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AP Biology Ch. 20 Slides for Test. DNA Cloning Plasmids -used to insert foreign DNA Recombinant plasmid is inserted into a bacteria Reproduction in bacteria.

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Presentation on theme: "AP Biology Ch. 20 Slides for Test. DNA Cloning Plasmids -used to insert foreign DNA Recombinant plasmid is inserted into a bacteria Reproduction in bacteria."— Presentation transcript:

1 AP Biology Ch. 20 Slides for Test

2 DNA Cloning Plasmids -used to insert foreign DNA Recombinant plasmid is inserted into a bacteria Reproduction in bacteria cell results in cloning of the plasmid including foreign gene Useful for making copies of a gene and producing a protein product

3 Making recombinant DNA Use bacterial restriction enzymes Cuts DNA at specific restriction sites Cuts covalent bonds between sugar phosphate backbone Making restriction fragments Most useful restriction enzymes cut DNA in a staggered way forming “sticky ends” DNA ligase seals the bonds between restriction fragments. Cloning vector is original plasmid carrying foreign gene into the host cell.

4 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. A bacterial artificial chromosome (BAC)is a large plasmid that can carry a large insert with many genes.

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

6 Fig DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA

7 Fig DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA DNA polymerase

8 Fig DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA DNA polymerase cDNA

9 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 This process is called nucleic acid hybridization

10 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 G 5 3 …… GGCCCTTTAAA C 3 5 CCGGGAAATTT

11 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

12 Fig Probe DNA Radioactively labeled probe molecules Film Nylon membrane Multiwell plates holding library clones Location of DNA with the complementary sequence Gene of interest Single-stranded DNA from cell Nylon membrane TECHNIQUE

13 Detecting DNA sequence by hybridization with a nucleic acid probe Results The location of the black spot on the photographic film identifies the clone containing the gene of interest. By using probes with different nucleotide sequences, researchers can screen the collection of bacterial clones for different genes.

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

15 Problems with expressing eukaryotic genes in bacterial host cells Scientists use an expression vector, a cloning vector that contains a highly active prokaryotic promoter This allows the bacteria to recognize the promoter and proceed to express the foreign gene. This allows synthesis of many eukaryotic proteins in bacteria cells. Another problem is the presence of introns in eukaryotic genes. Bacteria cells do not have RNA splicing machinery. This can be overcome by using cDNA which includes only the exons.

16 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

17 Amplifying DNA using Polymerase Chain reaction (PCR) PCR can produce many copies of a specific target segment of DNA Three-step cycle-heating--cooling--and replication Brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

18 Fig Genomic DNA TECHNIQUE Cycle 1 yields 2 molecules Denaturation Annealing Extension Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Target sequence Primers New nucleo- tides

19 Fig. 20-8a 5 Genomic DNA TECHNIQUE Target sequence 3 3 5

20 Fig. 20-8b Cycle 1 yields 2 molecules Denaturation Annealing Extension Primers New nucleo- tides

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

22 Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

23 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

24 Gel Electrophoresis and Southern Blotting Gel electrophoresis uses a gel as a molecular sieve to separate nucleic acids by size A current is applied to the gel that causes the charged molecules to move DNA is negatively charged and it moves towards a positive pole. Molecules are sorted into “bands” by their size

25 Fig. 20-9a Mixture of DNA mol- ecules of different sizes Power source Longer molecules Shorter molecules Gel Anode Cathode TECHNIQUE 1 2 Power source – + + –

26 Fig. 20-9b RESULTS

27 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

28 Fig Normal allele Sickle-cell allele Large fragment (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp (a) Dde I restriction sites in normal and sickle-cell alleles of  -globin gene Normal  -globin allele Sickle-cell mutant  -globin allele Dde I Large fragment 376 bp 201 bp 175 bp Dde I

29 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

30 Fig a TECHNIQUE Nitrocellulose membrane (blot) Restriction fragments Alkaline solution DNA transfer (blotting) Sponge Gel Heavy weight Paper towels Preparation of restriction fragmentsGel electrophoresis I II III DNA + restriction enzyme III Heterozygote II Sickle-cell allele I Normal  -globin allele 1 32

31 Fig b I II III Film over blot Probe detectionHybridization with radioactive probe Fragment from sickle-cell  -globin allele Fragment from normal  -globin allele Probe base-pairs with fragments Nitrocellulose blot 4 5 Radioactively labeled probe for  -globin gene

32 Studying the Expression of Interacting Groups of Genes 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

33 Fig TECHNIQUE Isolate mRNA. Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample. Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA fragments representing specific genes DNA microarray with 2,400 human genes DNA microarray

34 Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell Concept 20.3: Cloning organisms may lead to production of stem cells for research and other applications

35 Cloning Plants: Single-Cell Cultures 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

36 Fig EXPERIMENT Transverse section of carrot root 2-mg fragments Fragments were cultured in nu- trient medium; stirring caused single cells to shear off into the liquid. Single cells free in suspension began to divide. Embryonic plant developed from a cultured single cell. Plantlet was cultured on agar medium. Later it was planted in soil. A single somatic carrot cell developed into a mature carrot plant. RESULTS

37 Cloning Animals: Nuclear Transplantation In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell 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

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

39 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

40 Fig TECHNIQUE Mammary cell donor RESULTS Surrogate mother Nucleus from mammary cell Cultured mammary cells Implanted in uterus of a third sheep Early embryo Nucleus removed Egg cell donor Embryonic development Lamb (“Dolly”) genetically identical to mammary cell donor Egg cell from ovary Cells fused Grown in culture

41 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”

42 Fig

43 Problems Associated with Animal Cloning 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

44 Stem Cells of Animals A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem cells; these are able to differentiate into all cell types The adult body also has stem cells, which replace nonreproducing specialized cells

45 Fig Cultured stem cells Early human embryo at blastocyst stage (mammalian equiva- lent of blastula) Different culture conditions Different types of differentiated cells Blood cells Nerve cells Liver cells Cells generating all embryonic cell types Adult stem cells Cells generating some cell types Embryonic stem cells From bone marrow in this example

46 The aim of stem cell research is to supply cells for the repair of damaged or diseased organs

47 Concept 20.4: The practical applications of DNA technology affect our lives in many ways Many fields benefit from DNA technology and genetic engineering One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases

48 Human Gene Therapy Gene therapy is the alteration of an afflicted individual’s genes Gene therapy holds great potential for treating disorders traceable to a single defective gene Vectors are used for delivery of genes into specific types of cells, for example bone marrow Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

49 Fig Bone marrow Cloned gene Bone marrow cell from patient Insert RNA version of normal allele into retrovirus. Retrovirus capsid Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Inject engineered cells into patient

50 Pharmaceutical Products Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases

51 Host cells in culture can be engineered to secrete a protein as it is made This is useful for the production of insulin, human growth hormones, and vaccines Protein Production in Cell Cultures

52 Environmental Cleanup Genetic engineering can be used to modify the metabolism of microorganisms Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels

53 Agricultural Applications DNA technology is being used to improve agricultural productivity and food quality

54 Animal Husbandry Genetic engineering of transgenic animals speeds up the selective breeding process Transgenic animals are made by introducing genes from one species into the genome of another animal Beneficial genes can be transferred between varieties or species Protein Production by “Pharm” Animals and Plants Transgenic animals are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use “Pharm” plants are also being developed to make human proteins for medical use

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