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Avery, MacLeod, and McCarty 1944 • Used bacteria from Griffith’s mouse experiment • Denatured proteins in membrane and discovered that the DNA still could make other bacteria pathogenic Biotechnology – pg. 140 in Cliffs Ch. 20 in text Recombinant DNA – a combination of DNA segments from two different sources Can occur through transduction, conjugation, transformation Can also occur during crossing over during meiosis in eukaryotes Biotechnology – use of biological systems to produce products like medicine Often use bacteria and viruses in experiments and production of products Recombinant DNA technology Set of lab techniques for combining genes from different sources. Requires the “cutting” of DNA using restriction enzymes Restriction enzymes cut DNA at very specific sequences called restriction sites Using bacterial plasmids we can clone specific genes to produce proteins of interest Ex. Medicine, farming, oil clean up Creating Sticky Ends Animation: Restriction Enzymes Right-click slide / select “Play” © 2011 Pearson Education, Inc. Using Restriction Enzymes to cut DNA • Restriction Enzyme Video Figure 20.3-3 Restriction site 5 3 GAATTC CTTAAG DNA 5 3 1 Restriction enzyme cuts sugar-phosphate backbones. 5 3 5 3 5 Sticky 3 3 5 end 5 2 DNA fragment added 3 3 5 from another molecule cut by same enzyme. Base pairing occurs. 5 3 5 3 3 DNA ligase 3 5 G AATT C C TTAA G G AATT C C TTAA G 53 5 3 3 5 One possible combination seals strands 5 3 3 Recombinant DNA molecule 5 Recombinant DNA Figure 20.6-5 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA A A A A A A 3 5 3 T T T T T 5 DNA Primer strand A A A A A A 3 T T T T T 5 5 3 5 3 DNA polymerase 3 5 3 5 5 3 cDNA Figure 20.2 Bacterium 1 Gene inserted into plasmid Bacterial Plasmid chromosome Recombinant DNA (plasmid) Cell containing gene of interest Gene of interest 2 Plasmid put into bacterial cell DNA of chromosome (“foreign” DNA) Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed from gene of interest Gene of interest Protein harvested Copies of gene Basic research on gene 4 Basic research and various applications Basic research on protein Gene for pest Gene used to alter Protein dissolves Human growth resistance inserted bacteria for cleaning blood clots in heart hormone treats into plants up toxic waste attack therapy stunted growth DNA cloning 1. Use restriction enzyme to cut a sample of DNA in test tube – this will create fragments with sticky ends, some will have our gene of interest 2. Cut a plasmid (cloning vector) with one restriction site for the restriction enzyme – the plasmid will now have the same sticky ends (plasmid should also be resistant to antibiotic like ampicillin) 3. Mix the foreign DNA with the plasmids 4. Apply DNA ligase Transformation Time Place the engineered plasmid into bacterial culture (in test tube) Heat shock and let transformation occur Plate the bacteria and those that grow on ampicillin will have “transformed” with the foreign gene of interest Genomic Library At the end, the bacteria now contain our gene of interest – genomic library Now the gene can be transcribed and translated to make the protein of interest This DNA is without introns because it was made from mRNA using reverse transcriptase before the experiment. cDNA Figure 20.4 TECHNIQUE Bacterial plasmid R amp gene Hummingbird cell lacZ gene Restriction site Sticky ends Gene of interest Hummingbird DNA fragments Recombinant plasmids Nonrecombinant plasmid 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 Figure 20.5 Foreign genome Cut with restriction enzymes into either small large or Bacterial artificial fragments fragments chromosome (BAC) Large insert with many genes Recombinant plasmids (b) BAC clone Plasmid clone (a) Plasmid library (c) Storing genome libraries 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 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 © 2011 Pearson Education, Inc. • A probe can be synthesized that is complementary to the gene of interest • For example, if the desired gene is 5 CTCATCACCGGC 3 – Then we would synthesize this probe 3 GAGTAGTGGCCG © 2011 Pearson Education, Inc. 5 Figure 20.7 Radioactively labeled probe molecules TECHNIQUE Gene of interest Probe DNA Multiwell plates holding library clones Nylon membrane 5 3 CTCATCACCGGC GAGTAGTGGCCG 5 3 Singlestranded DNA from cell Location of DNA with the complementary sequence Film Nylon membrane Finding specific mutations Gel Electrophoresis • In humans, researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition • Genetic markers called SNPs (single nucleotide polymorphisms) occur on average every 100–300 base pairs © 2011 Pearson Education, Inc. Figure 20.16 DNA T Normal allele SNP C Disease-causing allele Figure 20.10 Normal -globin allele 175 bp DdeI Large fragment 201 bp DdeI Normal Sickle-cell allele allele DdeI DdeI Large fragment Sickle-cell mutant -globin allele 376 bp 376 bp DdeI 201 bp 175 bp Large fragment DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of the -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles Figure 20.11 TECHNIQUE DNA restriction enzyme Restriction fragments I II III Heavy weight Nitrocellulose membrane (blot) Gel Sponge I Normal II Sickle-cell III Heterozygote -globin allele allele 1 Preparation of restriction fragments I II III Radioactively labeled probe for -globin gene Nitrocellulose blot 4 Hybridization with labeled probe Alkaline solution 2 Gel electrophoresis Paper towels 3 DNA transfer (blotting) Probe base-pairs with fragments Fragment from sickle-cell -globin allele Fragment from normal - globin allele I II III Film over blot 5 Probe detection Gel Box Applications of Gene Technology DNA Fingerprint DNA Fingerprinting • DNA fingerprinting Making copies of DNA - PCR Polymerase chain reaction (PCR) makes copies of DNA in order to have enough sample to run many tests on. You take the sample of DNA, and heat them along with DNA polymerase and A,T,C,G “primers” They will make millions of copies of the sample. Figure 20.8 5 TECHNIQUE 3 Target sequence Genomic DNA 1 Denaturation 3 5 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleotides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Studying the Expression of Interacting Groups of Genes Automation has allowed scientists to measure the 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 © 2011 Pearson Education, Inc. Figure 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 a specific gene DNA microarray 4 Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. DNA microarray with 2,400 human genes Using reverse transcriptase in gene therapy Isolate mRNA and use an enzyme called reverse transcriptase to create DNA These artificial DNA molecules can be inserted via a virus into a patient’s cells, then into the patient. Gene Therapy in humans Gene technology in Farming Golden Rice Rice injected with DNA that codes for betacarotene that we use to make vitamin A DNA injection Cloning Plants: Single-Cell Cultures • One experimental approach is to see whether a differentiated cell can generate a whole organism • A totipotent cell is one that can generate a complete new organism • Plant cloning is used extensively in agriculture © 2011 Pearson Education, Inc. Figure 20.17 Cross section of carrot root 2-mg fragments Fragments were cultured in nutrient 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. Adult plant 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 © 2011 Pearson Education, Inc. TECHNIQUE Mammary cell donor Egg cell donor 1 Figure 20.19 Cultured mammary cells 2 Egg cell from ovary 3 Cells fused 4 Grown in culture Nucleus removed Nucleus from mammary cell Early embryo 5 Implanted in uterus of a third sheep Surrogate mother 6 Embryonic development RESULTS Lamb (“Dolly”) genetically identical to mammary cell donor DNA Sequencing • Relatively short DNA fragments can be sequenced by the dideoxy chain termination method, the first automated method to be employed • 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 © 2011 Pearson Education, Inc. Figure 20.12 TECHNIQUE DNA (template strand) 5 C 3 5 3 T G A C T T C G A C A A Primer Deoxyribonucleotides Dideoxyribonucleotides T 3 (fluorescently tagged) G T T 5 DNA polymerase dATP ddATP dCTP ddCTP dTTP ddTTP dGTP ddGTP P P P P P P G OH DNA (template C strand) T G A C T T C ddG C G ddC T A T C G G A T T T A T ddA G C T G T T ddA A G C T G T T ddG A A G C T G T T Shortest Direction of movement of strands Longest labeled strand Detector Laser Shortest labeled strand RESULTS Last nucleotide of longest labeled strand Last nucleotide of shortest labeled strand H Labeled strands ddT G A A G C T G T T G A C T G A A G C G ddC T G A A G C T G T T ddA C T G A A G C T G T T ddG A C T G A A G C T G T T 3 5 Longest Gene Sequencing • Sanger Method of Sequencing • DNA sequencing machine ad