* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Chapter 20: Biotechnology
Gene desert wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Gene expression profiling wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Gene expression wikipedia , lookup
Genome evolution wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
List of types of proteins wikipedia , lookup
Gene regulatory network wikipedia , lookup
Non-coding DNA wikipedia , lookup
Molecular evolution wikipedia , lookup
Point mutation wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
DNA vaccination wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
Restriction enzyme wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Genetic engineering wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Molecular cloning wikipedia , lookup
Genomic library wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School 1. What is Recombinant DNA? • Recombinant DNA are nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule • Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes 2. What is Biotechnology and Genetic Engineering? How is Genetic Engineering advancing our society? Give me two examples of genetic engineering not found in the book. • Genetic Engineering is the direct manipulation of genes for practical purposes • DNA technology has revolutionized Biotechnology, the manipulation of organisms or their genetic components to make useful products • An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes 3. How are Bacteria and Plasmids used for cloning? • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids • Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome • Cloned genes are useful for making copies of a particular gene and/or producing a protein product 3. How are Bacteria and Plasmids used for cloning? • Gene cloning involves using bacteria to make multiple copies of a gene • Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell • Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA • This results in the production of multiple copies of a single gene Fig. 20-2a Cell containing gene of interest Bacterium 1 Gene inserted into plasmid Bacterial chromosome Plasmid Recombinant DNA (plasmid) Gene of interest 2 2 Plasmid put into bacterial cell Recombinant bacterium DNA of chromosome Fig. 20-2b Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Gene of Interest Copies of gene Protein harvested 4 Basic research and Basic research on gene Gene for pest resistance inserted into plants various applications Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Basic research on protein Human growth hormone treats stunted growth 4. Gene cloning may be used for which two purposes? Give me an example of each. • Gene cloning is used to make many copies of a gene for research (such as a disease gene) and study or used to make a protein product (such as insulin). 5. How are Restriction Enzymes used for creating Recombinant DNA? Please use the words Restriction Site, Restriction Fragments, Sticky End, and DNA ligase in your answer. • Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites • A restriction enzyme usually makes many cuts, yielding restriction fragments • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments • DNA ligase is an enzyme that seals the bonds between restriction fragments Fig. 20-3-1 Restriction site DNA 1 5 3 3 5 Restriction enzyme cuts sugar-phosphate backbones. Sticky end Fig. 20-3-2 Restriction site DNA 1 5 3 3 5 Restriction enzyme cuts sugar-phosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. One possible combination Fig. 20-3-3 Restriction site DNA 1 5 3 3 5 Restriction enzyme cuts sugar-phosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. One possible combination 3 DNA ligase seals strands. Recombinant DNA molecule 6. Briefly describe the steps for gene cloning the B-Globin gene in Hummingbirds. • Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid: • • • • • • • The hummingbird genomic DNA and a bacterial plasmid are isolated Both are digested with the same restriction enzyme The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends Some recombinant plasmids now contain hummingbird DNA The DNA mixture is added to bacteria that have been genetically engineered to accept it The bacteria are plated on a type of agar that selects for the bacteria with recombinant plasmids This results in the cloning of many hummingbird DNA fragments, including the β-globin gene Fig. 20-4-1 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restriction site ampR gene Bacterial plasmid Sticky ends Gene of interest Hummingbird DNA fragments Fig. 20-4-2 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restriction site ampR gene Sticky ends Bacterial plasmid Gene of interest Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Fig. 20-4-3 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restriction site ampR gene Sticky ends Bacterial plasmid Gene of interest Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restriction site ampR gene Sticky ends Bacterial plasmid Gene of interest Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids RESULTS Colony carrying nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones 7. What is the Genomic Library? How is this used in research? • A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome • A genomic library that is made using bacteriophages is stored as a collection of phage clones Fig. 20-5a Foreign genome cut up with restriction enzyme or Recombinant phage DNA Bacterial clones (a) Plasmid library Recombinant plasmids (b) Phage library Phage clones 8. What is a Bacterial Artificial Chromosome? What is the advantage of using this for research? • A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert • BACs are another type of vector used in DNA library construction Fig. 20-5b Large plasmid Large insert with many genes BAC clone (c) A library of bacterial artificial chromosome (BAC) clones 1. What is PCR? What is it used for? • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA • A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules • This is a very useful method if the DNA source is very limited or impure as it can make many many copies of one segment in a short amount of time. Fig. 20-8a 5 TECHNIQUE 3 Target sequence Genomic DNA 3 5 Fig. 20-8b 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleotides Fig. 20-8c Cycle 2 yields 4 molecules Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence 2. What is Gel Electrophoresis? How is it used for research? • • • • • One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size A current is applied that causes charged molecules to move through the gel Molecules are sorted into “bands” by their size In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis • Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene • The procedure is also used to prepare pure samples of individual fragments Fig. 20-9a TECHNIQUE Mixture of DNA molecules of different sizes Power source – Cathode Anode + Gel 1 Power source – + Longer molecules 2 Shorter molecules Fig. 20-9b RESULTS Fig. 20-10 Normal -globin allele 175 bp DdeI Sickle-cell allele Large fragment 201 bp DdeI Normal allele DdeI DdeI Large fragment Sickle-cell mutant -globin allele 376 bp DdeI 201 bp 175 bp Large fragment 376 bp DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 3. Briefly describe the Southern blotting method? What is this used for? • A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization • Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel Fig. 20-11a TECHNIQUE DNA + restriction enzyme Restriction fragments I II III Nitrocellulose membrane (blot) Heavy weight Gel Sponge I Normal -globin allele II Sickle-cell III Heterozygote allele 1 Preparation of restriction fragments Alkaline solution 2 Gel electrophoresis Paper towels 3 DNA transfer (blotting) Fig. 20-11b Radioactively labeled probe for -globin gene I II III Probe base-pairs with fragments Fragment from sickle-cell -globin allele Fragment from normal -globin Nitrocellulose blot allele 4 Hybridization with radioactive probe I II III Film over blot 5 Probe detection 4. What two methods can be used to study gene changes in embryonic development? Briefly describe them? • Changes in the expression of a gene during embryonic development can be tested using • Northern blotting combines gel electrophoresis of mRNA followed by hybridization with a probe on a membrane • Identification of mRNA at a particular developmental stage suggests protein function at that stage • Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive • Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest • The products are run on a gel and the mRNA of interest identified • Both methods are used to compare mRNA from different developmental stages 5. What is the purpose and method of a DNA microarray assay? • Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays • DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions Fig. 20-15 TECHNIQUE 1 Isolate mRNA. 2 Make cDNA by reverse transcription, using fluorescently labeled nucleotides. 3 Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA fragments representing specific genes DNA microarray 4 Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample. DNA microarray with 2,400 human genes 6. What is the purpose and method of in vitro mutagenesis and RNA interference? • One way to determine function is to disable the gene and observe the consequences • Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function • When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype • Gene expression can also be silenced using RNA interference (RNAi) • Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene’s mRNA 7. When studying humans, what is the purpose of looking for a single nucleotide polymorphism? How does this aid us in finding and tracking human genetic diseases? • A single nucleotide polymorphism (SNP) is a single base pair site where a variation is found in at least 1% of the population. • Once a gene (especially disease genes) has been found to have a SNP shared by affected people and not unaffected people, the gene is sequenced. • The SNP and disease causing gene will most likely be inherited together because they are close to each other on the genome. Therefore, the SNP can be used as a marker for the disease. • Doctors can perform a sensitive microarray analysis to look for SNPs or by PCR. 8. How much of the human genome doesn’t code for a protein? • 98% of the human genome does not directly code for proteins.