* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download ch. 12 Biotechnology-notes-ppt
Epigenetics wikipedia , lookup
Genome (book) wikipedia , lookup
Metagenomics wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Genome evolution wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Human genome wikipedia , lookup
DNA profiling wikipedia , lookup
DNA polymerase wikipedia , lookup
Primary transcript wikipedia , lookup
Nutriepigenomics wikipedia , lookup
SNP genotyping wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Point mutation wikipedia , lookup
Genetic engineering wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Designer baby wikipedia , lookup
Microsatellite wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Genome editing wikipedia , lookup
DNA vaccination wikipedia , lookup
Epigenomics wikipedia , lookup
Genomic library wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Non-coding DNA wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
DNA supercoil wikipedia , lookup
Microevolution wikipedia , lookup
Molecular cloning wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Helitron (biology) wikipedia , lookup
Biotechnology Pre-AP Biology Ch.12 Ms. Haut DNA technology has many useful applications – The Human Genome Project – The production of vaccines, cancer drugs, and pesticides – Engineered bacteria that can clean up toxic wastes Copyright © 2003 Pearson Education, Inc. publishing 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 – 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) BACTERIAL PLASMIDS AND GENE CLONING •Plasmids are used to customize bacteria: An overview – Gene cloning is one application of DNA technology • Methods for studying and manipulating genetic material The Bacterial Chromosome • One double-stranded, circular molecule of DNA • Located in nucleoid region, so transcription and translation can occur simultaneously • Many also contain extrachromosomal DNA in plasmids Binary Fission Plasmids • Short, circular DNA molecules outside the chromosome • Carry genes that are beneficial but not essential • Replicate independently of chromosome R Plasmids • Contain genes that confer antibiotic resistance • Medical consequences:resistant strains of pathogens due to overuse of antibiotics Genetic Recombination Produces New Bacterial Strain • Transformation • Transduction • Conjugation Gene transfer occurs separately from bacterial reproduction Bacteria as Tools • Bacterial Transformation— – Uptake of DNA from the fluid surrounding the cell – Causes genetic recombination Transformation • Alteration of bacterial cell’s genotype by uptake of naked, foreign DNA from the environment Transformation • Biotech companies use this technique to artificially introduce foreign genes into bacterial genomes (human insulin, human growth hormone) Transduction • Gene transfer from one bacterium to another by a bacteriophage Conjugation • Direct transfer of genetic material between bacterial cells that are temporarily joined (bacterial sex) “male” “female” F- F+ Sex pili “Maleness” results from presence of F factor— segment of DNA in chromosome or in F plasmid Mating bridge Sex pili Donor cell (“male”) Figure 12.1C Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings Recipient cell (“female”) • The transferred DNA is then integrated into the recipient cell’s chromosome Donated DNA Degraded DNA Crossovers Recipient cell’s chromosome Figure 12.1D Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings Recombinant chromosome Bacterial plasmids can serve as carriers for gene transfer F factor (integrated) Male (donor) cell Origin of F replication Bacterial chromosome • An F factor is a DNA segment in bacteria that enables conjugation and contains an origin of replication Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings Figure 12.2A F factor starts replication and transfer of chromosome Recipient cell Only part of the chromosome transfers Recombination can occur – Researchers can insert desired genes into plasmids, creating recombinant DNA • Figure 12.1 And insert those plasmids into bacteria (transformation) E. coli – If the recombinant bacteria multiply into a clone • The foreign genes are also copied 1 Isolate DNA Human cell from two sources 2 Cut both Plasmid DNAs with the same restriction enzyme DNA Gene V Sticky ends 3 Mix the DNAs; they join by base-pairing 4 Add DNA ligase to bond the DNA covalently Recombinant DNA plasmid Gene V 5 Put plasmid into bacterium by transformation 6 Clone the bacterium Bacterial clone carrying many copies of the human gene •Restriction 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 – Creating recombinant DNA using restriction enzymes and DNA ligase Figure 12.2 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 •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 Phage clone Phage library CONNECTION •Recombinant cells and organisms can mass-produce gene products – Applications of gene cloning include • The mass production of gene products for medical and other uses Table 12.6 Genetically modified organisms are transforming agriculture • New genetic varieties of animals and plants are being produced – A plant with a new trait can be created using the Ti plasmid • Biotech companies can artificially induce transformation of bacteria • “Golden rice” has been genetically modified to contain beta-carotene – This rice could help prevent vitamin A deficiency Figure 12.18B Drought resistant corn Flavr Savr Tomato (CalGene) •Transgenic organisms – Are those that have had genes from other organisms inserted into their genomes – Different organisms, including bacteria, yeast, and mammals • Can be used for this purpose These sheep carry a gene for a human blood protein that is a potential treatment for cystic fibrosis Figure 12.6 CONNECTION •DNA technology is changing the pharmaceutical industry – DNA technology • Is widely used to produce medicines and to diagnose diseases DNA technology is changing the pharmaceutical industry and medicine • Hormones, cancer-fighting drugs, and new vaccines are being produced using DNA technology – This lab equipment is used to produce a vaccine against hepatitis B Figure 12.17 •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 •Diagnosis and Treatment of Disease – DNA technology • Is being used increasingly in disease diagnosis •Vaccines – DNA technology • Is also helping medical researchers develop vaccines Figure 12.7B Gene therapy may someday help treat a variety of diseases • Techniques for manipulating DNA have potential for treating disease by altering an afflicted individual’s genes • 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 – Progress is slow, however – There are also ethical questions related to gene therapy 3 Viral DNA inserts into chromosome Bone marrow cell from patient Bone marrow 4 Inject cells Figure 12.19 into patient DNA Fingerprinting • A method of developing a person’s DNA “profile,” similar to a fingerprint. • Pioneered in England in 1984 by Dr. Alec Jeffreys Dr. Alec Jeffreys First Forensic Use • First used by law enforcement in England in the mid-1980’s. • DNA evidence exonerated one man, and convicted another. • Described in The Blooding, by Joseph Wambaugh How does it work? • 99.9% of your DNA is the same as everyone else’s. • The 0.1% that differs are a combination of: – Gene differences (Differences in the genes themselves) – Differences in “polymorphic regions” between the genes on the DNA. How does it work? • Certain points between the genes on the DNA have repeating base sequences. – For example: ATTACGCGCGCGCGCGCGCTAGC – These are called variable nucleotide tandem repeats (VNTRs for short) How does it work? • Everyone has VNTRs at the same place in their DNA, but they are different lengths for different people. – For example: Person 1: ATTACGCGCGCGCGCGCGTAGC (7 repeats) Person 2: ATTACGCGCGCGCGTAGC (5 repeats) To Make a DNA Fingerprint… • First, we use restriction enzymes to chop the DNA up into millions of fragments of various lengths. – The restriction enzymes cut everyone’s DNA in the same place. – Some of the fragments contain VNTRs; some do not. The ones that do are different lengths for different people. Restriction Fragment Length Polymorphisms (RFLPs) • Polymorphisms are slight differences in DNA sequences as seen in individuals of the same species To Make a DNA Fingerprint… • Next, we use gel electrophoresis to sort the DNA fragments by size. Gel Electrophoresis • Method for sorting proteins or nucleic acids on the basis of their electric charge and size Gel Electrophoresis Electrical current carries negatively-charged DNA through gel towards positive electrode • Agarose gel sieves DNA fragments according to size • – Small fragments move farther than large fragments Gel Electrophoresis 1 To Make a DNA Fingerprint… Restriction fragment preparation Restriction fragments 2 Gel electrophoresis 3 Blotting 4 Radioactive probe Filter paper Probe 5 Detection of radioactivity (autoradiography) Film Figure 12.11C • Finally, a radioactive probe attaches to our VNTRs. Only the fragments with our VNTRs will show up on the gel. To Make a DNA Fingerprint… • Since VNTRS are different lengths in different people, this creates a DNA Fingerprint. Two uses for DNA Fingerprints... • Forensics DNA taken from crime scenes (blood, semen, hair, etc.) can be compared to the DNA of suspects. Real-life CSI! Two uses for DNA Fingerprints... • Forensics This is an example of a gel that might be used to convict a rape suspect. Compare the “Sperm DNA” to the “Suspect DNA.” Which suspect committed the rape? Two uses for DNA Fingerprints... • Paternity Testing Since all of our DNA markers came from either mommy or daddy, we can use DNA fingerprints to determine whether a child and alleged father are related…just like on Maury Povich! Interpreting DNA Fingerprints • A blood stain was found at a murder scene. The blood belongs to which of the seven possible suspects? Interpreting DNA Fingerprints • Who committed this rape? Interpreting DNA Fingerprints • These DNA fingerprints are from a mother, a child, and two possible biological fathers. Which one is the daddy? Interpreting DNA Fingerprints • Mother, father, and four children. Which child is from a different father? DNA Fingerprinting – 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 Base pairing indicates the gene of interest RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING •Nucleic acid probes identify clones carrying specific genes – DNA technology methods • Can be used to identify specific pieces of DNA CONNECTION •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 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 DNA of an unexpressed gene Using DNA Probes to Detect Harmful Alleles 1 Restriction fragment preparation I •Radioactive probes II III Restriction fragments – Can reveal DNA bands of interest on a gel •Detecting a harmful allele using restriction fragment analysis 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 I II III Polymerase Chain Reaction •The PCR method is used to amplify DNA sequences – Can be used to clone a small sample of DNA quickly, producing enough copies for analysis – Does not rely on cells for DNA replication Figure 12.14 PCR Reaction GENOMICS CONNECTION •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 CONNECTION •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 – Besides being interesting themselves • Table 12.17 Nonhuman genomes can be compared with the human genome Proteomics •Is the study of the full sets of proteins produced by organisms Biotechnology Explorer™ Protein Fingerprinting Instruction Manual Biotechnology Explorer™ Protein Fingerprinting Instruction Manual Could GM organisms harm human health or the environment? • Genetic engineering involves some risks – Possible ecological damage from pollen transfer between GM and wild crops – Pollen from a transgenic variety of corn that contains a pesticide may stunt or kill monarch caterpillars Figure 12.20A, B DNA technology raises important ethical questions • Our new genetic knowledge will affect our lives in many ways • The deciphering of the human genome, in particular, raises profound ethical issues – Many scientists have counseled that we must use the information wisely Figure 12.21A-C Analysis of Stained Gel • • Determine restriction fragment sizes – Create standard curve using DNA marker – Measure distance traveled by restriction fragments – Determine size of DNA fragments Identify the related samples Molecular Weight Determination Fingerprinting Standard Curve: Semi-log Distance (mm) 23,000 11.0 9,400 13.0 6,500 15.0 4,400 18.0 2,300 23.0 2,000 24.0 100,000 10,000 Size, base pairs Size (bp) B 1,000 100 0 5 10 15 Distance, mm 20 A 25 30 Acknowledgements • BIOLOGY: CONCEPTS AND CONNECTIONS 4th Edition, by Campbell, Reece, Mitchell, and Taylor, ©2003. These images have been produced from the originals by permission of the publisher. These illustrations may not be reproduced in any format for any purpose without express written permission from the publisher. • Unless otherwise noted, illustrations are credited to Pearson Education which have been borrowed from BIOLOGY: CONCEPTS AND CONNECTIONS 4th Edition, by Campbell, Reece, Mitchell, and Taylor, ©2001. These images have been produced from the originals by permission of the publisher. These illustrations may not be reproduced in any format for any purpose without express written permission from the publisher.