Electrophoresis made Easy Biotechnology

Chapter 13 2 Traditional Applications Biotechnology is applied biology Modern focus on genetic engineering, recombinant DNA technology, and analysis of biomolecules Modern focus on genetic engineering, recombinant DNA technology, and analysis of biomolecules

Chapter 13 3 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 Use of yeast to produce beer and wine in Egypt and Near East Selective breeding of plants Selective breeding of plants Selective breeding of animals Selective breeding of animals

Chapter 13 4 Genetic Engineering Genetic engineering refers to the modification of genetic material to achieve specific goals Learn more about cellular processes, including inheritance and gene expression Learn more about cellular processes, including inheritance and gene expression Provide better understanding and treatment of diseases, particularly genetic disorders 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 Generate economic and social benefits through production of valuable biomolecules and improved plants and animals for agriculture

Chapter 13 5 Recombinant DNA Genetic engineering utilizes recombinant DNA technology Splicing together of genes or portions of genes from different organisms Splicing together of genes or portions of genes from different organisms Recombinant DNA can be transferred to plants and animals Modified animals are called transgenic or genetically modified organisms (GMOs) Modified animals are called transgenic or genetically modified organisms (GMOs) Most modern biotechnology includes manipulation of DNA Most modern biotechnology includes manipulation of DNA

Chapter 13 6 Recombination in Nature Recombination in Nature Many natural processes recombine DNA: 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 Thus, eggs and sperm contain recombinant DNA

Chapter 13 7 Transformation Transformation Bacteria can naturally take up DNA from the environment ( transformation ) and integrate the new genes into the genome ( recombination )

Chapter µ m Recombination in Bacteria (a)(b)(c) Bacterium Chromosome Plasmid Plasmid replicates in cytoplasm DNA fragment incorporated into chromosome Plasmid transferred to new host DNA fragments transferred to new host

Chapter 13 9 Transformation Transformation Small circular DNA molecules ( plasmids ) carry supplementary genes Plasmid genes may allow bacteria to grow in novel environments Plasmid genes may allow bacteria to grow in novel environments Plasmid genes may enhance virulence of bacteria in establishing an infection Plasmid genes may enhance virulence of bacteria in establishing an infection Plasmid genes may confer resistance to antimicrobial drugs Plasmid genes may confer resistance to antimicrobial drugs

Chapter Viral Transfer of DNA Viral Transfer of DNA Viral life cycle 1.Viral particle invades host cell 2.Viral DNA is replicated 3.Viral protein molecules are synthesized 4.Offspring viruses are assembled and break out of the host cell

Chapter Viral Transfer of DNA Viral Transfer of DNA Viral transfer of DNA Viruses may package some genes from host cell into viral particles during assembly 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 Infection of new host cell injects genes from previous host, allowing for recombination

Chapter Viruses May Transfer Genes

Chapter Biotechnology and Forensics Forensics is the science of criminal and victim identification DNA technology has allowed forensic science to identify victims and criminals from trace biological samples Genetic sequences of any human individual are unique Genetic sequences of any human individual are unique DNA analysis reveals patterns that identify people with a high degree of accuracy DNA analysis reveals patterns that identify people with a high degree of accuracy

Chapter 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

Chapter 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 Template DNA, primer, nucleotides, and DNA polymerase are all in the reaction mix

Chapter Polymerase Chain Reaction Four steps of a PCR cycle 1.Template strand separation – The test tube is heated to o C to cause the double stranded template DNA to separate into single strands… 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…

Chapter Polymerase Chain Reaction 3.New DNA synthesis at targeted sequences The temperature is raised to o C where the heat-stable DNA polymerase synthesizes new DNA of the sequences targeted by the primers… 4.Repetition of the cycle The cycle is repeated automatically (by a thermocycler machine) for cycles, producing up to 1 billion copies of the original targeted DNA sequence

Chapter Polymerase Chain Reaction: (a) One PCR Cycle Original Double- helix DNA Separate DNA Strands 90 °C Primers & DNA polymerase bind 50 °C DNA Polymerase Primer DNA 72 °C DNA synthesized

Chapter Polymerase Chain Reaction: (b) Multiple PCR Cycles DNA fragment to be amplified 2 copies4 copies8 copies

Chapter 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

Chapter 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

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Chapter 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

Chapter Gel Electrophoresis Four steps of gel electrophoresis 1.DNA mixtures are placed into wells at one end of a slab of agarose gel 2.An electric current introduced through the gel causes the negatively-charged DNA fragments to migrate towards the positive electrode

Chapter 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 4.The invisible bands of DNA are made visible using stains or DNA probes

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Chapter RFLP: Gel Electrophoresis Direction of Migration Larger fragments move more slowly; smaller fragments move more rapidly

Chapter 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 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

Chapter C SR CI EC SR CI EM NE EM NE EC SR CI EC SR CI EM NE EM NE E DNA Fingerprint in Forensics SuspectsSuspects Q: Which suspect should be indicted? A: #3 is prime suspect

Chapter 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

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Chapter Restriction Enzymes Cut DNA Enzymes that create staggered cuts with “ sticky ends ” are the most useful in gene cloning

Chapter Splicing of DNA Fragments Sticky ends allow for splicing of a DNA fragment with another complementary fragment ( Bt – Bacillus thurengensis ) Bt gene can be cut out of the Bacillus chromosome with the same enzyme used to cut open the plasmid Bt gene can be cut out 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 Bt gene fragment ends can base-pair with sticky ends of the opened plasmid, adding gene to the plasmid circle

Chapter Splicing of DNA Fragments DNA ligase enzyme used next to permanently bond gene into plasmid

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Chapter Plasmids Are Used to Insert Genes The Ti plasmid from Agrobacterium tumefaciens is ideal for transferring genes into plant chromosomes

Chapter 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 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 A gene inserted into a Ti plasmid is therefore carried into the plant cell chromosomes by a natural process

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Chapter The Human Genome Project Findings Human genome contains ~25,000 genes Human genome contains ~25,000 genes New genes, including many disease-associated genes have been discovered 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 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 genes comprise 2% of all the DNA

Chapter The Human Genome Project Applications Improved diagnosis, treatment and cures of genetic disorders or predispositions 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 Comparison of our genome to those of other species will clarify the genetic differences that help to make us human

Chapter 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 Cystic fibrosis is a disease caused by any of 32 alleles out of 1000 total possible alleles

Chapter DNA Probes Arrays of single-stranded DNA complementary to each of the defective alleles can be bound to filter paper A person’s DNA sample is cut up and separated into single-strands The array is bathed in the DNA sample Strands of DNA binding to complementary sequence on the paper indicate presence of a defective allele in person’s genome

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Chapter 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

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Chapter Scientific Objections to GMOs Safety issues from eating GMOs Could ingestion of Bt protein in insect- resistant plants be dangerous to humans? Could ingestion of Bt protein in insect- resistant plants be dangerous to humans? Are transgenic fish producing extra growth hormone dangerous to eat? Are transgenic fish producing extra growth hormone dangerous to eat? Could GM crops cause allergic reactions? 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 Toxicology study of GM plants (2003) concluded that ingestion of current transgenic crops pose no significant health dangers

Chapter Scientific Objections to GMOs Environmental hazards posed by GMOs Pollen from modified plants can carry GM genes to the wild plant population Pollen from modified plants can carry GM genes to the wild plant population – Could herbicide resistance genes be transferred to weed species, creating superweeds? Could GM fish reduce biodiversity in the wild population if they escape? 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

Chapter 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) US found to lack adequate system to monitor changes in ecosystem wrought by GMOs (National Academy of Science Study 2003)

Chapter The Human Genome Should parents be given information about the genetic health of an unborn fetus? Should parents be allowed to select the genomes of their offspring? Embryos from in vitro fertilization are currently tested before implantation Embryos from in vitro fertilization are currently tested before implantation Many unused embryos are discarded Many unused embryos are discarded Should parents be allowed to design or correct the genomes of their offspring?

Chapter Hope through Gene Therapy

Chapter Human Cloning: Permanent Genetic Correction? Parents with genetic disease Zygote with defective gene Embryo with defective gene Baby with genetic disorder Cell Culture Viral vector with therapeutic gene Treated Culture Genetically corrected cell from culture Enucleated egg cell Genetically corrected egg cell Genetically corrected embryo clone Healthy baby

Chapter 13 The end see also the following related videos: 1. Plasmid Cloning 2. DNA Fingerprinting 3. Gene Therapy and many more! Plasmid CloningDNA FingerprintingGene TherapyPlasmid CloningDNA FingerprintingGene Therapy