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Chapter 20

Chapter 20. Biotechnology. 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

reuben-reilly
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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 • 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

  3. Fig. 20-1 An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes (Research Method Fig. 20.15)

  4. 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 • 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 producing a protein product • 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

  5. Fig. 20-2a Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 2 Plasmid put intobacterial cell Recombinantbacterium

  6. Fig. 20-2b Recombinantbacterium Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest 3 Protein expressedby gene of interest Gene ofInterest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste

  7. Using Restriction Enzymes to Make Recombinant DNA • 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

  8. Fig. 20-3-1 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end

  9. Fig. 20-3-2 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

  10. 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

  11. 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 • 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

  12. Fig. 20-4-1 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Isolate plasmid DNA from bacterial cells and DNA from hummingbird cells. The hummingbird DNA contains the gene of interest. Cut both DNA samples with the same restriction enzyme, one that makes a single cut within the lacZ gene and many cuts within the hummingbird DNA The plasmid has been engineered to carry two genes; ampR, which makes E. coli cells resistant to the antibiotic ampicillin, and lacZ, which encodes an enzyme that hydrolyzes the sugar lactose. This enzyme can also hydrolyze a similar synthetic molecule to form a blue product.

  13. 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 Mix the cut plasmids and DNA fragments. Some join by base pairing; add DNA ligase to seal them together. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant plasmids Grows white because lacZ gene has been cut Grows blue because lacZ gene is intact

  14. Fig. 20-4-3 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Mix the DNA with bacterial cells that have a mutation in their own lacZ gene. (This gene is normally found on the chromosomal DNA not plasmid DNA.) Some cell take up a recombinant plasmid or other DNA molecule by transformation. Some cells may not take up and plasmids (unlikely) and will not grow on a growth medium that contains ampicillin. Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids

  15. 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 Recombinant plasmids Plate the bacteria on agar containing ampicillin and X-gal, a molecule resembling lactose. Incubate until colonies grow. Bacteria carryingplasmids RESULTS Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non-recombinant plasmidwith intact lacZ gene One of manybacterial clones

  16. Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form a colony. Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal, forming a blue product. Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because hey cannot hydrolyze X-gal. Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids RESULTS Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non-recombinant plasmidwith intact lacZ gene Which white colony carries the gene of interest? See Fig. 20.7 One of manybacterial clones

  17. Fig. 20-5a Foreign genomecut up withrestrictionenzyme or Recombinantphage DNA Bacterial clones Recombinantplasmids Phageclones 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

  18. Fig. 20-5b Large insertwith many genes Large plasmid 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 BACclone (c) A library of bacterial artificial chromosome (BAC) clones

  19. 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

  20. Fig. 20-6-1 DNA innucleus mRNAs in cytoplasm 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 mRNA is used as a template for the first strand because it contains only exons. Bacteria do not contain splisosomes.

  21. Fig. 20-6-2 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail Reverse transcriptase makes the first DNA strand using the RNA as a template and a stretch of dT’s as a DNA primer mRNA Primer DNAstrand

  22. Fig. 20-6-3 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA mRNA is degraded by another enzyme.

  23. Fig. 20-6-4 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase synthsizes the second strand, using a primer in the reaction mixture. DNA polymerase

  24. Fig. 20-6-5 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA The result is cDNA, which carries the complete coding sequence of the gene but no introns. DNA polymerase cDNA

  25. 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 • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene • This process is called nucleic acid hybridization … … 5 G G C T A A C T T A G C 3 3 C C G A T T G A A T C G 5 • 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

  26. Fig. 20-7 • TECHNIQUE Radioactivelylabeled probemolecules ProbeDNA Gene ofinterest Multiwell platesholding library clones Single-strandedDNA from cell Film Nylon membrane Nylonmembrane Location ofDNA with thecomplementarysequence This is the technique used to identify the gene of interest in bacterial cloning. (Fig. 20.4)

  27. 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

  28. 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

  29. Fig. 20-8a Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) 5 3 TECHNIQUE Targetsequence Genomic DNA 3 5 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

  30. Fig. 20-8b 1 5 3 Denaturation Heat briefly to separate DNA strands 3 5 2 Annealing Cool to allow primers to form hydrogen bonds with ends of target sequence Cycle 1yields 2 molecules Primers 3 Extension DNA polymerase adds nucleotides to the 3’ end of each primer. Newnucleo-tides

  31. Fig. 20-8c Cycle 2yields 4 molecules

  32. Fig. 20-8d Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

  33. 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 • 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

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

  35. Fig. 20-9b • 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

  36. 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

  37. Fig. 20-11a • 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 TECHNIQUE Heavyweight Restrictionfragments I II III DNA + restriction enzyme Nitrocellulosemembrane (blot) Gel Sponge I Normal-globinallele II Sickle-cellallele III Heterozygote Papertowels Alkalinesolution 3 DNA transfer (blotting) With the gel arranged as shown above, capillary action pulls the alkaline solution upward through the gel, transferring the DNA to a nitrocellulose membrane, producing the blot; the DNA is denatured in the process. The single strands of DNA stuck to the nitro- cellulose are positioned in bands corresponding to those on the gel. 2 1 Preparation of restriction fragments. Each DNA sample is mixed with the restriction enzyme. Digestion of each sample yields a mixture of thousands of restriction fragments. Gel electrophoresis The restriction fragments in each sample are separated by electrophoresis, forming a characteristic pattern of bands.

  38. Fig. 20-11b 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 5 Probe detection A sheet of photographic Film is laid over the blot. The radioactivity in the Bound probe exposes the Film to form an image Corresponding to those bands Containing DNA that base-paired With the probe. Nitrocellulose blot 4 Hybridization with radioactive probe The nitrocellulose blot is exposed to a solution containing a radioactively labeled probe. Probe molecules attach by base- paring to any restriction fragments containing a part of the gene

  39. Fig. 20-11 TECHNIQUE Heavyweight Restrictionfragments I II III Nitrocellulosemembrane (blot) DNA + restriction enzyme Gel Sponge I Normal-globinallele II Sickle-cellallele III Heterozygote Papertowels Alkalinesolution 2 1 3 Preparation of restriction fragments DNA transfer (blotting) Gel electrophoresis Because the band patterns are different, this method can be used to identify different genotypes. (homozygous dominant, heterozygotes, or recessive) 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 4 5 Probe detection Hybridization with radioactive probe

  40. DNA Sequencing • Relatively short DNA fragments can be sequenced by the dideoxy chain termination method • Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths • Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment • The DNA sequence can be read from the resulting spectrogram

  41. The fragment of DNA to be sequenced is denatured into single strands and incubated in a test tube with the necessary ingredients for DNA synthesis; a primer designed to base pair with the know # end of the template strand, DNA polymerase, the four deoxyribonucleotides, and the four dideoxyribonucleotides, each tagged with a specific flourescent molecule. TECHNIQUE DNA(template strand) Primer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged) dATP ddATP dCTP ddCTP dTTP ddTTP DNA polymerase dGTP ddGTP

  42. Fig. 20-12b TECHNIQUE DNA (template strand) Labeled strands Synthesis of each new strand continues until a ddNTP is inserted. This prevents further elongation. Eventually, a set of labeled strands of various lenghts is generated. Shortest Longest Directionof movementof strands Longest labeled strand Detector Laser Shortest labeled strand RESULTS Last baseof longestlabeledstrand Last baseof shortestlabeledstrand

  43. Fig. 20-12b TECHNIQUE DNA (template strand) Labeled strands The labeled strands are separated and a flourescence detector senses the color of each flourescent tag. Shortest Longest Directionof movementof strands Longest labeled strand Detector Laser Shortest labeled strand RESULTS Last baseof longestlabeledstrand Last baseof shortestlabeledstrand

  44. Fig. 20-14 • In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism 50 µm

  45. Concept 20.3: Cloning organisms may lead to production of stem cells for research and other applications • Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell • One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism • A totipotent cell is one that can generate a complete new organism (Fig. 20.16) • In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell (Fig. 20.17) • Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg • However, the older the donor nucleus, the lower the percentage of normally developing tadpoles

  46. Fig. 20-16 EXPERIMENT RESULTS Transversesection ofcarrot root 2-mgfragments A singlesomaticcarrot celldevelopedinto a maturecarrot plant. Fragments werecultured in nu-trient medium;stirring causedsingle cells toshear off intothe liquid. Singlecellsfree insuspensionbegan todivide. Embryonicplant developedfrom a culturedsingle cell. Plantlet wascultured onagar medium. Later it wasplantedin soil.

  47. Fig. 20-17 Frog egg cell Frog embryo Frog tadpole EXPERIMENT UV Fully differ- entiated (intestinal) cell Less differ-entiated cell Donornucleustrans-planted Donor nucleus trans- planted Enucleated egg cell Egg with donor nucleus activated to begin development RESULTS Most develop into tadpoles Most stop developing before tadpole stage

  48. Reproductive Cloning of Mammals • In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell • Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus • Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs • CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent” (Color patterns are different because of random chromosome inactivation, which is a normal occurrence during embryonic development – fig. 15.8) • In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth • Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development

  49. Fig. 20-18 TECHNIQUE Mammarycell donor Egg celldonor 2 1 Egg cellfrom ovary Nucleusremoved Cells fused 3 Culturedmammary cells 3 Nucleus frommammary cell Grown inculture 4 Early embryo Implantedin uterusof a thirdsheep 5 Surrogatemother Embryonicdevelopment 6 Lamb (“Dolly”)genetically identical tomammary cell donor RESULTS

  50. Fig. 20-19

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