* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Recombinant Technology
Comparative genomic hybridization wikipedia , lookup
Genome (book) wikipedia , lookup
DNA profiling wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
DNA polymerase wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Metagenomics wikipedia , lookup
SNP genotyping wikipedia , lookup
Human genome wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Genome evolution wikipedia , lookup
Nutriepigenomics wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Point mutation wikipedia , lookup
Primary transcript wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Microsatellite wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Genetic engineering wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
DNA supercoil wikipedia , lookup
Epigenomics wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
DNA vaccination wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Designer baby wikipedia , lookup
Non-coding DNA wikipedia , lookup
Genome editing wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Genomic library wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Molecular cloning wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Microevolution wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Helitron (biology) wikipedia , lookup
Chapter 12 DNA Technology and Genomics PowerPoint Lectures for Biology: Concepts and Connections, Fifth Edition – Campbell, Reece, Taylor, and Simon Lectures by Chris Romero Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings DNA and Crime Scene Investigations • Many violent crimes go unsolved – For lack of enough evidence • If biological fluids are left at a crime scene – DNA can be isolated from them Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • DNA fingerprinting is a set of laboratory procedures – That determines with near certainty whether two samples of DNA are from the same individual – That has provided a powerful tool for crime scene investigators Investigator at one of the crime scenes (above), Narborough, England (left) Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings BACTERIAL PLASMIDS AND GENE CLONING 12.1 Plasmids are used to customize bacteria: An overview • Gene cloning is one application of DNA technology – Methods for studying and manipulating genetic material Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Researchers can insert desired genes into plasmids, creating recombinant DNA – And insert those plasmids into bacteria Bacterium Cell containing gene of interest 1 Plasmid isolated 2 DNA isolated 3 Gene inserted into plasmid Plasmid Bacterial chromosome Recombinant DNA (plasmid) DNA Gene of 4 Plasmid put into interest bacterial cell Recombinant bacterium 5 Cell multiplies with gene of interest Copies of gene Clone of cells Gene for pest resistance inserted into plants Figure 12.1 Copies of protein Gene used to alter bacteria for cleaning up toxic waste Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Protein used to make snow form at higher temperature Protein used to dissolve blood clots in heart attack therapy • If the recombinant bacteria multiply into a clone – The foreign genes are also copied Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.2 Enzymes are used to “cut and paste” DNA • The tools used to make recombinant DNA are – Restriction enzymes, which cut DNA at specific sequences – DNA ligase, which “pastes” DNA fragments together Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Creating recombinant DNA using restriction enzymes and DNA ligase Restriction enzyme recognition sequence 1 GAATTC CTTAAG DNA Restriction enzyme cuts the DNA into fragments 2 Sticky end Addition of a DNA fragment from another source 3 Two (or more) fragments stick together by base-pairing 4 G A AT T C C T TA A G G A AT T C C T TA A G DNA ligase pastes the strand 5 Figure 12.2 Recombinant DNA molecule Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.3 Genes can be cloned in recombinant plasmids: A closer look • Bacteria take the recombinant plasmids from their surroundings – And reproduce, thereby cloning the plasmids and the genes they carry Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Cloning a gene in a bacterial plasmid E.coli Human cell Isolate DNA 1 from two sources Cut both DNAs 2 with the same restriction enzyme Plasmid DNA Gene V Sticky ends 3 Mix the DNAs; they join by base-pairing 4 Add DNA ligase to bond the DNA covalently Gene V Recombinant DNA plasmid 5 Put plasmid into bacterium by transformation 6 Clone the bacterium Recombinant bacterium Figure 12.3 Bacterial clone carrying many copies of the human gene Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.4 Cloned genes can be stored in genomic libraries • Genomic libraries, sets of DNA fragments containing all of an organism’s genes – Can be constructed and stored in cloned bacterial plasmids or phages Genome cut up with restriction enzyme Recombinant plasmid Recombinant phage DNA or Bacterial clone Figure 12.4 Plasmid library Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Phage clone Phage library 12.5 Reverse transcriptase helps make genes for cloning • Reverse transcriptase can be used to make smaller, complementary DNA (cDNA) libraries – Containing only the genes that are transcribed by a particular type of cell Cell nucleus Exon Intron DNA of eukaryotic gene Exon Intron Exon 1 Transcription RNA transcript 2 RNA splicing (removes introns) mRNA Test tube Reverse transcriptase 3 Isolation of mRNA from cell and addition of reverse transcriptase; synthesis of DNA strand cDNA strand 4 Breakdown of RNA 5 Synthesis of second DNA strand Figure 12.5 cDNA of gene (no introns) Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.6 Recombinant cells and organisms can massproduce gene products • Applications of gene cloning include – The mass production of gene products for medical and other uses Table 12.6 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Different organisms, including bacteria, yeast, and mammals – Can be used for this purpose Figure 12.6 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.7 DNA technology is changing the pharmaceutical industry • DNA technology – Is widely used to produce medicines and to diagnose diseases Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Therapeutic hormones • In 1982, humulin, human insulin produced by bacteria – Became the first recombinant drug approved by the Food and Drug Administration Figure 12.7A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Diagnosis and Treatment of Disease • DNA technology – Is being used increasingly in disease diagnosis Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Vaccines • DNA technology – Is also helping medical researchers develop vaccines Figure 12.7B Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING 12.8 Nucleic acid probes identify clones carrying specific genes • DNA technology methods – Can be used to identify specific pieces of DNA Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • A nucleic acid probe – Is a short, single-stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA – Can tag a desired gene in a library Radioactive probe (DNA) Mix with singlestranded DNA from various bacterial (or phage) clones Single-stranded DNA Figure 12.8 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Base pairing indicates the gene of interest CONNECTION 12.9 DNA microarrays test for the expression of many genes at once • DNA microarray assays – Can reveal patterns of gene expression in different kinds of cells Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • DNA microarray DNA microarray Each well contains DNA from a particular gene 1 mRNA isolated Reverse transcriptase and fluorescent DNA nucleotides 2 cDNA made from mRNA Actual size (6,400 genes) 4 Unbound cDNA rinsed away Fluorescent spot 3 cDNA applied to wells Nonfluorescent spot cDNA DNA of an expressed gene Figure 12.9 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings DNA of an unexpressed gene 12.10 Gel electrophoresis sorts DNA molecules by size Mixture of DNA molecules of different sizes – – Longer molecules Power source Gel + Shorter molecules + Figure 12.10 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Completed gel 12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings How Restriction Fragments Reflect DNA Sequence • Restriction fragment length polymorphisms (RFLPs) – Reflect differences in the sequences of DNA samples Crime scene Suspect w Cut C C G G G G C C z A C G G T G C C C C G G G G C C x Cut y Figure 12.11A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings C C G G G G C C Cut y DNA from chromosomes • After digestion by restriction enzymes – The fragments are run through a gel 1 – 2 Longer fragments z x w Shorter fragments Figure 12.11B y + Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings y Using DNA Probes to Detect Harmful Alleles • Radioactive probes – Can reveal DNA bands of interest on a gel Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Detecting a harmful allele using restriction fragment analysis 1 Restriction fragment preparation I II III Restriction fragments 2 Gel electrophoresis I II III 3 Blotting Filter paper 4 Radioactive probe Radioactive, singlestranded DNA (probe) Probe 5 Detection of radioactivity (autoradiography) I II III Film Figure 12.11C Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings I II III CONNECTION 12.12 DNA technology is used in courts of law Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • DNA fingerprinting can help solve crimes Defendant’s blood Blood from defendant’s clothes Victim’s blood Figure 12.12A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Figure 12.12B CONNECTION 12.13 Gene therapy may someday help treat a variety of diseases • Gene therapy – Is the alteration of an afflicted individual’s genes Cloned gene (normal allele) 1 Insert normal gene into virus Viral nucleic acid Retrovirus 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome Bone marrow cell from patient Bone marrow Figure 12.13 4 Inject cells into patient Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Gene therapy – May one day be used to treat both genetic diseases and nongenetic disorders • Unfortunately, progress is slow Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.14 The PCR method is used to amplify DNA sequences • The polymerase chain reaction (PCR) – Can be used to clone a small sample of DNA quickly, producing enough copies for analysis Initial DNA segment 1 Figure 12.14 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 2 4 8 Number of DNA molecules GENOMICS CONNECTION 12.15 The Human Genome Project is an ambitious application of DNA technology • The Human Genome Project, begun in 1990 and now largely completed, involved – Genetic and physical mapping of chromosomes, followed by DNA sequencing Figure 12.15 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • The data are providing insight into – Development, evolution, and many diseases Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.16 Most of the human genome does not consist of genes • The haploid human genome contains about 25,000 genes – And a huge amount of noncoding DNA Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Much of the noncoding DNA consists of repetitive nucleotide sequences – And transposons that can move about within the genome Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.17 The science of genomics compares whole genomes • The sequencing of many prokaryotic and eukaryotic genomes – Has produced data for genomics, the study of whole genomes Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Besides being interesting themselves – Nonhuman genomes can be compared with the human genome Table 12.17 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Proteomics – Is the study of the full sets of proteins produced by organisms Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings GENETICALLY MODIFIED ORGANISMS CONNECTION 12.18 Genetically modified organisms are transforming agriculture Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Recombinant DNA technology – Can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms Agrobacterium tumefaciens DNA containing gene for desired trait Ti plasmid 1 Insertion of gene into plasmid using restriction enzyme and DNA ligase T DNA Restriction site Plant cell Recombinant Ti plasmid 2 Introduction Regeneration into plant of plant cells in culture T DNA carrying new Plant with new trait gene within plant chromosome Figure 12.18A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 3 • Transgenic organisms – Are those that have had genes from other organisms inserted into their genomes Figure 12.18B Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • A number of important crops and plants – Are genetically modified Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.19 Could GM organisms harm human health or the environment? • Development of GM organisms – Requires significant safety measures Figure 12.19A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Genetic engineering involves risks – Such as ecological damage from GM crops Figure 12.19B Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.20 Genomics researcher Eric Lander discusses the Human Genome Project • Genomics pioneer Eric Lander – Points out that much remains to be learned from the Human Genome Project Figure 12.20 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.17 The science of genomics compares whole genomes • The sequencing of many prokaryotic and eukaryotic genomes – Has produced data for genomics, the study of whole genomes Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings HOMEWORK Read Cohen’s work for plasmids and answer the questions on the following website http://www.stanford.edu/class/bio11n/html/week1/ papertopic.htm Read Villa-Komaroff et al. work and answer the questions on the following website http://www.stanford.edu/class/bio11n/html/week2/ papertopic.htm Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings READING http://www.dnaftb.org/dnaftb/ Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings