Topic 5: DNA Technology and Genomics Unit 4, Topic 5 - Genetic Engineering Topic 5: DNA Technology and Genomics

Unit 4, Topic 5 - Genetic Engineering OBJ 19 Recombinant DNA One purpose: To insert genes of one organism into bacteria to harvest protein Important players: Gene of interest (i.e. insulin, hGH, etc) Restriction enzymes Plasmid Bacteria

Making Recombinant DNA Unit 4, Topic 5 - Genetic Engineering OBJ 19 Making Recombinant DNA Isolate gene - Cut out the gene of interest Cut the plasmid Insert gene into the plasmid Insert the plasmid into bacteria (bacterial transformation-discovered by Freddy G) Grow bacteria and harvest the protein

Unit 4, Topic 5 - Genetic Engineering OBJ 19 Isolating the gene Use special proteins to cut the DNA strand at specific places Restriction enzymes: Target very specific base sequences Are found in more than 100 different varieties Are used in nature to protect bacteria from foreign invaders

Unit 4, Topic 5 - Genetic Engineering OBJ 19 Restriction Enzymes Each restriction enzyme recognizes a very specific nucleotide sequence EcoR1 recognizes: GAATTC CTTAAG The enzyme cuts it: G AATTC CTTAA G

Unit 4, Topic 5 - Genetic Engineering OBJ 19 Unit 4, Topic 5 - Genetic Engineering

Unit 4, Topic 5 - Genetic Engineering OBJ 19 Plasmids Plasmids - self-replicating rings of DNA containing 2-30 genes, found in bacterial cells Plasmid and gene cut with same restriction enzymes – Why? Plasmid and gene have complementary “sticky” ends

OBJ 19

Unit 4, Topic 5 - Genetic Engineering OBJ 19 How could you tell if the transformation really took place? The gene of interest is attached to a promoter and some kind of marker

How could you tell if the transformation really took place? Obj 19 How could you tell if the transformation really took place? Green Florescent Protein Source bioluminescent jellyfish Used as a visual marker of transformation

Insulin Production Obj 20

Pest-resistant crops (Bt toxin) Unit 4, Topic 5 - Genetic Engineering OBJ 20 Pest-resistant crops (Bt toxin) Bt Corn – Transgenic corn that contains a bacterial gene for the Bt toxin Bacillus thuringiensis – soil bacteria that produces a toxin that kills the Lepidoptera larvae

Gene Therapy Mutated forms of genes are replaced by functional genes. Obj 20 Gene Therapy Mutated forms of genes are replaced by functional genes. Currently used to treat some forms of… Leukemia Parkinson’s

Other Recombinant DNA Applications Unit 4, Topic 5 - Genetic Engineering OBJ 20 Other Recombinant DNA Applications Herbicide-resistant crops (glyphosate) Fast-growing fish (salmon & pout genes) Green-glowing aquarium fish (jellyfish genes) Spider silk expressed in goats milk!

Genetic Markers Regions of DNA that vary from person to person Obj 21 Genetic Markers Regions of DNA that vary from person to person Used to distinguish between DNA samples DNA Profiling (DNA fingerprinting)– identifies unique sections of DNA Same area of DNA strand from different people (sources), different length of sequence.

DNA Profiling (DNA ‘fingerprinting’) Unit 4, Topic 5 - Genetic Engineering OBJ 21 DNA Profiling (DNA ‘fingerprinting’) Purpose: Forensic science (crime scenes) Determining paternity Research (disease discovery and diagnosis) – targets coding regions

Polymerase Chain Reaction (PCR) Obj 22 Method used to copy DNA Samples Trace evidence can be amplified so many tests can be done Primers – small sections of DNA Selected to target certain DNA markers or genes

PCR - Steps Obj 22 Add Sample DNA, primers, DNA (Taq) polymerase to tube and free DNA nucleotide bases. DNA polymerase from bacteria Thermus aquaticus – lives in high temperature environments Increase sample Temperature to 94 oC Hot enough to separate interrupt H-bonds in DNA but not degrade the strands Reduce temperature to 54 oC – Primers base pair to open DNA Strands Increase Heat to72 oC – Optimal functional temperature of Taq polymerase, DNA replication!!!! Repeat Cycle about 40 times. 240 = ????????? Thermocycler

Unit 4, Topic 5 - Genetic Engineering OBJ 23 Gel Electrophoresis Laboratory method for comparing different samples of DNA Allows us to visualize DNA variations in.. Genetic markers – DNA profiling Genes – genetic tests

Gel Electrophoresis Method Unit 4, Topic 5 - Genetic Engineering OBJ 23 Gel Electrophoresis Method DNA samples are “digested” with specific restriction enzymes Since the samples vary, the number and length of DNA fragments created will vary Example: 2 different plasmids

Gel Electrophoresis Method Unit 4, Topic 5 - Genetic Engineering OBJ 23 Gel Electrophoresis Method DNA samples are “loaded” into the “wells” of an agarose gel that is submerged in buffer Electrical current is run through the buffer solution. DNA is negative!! Phosphate groups PO4- DNA fragments are pulled toward the positive pole Smaller fragments travel farther than larger fragments

Unit 4, Topic 5 - Genetic Engineering OBJ 23 Unit 4, Topic 5 - Genetic Engineering

Unit 4, Topic 5 - Genetic Engineering Example Gel Unit 4, Topic 5 - Genetic Engineering OBJ 23 Wells Bands of Larger Fragments Number of base pairs (bp) Bands of Smaller Fragments DNA Ladder - DNA sample with known fragment sizes

Unit 4, Topic 5 - Genetic Engineering Paternity cases OBJ 24 DNA tests that targets specific regions of DNA Genetic overlap is identified by sharing some bands with the biological Mother and Father

Which DNA segments are used for DNA profiling? Unit 4, Topic 5 - Genetic Engineering OBJ 24 Which DNA segments are used for DNA profiling? Short tandem repeats (STRs) are short sequences repeated many times in a row 13 different STR sites is enough for an individual profile PCR is used to amplify these sights and gel electrophoresis can separate the DNA segments of varying length.

Which DNA segments are used for DNA profiling? Unit 4, Topic 5 - Genetic Engineering OBJ 24 Which DNA segments are used for DNA profiling? Single nucleotide polymorphisms (SNPs) are single base-pair variations (like a substitution mutation) Restriction fragment length polymorphisms (RFLPs) are SNPs that change the length of DNA fragments created by restriction enzymes

Unit 4, Topic 5 - Genetic Engineering OBJ 24 RFLP - Example Restriction Fragment Length Polymorphisms (RFLPs) -

OBJ 24 RFLP – Example 2

Microarray Method for studying expression profiles of different types of cells. Ex. Pancreas vs. Hemoglobin Ex. Cancer cells vs. healthy cells

Reproductive vs. Therapeutic Cloning Unit 4, Topic 5 - Genetic Engineering Reproductive vs. Therapeutic Cloning

Gene Therapy?

Unit 4, Topic 5 - Genetic Engineering Gene Therapy Purpose: To alter an afflicted individual’s genes and replace defective genes with normal ones

Unit 4, Topic 5 - Genetic Engineering

Unit 4, Topic 5 - Genetic Engineering Gene Therapy Technical issues: Can we ensure that cells will make the right amount of the protein, at the right time, in the right place? Can we ensure the gene’s insertion doesn’t harm the cell’s normal function?

Unit 4, Topic 5 - Genetic Engineering Gene Therapy Ethical issues: Will it lead to eugenics, an effort to control the genetic makeup of humans? Should we try to eliminate genetic defects in our children and their descendents? Genetic variety is necessary for survival of a species over time!