antimicrobial drugs
Viral Transfer of DNA • Viral life cycle 1. 2. 3. 4. Viral particle invades host cell Viral DNA is replicated Viral protein molecules are synthesized Offspring viruses are assembled and break out of the host cell
Viral Transfer of DNA • Viral transfer of DNA – Viruses may package some genes from host cell into viral particles during assembly – Infection of new host cell injects genes from previous host, allowing for recombination
Section 13. 3 Outline • 13. 3 Biotechnology in Forensics – How Biotechnology Revolutionized Forensics – Amplification of DNA by Polymerase Chain Reaction – Gel Electrophoresis: Separation of DNA Fragments – DNA Probes Are Used to Highlight Bands in a Gel – DNA Fingerprinting
Biotechnology and Forensics • Forensics is the science of criminal and victim identification
Biotechnology and Forensics • DNA technology has allowed forensic science to identify victims and criminals from trace biological samples – Genetic sequences of any human individual are unique – DNA analysis reveals patterns that identify people with a high degree of accuracy
Polymerase Chain Reaction • • Forensic technicians typically have very little DNA with which to perform analyses Polymerase Chain Reaction (PCR) produces virtually unlimited copies of a very small DNA sample
Polymerase Chain Reaction • • PCR requires small pieces of DNA (called primers) that are complementary to the gene sequences targeted for copying A PCR “run” is basically DNA replication in a tiny test tube – Template DNA, primer, nucleotides, and DNA polymerase are all in the reaction mix
Polymerase Chain Reaction • Four steps of a PCR cycle 1. Template strand separation – The test tube is heated to 90 -95 o. C to cause the double stranded template DNA to separate into single strands…
Polymerase Chain Reaction • Four steps of a PCR cycle 2. Binding of the primers – The temperature is lowered to 50 o. C to allow the primer DNA segments to bind to the targeted gene sequences through hydrogen bonding…
Polymerase Chain Reaction • Four steps of a PCR cycle 3. New DNA synthesis at targeted sequences – The temperature is raised to 70 -72 o. C where the heat-stable DNA polymerase synthesizes new DNA of the sequences targeted by the primers…
Polymerase Chain Reaction • Four steps of a PCR cycle 4. Repetition of the cycle – The cycle is repeated automatically (by a thermocycler machine) for 20 -30 cycles, producing up to 1 billion copies of the original targeted DNA sequence
Polymerase Chain Reaction • • Choice of primers determines which sequences are amplified (copied) Forensic scientists focus on short tandem repeats (STRs) found within the human genome
Polymerase Chain Reaction • • • STRs are repeated sequences of DNA within the chromosomes that do not code for proteins STRs vary greatly between different human individuals A match of 10 different STRs between suspect and crime scene DNA virtually proves the suspect was at the crime scene
Gel Electrophoresis • • Mixtures of DNA fragments can be separated on the basis of size Gel electrophoresis is a technique used to spread out different-length DNA fragments in a mixture
Gel Electrophoresis • Four steps of gel electrophoresis 1. DNA mixtures are placed into wells at one end of a slab of agarose gel
Gel Electrophoresis • Four steps of gel electrophoresis 2. An electric current introduced through the gel causes the negatively-charged DNA fragments to migrate towards the positive electrode
Gel Electrophoresis • Four steps of gel electrophoresis 3. Short DNA fragments move more easily through the three-dimensional meshwork of fibers between the gel – Short DNA fragments migrate farther than long DNA fragments so the mixture is separated into bands of DNA of specific lengths
Gel Electrophoresis • Four steps of gel electrophoresis 4. The invisible bands of DNA are made visible using stains or DNA probes
DNA Probes • DNA probes are short single-stranded DNA fragments used to identify DNA in a gel pattern – Probe sequence is complementary to a DNA fragment somewhere in the gel pattern
DNA Probes • DNA probes – Probes identify the location of a gene sequence by hydrogen-bonding to the band containing it
DNA Probes • DNA probes – Probes may have colored molecules attached to them to allow for visual identification of the bands to which they bind – Gel DNA pattern is usually transferred to piece of nylon paper before probing
DNA Fingerprinting • • DNA from a crime scene sample can be amplified by PCR and run on a gel with suspect DNAs Short tandem repeats (STRs) in the gel DNA can be identified by DNA probes
DNA Fingerprinting • • Distinctive pattern of STR numbers and lengths are fairly unique to a specific individual (forming a DNA fingerprint) DNA fingerprint from crime scene can be matched with DNA fingerprint of suspect
Section 13. 4 Outline • 13. 4 Biotechnology in Agriculture – Many Crops Are Genetically Modified – Cloning of the Desired Gene – Restriction Enzymes Cut DNA at Specific Places – Splicing of DNA Fragments Is Aided by Sticky Ends
Section 13. 4 Outline • 13. 4 Biotechnology in Agriculture (continued) – Plasmids Are Used to Insert Genes Into a Plant Cell – GM Plants May Be Engineered to Produce Medicines – GM Animals May Be Useful in Agriculture and Medicine
Many Crops Are Genetically Modified • One third to three-quarters of corn, cotton, and soybeans grown in the US are genetically modified
Many Crops Are Genetically Modified • Crop plants are commonly modified to improve insect and herbicide resistance – Herbicide resistant crops withstand applications of weed-killing chemicals – Bt gene (from Bacillus thuringiensis bacterium) can be inserted into plants to produce insect-killing protein in crops
Cloning of the Desired Gene • Modifying a plant genetically begins with gene cloning 1. Desired gene is first isolated from organism containing it • Desired gene may alternately be synthesized in the laboratory
Cloning of the Desired Gene • Modifying a plant genetically begins with gene cloning 2. Gene is next inserted into a small DNA circle called a plasmid which replicates itself autonomously in bacterial cells
Restriction Enzymes Cut DNA • • A DNA sequence (e. g. a gene) can be removed from a chromosome using special enzymes Restriction enzymes are nucleases that cut DNA at specific nucleotide sequences
Restriction Enzymes Cut DNA • Enzymes that create staggered cuts with “sticky ends” are the most useful in gene cloning
Splicing of DNA Fragments • Sticky ends allow for splicing of a DNA fragment with another complementary fragment – Bt gene can be cut of the Bacillus chromosome with the same enzyme used to cut open the plasmid – Bt gene fragment ends can base-pair with sticky ends of the opened plasmid, adding gene to the plasmid circle
Splicing of DNA Fragments • DNA ligase enzyme used next to permanently bond gene into plasmid
Plasmids Are Used to Insert Genes • The Ti plasmid from Agrobacterium tumefaciens is ideal for transferring genes into plant chromosomes
Plasmids Are Used to Insert Genes • Agrobacterium infects plant cells and inserts its small Ti plasmid into a plant chromosome in the nucleus – Pathogenic effects of certain tumor-causing Ti plasmid genes can be disabled – A gene inserted into a Ti plasmid is therefore carried into the plant cell chromosomes by a natural process
GM Plants and Medicines • Medically useful genes can be inserted into plants—example: – Potatoes have been engineered to produce harmless hepatitis B virus and E. coli proteins, stimulating an immune response when eaten
GM Plants and Medicines • Medically useful genes can be inserted into plants—example: – Plants could be engineered to produce human antibodies, conferring passive immunity to microbial infection merely by eating the plant
GM Animals • Transgenic (genetically modified or GM) animals can be engineered by incorporating genes into chromosomes of a fertilized egg
GM Animals • Healthy transgenic animals are difficult to engineer – Growth hormone genes have been inserted into pigs and fish species but some abnormalities have been observed • Animals like sheep might be engineered to produce medically important proteins in their milk
Section 13. 5 Outline • 13. 6 Biotechnology and the Human Genome – Findings and Applications of the Human Genome Project
The Human Genome Project • Findings – Human genome contains ~25, 000 genes – New genes, including many disease-associated genes have been discovered – Has determined the nucleotide sequence of all the DNA in our entire set of genes, called the human genome – The genes comprise 2% of all the DNA
The Human Genome Project • Applications – Improved diagnosis, treatment and cures of genetic disorders or predispositions – Comparison of our genome to those of other species will clarify the genetic differences that help to make us human
Section 13. 6 Outline • 13. 6 Biotechnology in Medicine – DNA Technology Can Be Used to Diagnose Inherited Disorders – Restriction Enzyme Fragment Analysis – Identification of Defective Alleles with DNA Probes – DNA Technology Can Be Used to Treat Disease
Diagnosis of Inherited Disorders • • Potential parents can learn if they are carriers of a heritable disorder through testing Alleles for defective genes differ from normal, functional genes in nucleotide sequence
Diagnosis of Inherited Disorders • Two methods employed to detect a defective allele in a person’s DNA sample – Restriction enzyme fragment analysis – Identification of defective alleles with DNA probes
Restriction Enzyme Fragment Analysis • A particular restriction enzyme may cut two different alleles of a gene differently – Differences in nucleotide sequence within genes produces different numbers of cutting sites and different lengths of fragments
Restriction Enzyme Fragment Analysis – Differences in restriction enzyme fragments between genes are known as restriction fragment length polymorphisms (RFLPs) – RFLP differences are revealed in gel electrophoresis
Restriction Enzyme Fragment Analysis • • Before PCR, restriction fragment differences were used to generate DNA fingerprints in forensics RFLP analysis is now commonly used to identify the presence of the sickle-cell anemia gene in a person’s DNA
DNA Probes • • Defective alleles can also be identified using DNA probes DNA probing is especially useful where there are many different alleles at a single gene locus – Cystic fibrosis is a disease caused by any of 32 alleles out of 1000 total possible alleles
DNA Probes • Arrays of single-stranded DNA complementary to each of the defective alleles can be bound to filter paper 1. A person’s DNA sample is cut up and separated into single-strands 2. The array is bathed in the DNA sample 3. Strands of DNA binding to complementary sequence on the paper indicate presence of a defective allele in person’s genome
DNA Probes • • • An expanded version of this type of DNA analysis is known as a microarray A microarray contains up to thousands of probes for a variety of disease-related alleles Microarray analysis has the potential to comprehensively identify disease susceptibility
Disease Treatment • Treatments using DNA technology – Administration of proteins to treat but not cure a disorder • Human insulin produced inexpensively and rapidly in recombinant bacteria for diabetics • Growth hormone and blood clotting factors produced safely and inexpensively in recombinant bacteria
Disease Treatment • Treatments using DNA technology – Replacing defective genes to possibly cure a disorder • Replacement of defective cystic fibrosis allele using a virus to carry in a functional gene sequence into patient lung cells • Defective bone marrow cell DNA replacement by functional gene in severe combined immune deficiency (SCID) patients
Section 13. 7 Outline • 13. 7 Biotechnology and Ethics – Issues Surrounding GM Organisms in Agriculture – Scientific Objections to Genetically Modified Organisms – Ethics of Using Biotechnology on the Human Genome
GM Organisms in Agriculture • The goal of breeding or genetically modifying plants or livestock is to make them more productive, efficient, or useful
GM Organisms in Agriculture • Genetic modification differs from selective breeding (“traditional biotechnology”) – Genetic engineering is much more rapid – Genetic engineering can transfer genes between species – Genetic engineering can produce new genes never seen before on Earth
GM Organisms in Agriculture • Benefits of genetically modified plants – Transgenic crops decrease applications of pesticides, saving fuel, labor, and money – GM plants can be sold at a lower price due to farm savings – Genetically engineered crops can deliver greater amounts of vitamins • e. g. “golden rice” which produces vitamin A
Scientific Objections to GMOs • Safety issues from eating GMOs – Could ingestion of Bt protein in insect-resistant plants be dangerous to humans? – Are transgenic fish producing extra growth hormone dangerous to eat?
Scientific Objections to GMOs • Safety issues from eating GMOs – Could GM crops cause allergic reactions? • USDA now monitors GM foods for allergic potential – Toxicology study of GM plants (2003) concluded that ingestion of current transgenic crops pose no significant health dangers
Scientific Objections to GMOs • Environmental hazards posed by GMOs – Pollen from modified plants can carry GM genes to the wild plant population • Could herbicide resistance genes be transferred to weed species, creating superweeds?
Scientific Objections to GMOs • Environmental hazards posed by GMOs – Could GM fish reduce biodiversity in the wild population if they escape? • Reduced diversity in wild fish makes them more susceptible to catastrophic disease outbreaks
Scientific Objections to GMOs • Environmental hazards posed by GMOs – US found to lack adequate system to monitor changes in ecosystem wrought by GMOs (National Academy of Science Study 2003)
The Human Genome • Should parents be given information about the genetic health of an unborn fetus?
The Human Genome • Should parents be allowed to select the genomes of their offspring? – Embryos from in vitro fertilization are currently tested before implantation – Many unused embryos are discarded
The Human Genome • Should parents be allowed to design or correct the genomes of their offspring?
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