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Chapter 20

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Chapter 20

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  1. Chapter 20 Biotechnology

  2. Overview: The DNA Toolbox • Sequencing of the human genome was completed by 2007 • DNA sequencing has depended on advances in technology, starting with making recombinant DNA • In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule

  3. Overview: The DNA Toolbox • Methods for making recombinant DNA are central to genetic engineering, 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

  4. Overview: The DNA Toolbox • Biotechnology • Actually has a long history • Selective breeding • Using microorganisms to make wine and cheese • An example of modern DNA technology is the microarray, a measurement of gene expression of thousands of different genes

  5. Fig. 20-1

  6. Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment • To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning • This was one of the advances that changed biotechnology and actually created the field we call genetic engineering

  7. DNA Cloning and Its Applications: A Preview • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids • Don’t always use plasmids – will see this later • Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome

  8. DNA Cloning and Its Applications: A Preview • 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

  9. Fig. 20-2 Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid 1. Gene of interest 2. Into plasmid from bacteria 3. Plasmid back into bacteria 4. Recombinant bacteria 5. Grow lots of bacteria 6. Purify/study gene of interest or Its protein product Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 Plasmid put intobacterial cell Recombinantbacterium 3 Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest Gene ofInterest Protein expressedby gene of interest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene How do we “cut out” The gene we want? Gene used to alter bacteria for cleaning up toxic waste Gene for pest resistance inserted into plants Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth

  10. Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites • ‘Molecular scissors’ • Naturally present in bacteria as a defense against foreign DNA – such as phages • They “restrict” growth of phage DNA • Bacterial DNA is protected by methylation Animation: Restriction Enzymes

  11. Fig. 20-3-1 Restriction site 5 3 3 5 DNA Restriction Enzyme cuts at a specific DNA sequence Restriction enzymecuts sugar-phosphatebackbones. 1 Most useful ones leave what Are called “sticky ends” Sticky end

  12. Using Restriction Enzymes to Make Recombinant DNA • 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 Animation: Restriction Enzymes

  13. Fig. 20-3-2 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end This is a weak interaction held together only by H bonding between the base pairs DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination

  14. Using Restriction Enzymes to Make Recombinant DNA • DNA ligase is an enzyme that seals the bonds between restriction fragments • DNA ligase forms bonds in the sugar – phosphate backbone of DNA

  15. Fig. 20-3-3 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination DNA ligaseseals strands. 3 Recombinant DNA molecule

  16. Using Restriction Enzymes to Make Recombinant DNA • A restriction enzyme usually makes many cuts, yielding restriction fragments • Try to use an enzyme that makes the fewest cuts • You buy these in a catalog • Lots of modified “genetically engineered” forms available now

  17. Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there

  18. Producing Clones of Cells Carrying Recombinant Plasmids • 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 Animation: Cloning a Gene

  19. Fig. 20-4-1 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments

  20. Fig. 20-4-2 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids How do we separate the recombinant from the non recombinant plasmids?

  21. Producing Clones of Cells Carrying Recombinant Plasmids • 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

  22. Fig. 20-4-3 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids

  23. Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid This method is called Blue/white screening Clone into and disrupt The lac Z gene Colonies will be white Cannot metabolize XGAL Recombinant plasmids Bacteria carryingplasmids RESULTS Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non-recombinant plasmidwith intact lacZ gene One of manybacterial clones

  24. Storing Cloned Genes in DNA Libraries • 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 • Can also construct libraries from bacterial artificial chromosomes – stripped down plasmids – can have huge inserts

  25. Fig. 20-5 Foreign genomecut up withrestrictionenzyme Large insertwith many genes Large plasmid or BACclone Recombinantphage DNA Bacterial clones Recombinantplasmids Phageclones (a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones

  26. Storing Cloned Genes in DNA Libraries • A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell • A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells

  27. Fig. 20-6-1 DNA innucleus mRNAs in cytoplasm Isolate mRNA Difficult Very unstable

  28. Fig. 20-6-2 DNA innucleus mRNAs in cytoplasm Copy RNA to DNA Reverse Transcriptase Reversetranscriptase Poly-A tail mRNA Primer DNAstrand

  29. Fig. 20-6-3 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail Degrade RNA RNase mRNA Primer DNAstrand DegradedmRNA

  30. Fig. 20-6-4 DNA innucleus mRNAs in cytoplasm DNA polymerase synthesizes the second strand (using a primer) Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase

  31. Fig. 20-6-5 DNA innucleus mRNAs in cytoplasm ds CDNA product Called cDNA for complementary DNA Complementary to mRNA Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase cDNA

  32. Screening a Library for Clones Carrying a Gene of Interest • So now we have these libraries • How do we find the gene we are interested in • Say we have identified a gene in a fruit fly that is involved in a pathway we are interested in and we want to look and see if there is a corresponding gene in humans

  33. Screening a Library for Clones Carrying a Gene of Interest • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene • Since we know the sequence of the fruit fly gene we can make a short primer and use this to “probe” our library • This process is called nucleic acid hybridization

  34. Screening a Library for Clones Carrying a Gene of Interest • A probe can be synthesized that is complementary to the gene of interest • For example, if the desired gene is – Then we would synthesize this probe … … 5 G G C T A A C T T A G C 3 C C G A T T G A A T C G 5 3

  35. Screening a Library for Clones Carrying a Gene of Interest • The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest • Once identified, the clone carrying the gene of interest can be cultured

  36. Expressing Cloned Eukaryotic Genes • After a gene has been cloned, its protein product can be produced in larger amounts for research • Cloned genes can be expressed as protein in either bacterial or eukaryotic cells

  37. Bacterial Expression Systems • Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells • To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter • Also bacteria can’t deal with introns so use a cDNA form of the gene

  38. Eukaryotic Cloning and Expression Systems • The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems • YACs behave normally in mitosis and can carry more DNA than a plasmid • Eukaryotic hosts can provide the post-translational modifications that many proteins require

  39. Eukaryotic Cloning and Expression Systems • One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes • Alternatively, scientists can inject DNA into cells using microscopically thin needles • Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination

  40. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) • 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

  41. Fig. 20-8 3 5 TECHNIQUE Targetsequence 3 5 Genomic DNA 1 5 3 Denaturation 5 3 2 Annealing Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides Cycle 2yields 4 molecules Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

  42. Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene • DNA cloning allows researchers to • Compare genes and alleles between individuals • Locate gene expression in a body • Determine the role of a gene in an organism • Several techniques are used to analyze the DNA of genes

  43. Gel Electrophoresis and Southern Blotting • 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 Video: Biotechnology Lab

  44. Fig. 20-9a TECHNIQUE Powersource Mixture ofDNA mol-ecules ofdifferentsizes Anode Cathode – + Gel 1 Powersource – + Longermolecules 2 Shortermolecules

  45. Fig. 20-9b RESULTS

  46. Gel Electrophoresis and Southern Blotting • 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

  47. Fig. 20-10 Normal -globin allele Normalallele Sickle-cellallele 175 bp Large fragment 201 bp DdeI DdeI DdeI DdeI Largefragment Sickle-cell mutant -globin allele 376 bp 201 bp175 bp Large fragment 376 bp DdeI 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

  48. Gel Electrophoresis and Southern Blotting • 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

  49. Fig. 20-11a Restriction digest of the entire genome would give so many bands you would not be able to distinguish them TECHNIQUE Heavyweight Restrictionfragments I II III DNA + restriction enzyme Nitrocellulosemembrane (blot) Gel Sponge I Normal-globinallele II Sickle-cellallele III Heterozygote Papertowels Alkalinesolution 2 3 1 Preparation of restriction fragments Gel electrophoresis DNA transfer (blotting)

  50. Fig. 20-11b Transfer the gel to a membrane and probe with radioactively labeled primer that binds to just the gene you are interested in Radioactively labeledprobe for -globin gene Probe base-pairswith fragments I II III I II III Fragment fromsickle-cell-globin allele Film overblot Fragment fromnormal -globin allele Nitrocellulose blot 5 4 Hybridization with radioactive probe Probe detection