Chapter 14: Genetic Engineering -Modification of the DNA of an organism to produce new genes with new characteristics

Biotechnology Use of organisms to benefit humanity

Recombinant DNA technology DNA from different organisms is spliced together Allows scientists to make many copies of any DNA segment (clone) Can introduce foreign DNA into cells of microorganisms

Recombinant DNA technology Restriction enzymes – cut DNA Bacteria produce for defense against viruses Vector – transports DNA into a cell Ex: bacteriophage Plasmid – separate, smaller circular DNA that maybe be present and able to replicate inside bacteria Transformation – uptake of foreign DNA by cells How plasmids can get into bacteria

Bacterial Conjugation and Recombination

Recombinant DNA technology Palindromic sequences – reads the same as complement, in opposite direction AAGCTT TTCGAA Many restriction enzymes cut these sequences Restriction enzymes cut on a stagger  sticky ends (can pair with complementary single-stranded end of other DNA cut with same enzyme) DNA Ligase – links 2 fragments  recombinant DNA

Restriction enzyme cuts sugar-phosphate backbones. Fig. 20-3-3 Restriction site DNA 5 3 3 5 1 Restriction enzyme cuts sugar-phosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA One possible combination 3 DNA ligase seals strands. Recombinant DNA molecule

Restriction Enzymes

Steps of Creating a Recombinant DNA Plasmid (Basic) 1. Plasmids and desired DNA cut by same restriction enzyme 2. Mix 2 types of DNA so sticky ends pair 3. DNA ligase forms bonds between fragments

Figure 20.2 A preview of gene cloning and some uses of cloned genes Cell containing gene of interest Bacterium 1 Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome 2 Plasmid put into bacterial cell Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Copies of gene Protein harvested Figure 20.2 A preview of gene cloning and some uses of cloned genes 4 Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

Cloning a Gene

Fig. 20-4-4 TECHNIQUE Hummingbird cell Bacterial cell lacZ gene Restriction site Sticky ends Gene of interest ampR gene Bacterial plasmid Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids Figure 20.4 Cloning genes in bacterial plasmids RESULTS Colony carrying non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones

Cloning DNA Genome – total DNA per cell Genomic library – collection of DNA fragments more or less representative of all DNA in genome Genetic Probe – single stranded DNA or RNA that is radioactively labeled and can attach to target sequence by base pairing rules

– Then we would synthesize this probe 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 3 C C G A T T G A A T C G 5

DNA Probe

Using a DNA probe

Polymerase Chain Reaction (PCR) Can amplify a small sample of DNA quickly DNA replication in vitro 2 strands separated by heating so special heat-resistant DNA polymerase called Taq polymerase used (thermophile) MAJOR BONUS: Only specific sequences can be replicated Study: crime scenes, archaeological remains

PCR

Gel Electrophoresis Separates fragments like DNA, RNA or polypeptides (they carry charge and can migrate in an electrical field RNA and DNA (-) --- so they move to (+) pole Smaller fragments go further Compare sample to standard Usually “blot” - transfer DNA from gel to nitrocellulose filter for further analysis DNA Fingerprinting

Gel Electrophoresis

Mixture of DNA mol- ecules of different sizes Fig. 20-9a TECHNIQUE Power source Mixture of DNA mol- ecules of different sizes – Cathode Anode + Gel 1 Power source Figure 20.9 Gel electrophoresis – + Longer molecules 2 Shorter molecules

Fig. 20-9b RESULTS Figure 20.9 Gel electrophoresis

DNA Fingerprint

Transgenic Organisms Plants and animals in which foreign genes have been incorporated Animals Inject DNA into nucleus of egg or stem cell Eggs implanted in uterus; stem cells injected into blastocysts + then implanted into foster mother Plants Disease resistance Pesticide resistance

Transgenics

From bone marrow in this example Fig. 20-20 Embryonic stem cells Adult stem cells Early human embryo at blastocyst stage (mammalian equiva- lent of blastula) From bone marrow in this example Cells generating all embryonic cell types Cells generating some cell types Cultured stem cells Different culture conditions Figure 20.20 Working with stem cells Different types of differentiated cells Liver cells Nerve cells Blood cells

TECHNIQUE RESULTS Mammary cell donor Egg cell donor Fig. 20-18 TECHNIQUE Mammary cell donor Egg cell donor 1 2 Egg cell from ovary Nucleus removed Cultured mammary cells 3 Cells fused 3 Nucleus from mammary cell 4 Grown in culture Early embryo Figure 20.18 Reproductive cloning of a mammal by nuclear transplantation For the Discovery Video Cloning, go to Animation and Video Files. 5 Implanted in uterus of a third sheep Surrogate mother 6 Embryonic development Lamb (“Dolly”) genetically identical to mammary cell donor RESULTS

Fig. 20-19 Figure 20.19 CC, the first cloned cat, and her single parent

Cloning Video

GE Plants

Application of GE Human proteins Human treatments for disease Vaccines Insulin Hormones - HGH Human treatments for disease Multiple sclerosis, certain cancers, heart attacks, forms of anemia Vaccines

Fig. 20-23 Figure 20.23 Goats as “pharm” animals

Fig. 20-24 (a) This photo shows Earl Washington just before his release in 2001, after 17 years in prison. Source of sample STR marker 1 STR marker 2 STR marker 3 Figure 20.24 STR analysis used to release an innocent man from prison For the Discovery Video DNA Forensics, go to Animation and Video Files. Semen on victim 17, 19 13, 16 12, 12 Earl Washington 16, 18 14, 15 11, 12 Kenneth Tinsley 17, 19 13, 16 12, 12 (b) These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder.

Forensics