* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Chapter 20 Notes: DNA Technology
Gene expression wikipedia , lookup
Genome evolution wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Molecular evolution wikipedia , lookup
Restriction enzyme wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
DNA vaccination wikipedia , lookup
Non-coding DNA wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
DNA supercoil wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Molecular cloning wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Chapter 20 Notes: DNA Technology Understanding & Manipulating Genomes • 1995: sequencing of the first complete genome (bacteria) • 2003: sequencing of the Human Genome mostly completed • These accomplishments depended on new technology: – Recombinant DNA: DNA from 2 sources (often 2 species) are combined in vitro into the same DNA molecule • Called Genetic engineering: direct manipulation of genes for practical purposes DNA technology has launched a revolution in the are of: BIOTECHNOLOGY: the use of living organisms or their components to do practical tasks -microorganisms to make wine/cheese -selective breeding of livestock -production of antibiotics -agriculture -criminal law **Practical goal of biotech = improvement of human health and food production Ch 20 looks at: 1. Main techniques for manipulating DNA 2. How genomes are analyzed & compared at the DNA level 3. Practical applications of DNA technology (including social & ethical issues) “Toolkit” for DNA technology involves: -DNA vectors -host organisms - restriction enzymes VECTORS = carriers for moving DNA from test tubes back into cells -bacterial plasmids (small, circular DNA molecules that replicate within bacterial cells) -viruses HOST ORGANISMS: bacteria are commonly used as hosts in genetic engineering because: 1) DNA can easily be isolated from & reintroduced into bacterial cells; 2) bacterial cultures grow quickly, rapidly replicating any foreign genes they carry. RESTRICTION ENZYMES = enzymes that recognize and cut short, specific nucleotide sequences (called restriction sites) -in nature, these enzymes protect the bacterial cell from other organisms by cutting up their foreign DNA Restriction Enzymes (cont.)… most restriction sequences are symmetrical in that the same sequence of 4-8 nucleotides is found on both strands, but run in opposite directions restriction enzymes usually cut phosphodiester bonds of both strands in a staggered manner producing single stranded “sticky ends” Restriction Enzymes (cont.)… “sticky ends” of restriction fragments are used in the lab to join DNA pieces from different sources (complementary base pairing) *RECOMBINANT DNA unions of different DNA sources can be made permanent by adding DNA ligase enzyme (form covalent bonds between bases) DNA Technologies: 1) Cloning 2) DNA fingerprinting (profiling) 3) Microarray 4) Gene therapy Steps Involved in Cloning a Human Gene: Human gene plasmid 1) Isolate human gene to clone (ex: insulin); 2) Isolate plasmid from bacterial cell; 3) cut both DNA samples with the same restriction enzyme to open up bacterial plasmid & create sticky ends on bath samples; 4) Mix the cut plasmids and human DNA genes & seal with DNA ligase; Cloning a Human Gene (cont.)… 5) Insert recombinant DNA plasmid back into bacterial cell; 6) As bacterial cell reproduces, it makes copies of the desired gene; -grow cells on a petri dish 7) Identify cell clones carrying the gene of interest. -HOW? Which ones took up the gene & are making insulin? -Add a 2nd gene besides insulin; add one for antibiotic resistance & then grow bacteria on that antibiotic LE 20-4_3 Bacterial cell Isolate plasmid DNA and human DNA. lacZ gene (lactose breakdown) Human cell Restriction site ampR gene (ampicillin resistance) Cut both DNA samples with the same restriction enzyme. Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone Why can bacteria produce insulin through recombinant DNA technology? The genetic code is universal!!!! DNA Fingerprinting Involves: The Polymerase Chain Reaction (PCR) allows any piece of DNA to be quickly amplified (copied many times) in vitro (artificial environment). DNA is incubated under appropriate conditions with special primers & DNA polymerase molecules PCR (continued)… BILLIONS of copies of DNA are produced in just a few hours (enough to use for testing) In 6 cycles of PCR: cycle 1: 2 copies cycle 2: 4 copies cycle 3: 8 copies cycle 4: 16 copies cycle 5: 32 copies cycle 6: 64 copies cycle 20: 1,048,576!! Polymerase Chain Reaction (PCR) PCR is highly specific; primers determine the DNA sequence to be amplified only minute amounts of DNA are needed Remember these? Starting materials for PCR: • DNA to be copied • Nucleotides • Primers • Taq polymerase (DNA polymerase isolated from bacteria living in hot springs…their enzymes can withstand high temps!) Steps of PCR: 1) Heat to separate DNA strands (95ºC); 2) Cool to allow primers to bind (55ºC); 3) Heat slightly so that DNA polymerase extends the 3’ end of each primer (72ºC) 4) Repeat steps #1-3 many times!!! Applications of PCR: DNA from tiny amounts of tissue or semen found at crime scene; DNA from single embryonic cells for prenatal diagnosis; DNA or viral genes from cells infected with difficult to detect viruses such as HIV; used extensively in Human Genome Project to produce linkage maps without the need for large family pedigree analysis. PCR works like a copying machine for DNA! Analysis of Cloned DNA: Gel electrophoresis separates macromolecules (nucleic acids or proteins) based on size, charge, or other property (DNA cut with restriction enzymes) for linear molecules like DNA, separation depends mainly on size a mixture of DNA fragments will be sorted into bands, each consisting of DNA molecules of the same length YOUR DNA MY DNA - + The DNA (-) is loaded at the top in a well Steps Involved in DNA Fingerprinting: 1) Collect DNA from a sample; 2) Perform PCR if necessary to make more DNA; 3) Cut DNA apart using RE’s which will recognize specific base sequences **DNA will be cut at different places for different people, therefore producing different size fragments (Restriction Fragment Length Polymorphisms… a.k.a. RFLP’s- noncoding regions between genes) DNA Fingerprinting (cont.)… 4) Electrophoresis is used to align DNA pieces on gel called agarose; electric current is used to actually move DNA pieces across the gel; 5)DNA fragments are chemically separated and transferred from the gel to a sheet of nylon and which is then exposed to radioactive probes which will stick to DNA complementary base pairs; 1 method of visualizing the DNA DNA Fingerprinting (cont.)… 6) Nylon sheet is placed against x-ray film and developed; when 2 bases join, a black band will appear; 7) Banding patterns can then be compared. Triangles: RE cut site “a” allele lost cleavage site #2 due to a mutation; results in a longer fragment Dark line represents a probe. Analysis and inheritance of allelic RFLP fragments (NIH). Sample Sample 1 2 DNA_DetectivePC.exe DNA Microarray Assays • Technique used to study how genes act together to produce & maintain a functioning organism – Which genes are transcribed in different situations such as different tissues or at different stages of development • Consists of tiny amounts of a large number of single strand-stranded DNA fragments representing different genes fixed to a glass slide in a tightly spaced array (grid) – Aka: DNA chip Microarray Technique 1. Isolate mRNA 2. Make cDNA by reverse transcription, using fluorescently labeled nucleotides 3. Apply the cDNA mixture to a microarray; the cDNA will hybridize with any complementary DNA on the microarray 4. Rinse off excess cDNA & scan for fluorescence • • each fluorescent spot represents a gene being expressed The intensity of fluorescence indicates level of expression LE 20-14 Tissue sample Isolate mRNA. Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a microscope slide on which copies of singlestranded DNA fragments from the organism’s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. mRNA molecules Labeled cDNA molecules (single strands) DNA microarray Size of an actual DNA microarray with all the genes of yeast (6,400 spots) Practical Applications of DNA Microarrays • Track gene expression changes from initial development throughout an organisms lifetime • Contribute to better understanding of certain diseases & suggest new diagnostic techniques or therapies Gene Therapy •Alteration of an afflicted individual’s genes •Experimental: in theory,a normal allele of a defective gene could be inserted into the somatic cells of the tissue affected by the disorder •Somatic cells must multiply throughout patients life (ex: bone marrow cells) LE 20-16 Cloned gene Insert RNA version of normal allele into retrovirus. Viral RNA Retrovirus capsid Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Inject engineered cells into patient. Bone marrow Practical Applications of DNA Technology: Medicine / Pharmaceutical 1) human gene therapy 2) pharmaceutical products -insulin, growth hormone, TPA (dissolves blood clots), proteins that mimic cell surface receptors for viruses like HIV 3) diagnosis of disease LE 20-9 Normal -globin allele 175 bp Ddel 201 bp Ddel Large fragment Ddel Ddel Sickle-cell mutant -globin allele 376 bp Ddel Large fragment Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of -globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles Applications of DNA Technology… Forensic uses DNA fingerprints: paternity, criminal cases Environmental uses: microorganisms engineered to break down sewage, oil spills, etc. Applications of DNA Technology… Agricultural uses (“Pharm” Animals and Plants) 1) livestock -bGH (bovine growth hormone) to enhance milk production -sheep that carry a gene for human blood protein -genes that cause devp’t of larger muscles in cattle -salmon that grow larger due to a modified growth hormone 2) genetically engineered plants - insecticide/herbicide resistance - decaying of ripening/resistance to spoiling - transgenic rice that contain beta-carotene to solve the vitamin A deficiency in poorer countries Regular Tomato Flavor Savr Tomato LE 20-19 Agrobacterium tumefaciens Ti plasmid Site where restriction enzyme cuts T DNA DNA with the gene of interest Recombinant Ti plasmid Plant with new trait Why is this a restriction enzyme site? A. Restriction enzymes bind to special hydrogen bond sites B. Restriction enzymes cut at GAATTC C. Restriction enzymes cut at CTTAAG D. Restriction enzymes recognize specific reverse order sequences What are “sticky ends?” A. B. C. D. Pieces of DNA that are run through a gel in order to give a unique banding pattern Single-stranded DNA ends that are available to hydrogen bond to a complimentary single strand DNA bases that are added to a PCR machine so that multiple exact copies of a DNA sequence can be produced Segments of DNA that act as a probe in order to diagnose a genetic condition What is this called? A. B. C. D. RFLP (restriction length polymorphism) PCR (polymerase chain reaction) Clone Recombinant DNA plasmid Human gene What is the purpose of PCR? A. B. C. D. To make billions of identical copies of a specific DNA segment To separate out DNA segments by size To help make medical products such as insulin To help cure diseases such as Cystic Fibrosis What is the basis for DNA movement in gel electrophoresis? A. DNA has a (+) charge so it moves towards a (-) electrical terminal B. DNA has a (+) charge so it moves towards a (+) electrical terminal C. DNA has a (-) charge so it moves towards a (+) electrical terminal D. DNA has a (-) charge so it moves towards a (-) electrical terminal Which of the following is NOT an application of biotechnology? A. Agricultural products such as bovine growth hormone B. Pharmaceutical products such as insulin C. Environmental uses; cleaning up oil spills D. Medical uses; to help parents have children with specific traits E. Medical uses; to help diagnose some diseases Vocabulary terms Transcription is the process of synthesizing RNA using a DNA duplex as template. Translation is the process of synthesizing a protein using an mRNA molecule as a guide. Gene expression refers to the transcription and translation of a gene or set of genes. Gene regulation refers to the control of gene expression. more vocab… Hybridization is the process by which two complementary strands of nucleic acid base pair to one another to form a duplex. If two strands of nucleic acid are not complementary, they will not hybridize to form a duplex. Gene knockouts are experiments in which a gene is deleted from the genome of an organism. Knockouts are used to gain information about the function of a gene. still more vocab… Transcriptional regulation is the control of gene expression at the level of transcription. RNA interference (RNAi) is the phenomenon in which experimentally introduced double-stranded RNA leads to loss of expression of the corresponding cellular gene.