DNA Technology & Genomics Chapter 20 DNA Technology & Genomics

Biotechnology Terms Biotechnology Genetic Engineering Recombinant DNA Process of manipulating organisms or their components to make useful products Genetic engineering + tissue/cell culturing technologies Genetic Engineering Manipulation of individual genes or entire genomes Insulin (insulin  E. coli bacteria OR yeast) & GMO (Genetically Modified Organism) Recombinant DNA Artificially created DNA Typically, DNA is integrated from another species

Biotechnology Terms (Page 2) Gene Cloning Laboratory production of multiple copies of DNA segment Therapeutic cloning – embryonic stem cells Spinal cord injuries Reproductive (organismal) cloning – Dolly the sheep Restriction Enzymes Enzymes that cut DNA at specific locations Usually, derived from bacteria Cut sites of DNA = restriction fragments Sticky ends – restriction fragments usually have one end longer than the other

Quick Assignment Relate the 6 terms just discussed in a concept map. Be prepared to defend your arrangement

Cloning Process 5 steps (first 2) 1. Identify & isolate the gene of interest Involves finding a cloning vector – plasmid or organism used to carry the DNA sequence to be cloned 2. Cut gene of interest from original site & open up vector’s DNA using a ________ ________ This ensures matching sticky ends on gene of interest & vector DNA

Cloning Process (Page 2) 5 steps (3-4) 3. Combine the 2 DNA pieces (into a recombinant plasmid?) Recombinant plasmid – plasmid + DNA fragments Sealed together using DNA Ligase Remember: we used ________ ________ to cut gene of interest from original site & cut vector’s DNA This ensures matching sticky ends on gene of interest & vector DNA 4. Transfer the vector (recombinant plasmid) into a host cell Usually involves bacterial transformation

Bacteria & Genetic Recombination Conjugation Bacterial Sex Genetic material is exchanged by direct contact Transduction Phage transfer of DNA Involves a phage vector Phage moves the DNA from bacterium to other bacterium

Bacteria & Genetic Recombination Transformation Uptake of exogenous DNA Griffith’s experiment - pathogenic DNA was transferred to benign bacteria Most common method for genetic engineering

Step 5 Select for transformed cells Link the gene of interest with a reporter gene Such as pBLU or pGLO pBLU = Blue coloration pGLO = fluorescent green under UV light In Lab 6, we will insert the coloration gene and an ampicillin resistance gene to select for transformed cells

At this point… You know which cells have the gene of interest You can identify the cells that have the gene of interest Now what? You need to extract the gene of interest How would you do that?

Nucleic Acid Hybridization Detects the gene of interest Uses a short, single stranded DNA or RNA called a nucleic acid probe The nucleic acid probe is complementary to a known sequence in the gene of interest Usually attach a radioactive isotope or fluorescent tag protein so that it is detectable

Genomic Libraries Nucleic Acid Hybridization repeated many times produces a genomic library Thousands of recombinant clones Each has a piece of the original genome being studied

cDNA Library cDNA = complementary DNA mRNA is extracted from cells Use what enzyme to make DNA from this mRNA? Then make another strand of DNA using what enzyme? cDNA library is only a portion of the genome Portion that codes for mRNA Exons? Introns? tRNA? rRNA?

Microarray Assay Genome-wide study of gene expression Different genes are in each well Identifies gene interactions + provides clues to gene functions Take samples throughout development + assay to determine which genes are expressed and at what stages Detect patterns of expression throughout development Detect likely response to a pathogenic agent

PCR 3 Steps Polymerase Chain Reaction Thermal cycling Amplification of DNA 3 Steps Denaturation (Heating) Annealing (Cooling) Primer formation Extension DNA polymerase adds nucleotides at 3’ end

Gel Electrophoresis  DNA is negatively charged so it moves AWAY from the (-) cathode toward the (+) anode

Southern Blotting Used to detect specific DNA sequences Useful for comparing samples Combines gel electrophoresis + nucleic acid hybridization

DNA Technology affects us… Disease Diagnosis PCR used to detect traces of viral DNA or RNA in sample RFLP (Restriction Fragment Length Polymorphisms) Different alleles have different RFLPs Gene Therapy – alter afflicted genes Pharmaceutical Production – Insulin production Forensic Application – DNA fingerprints

Page 2 Environmental cleanup Agricultural applications Genetically engineered microbes Detoxification of specific wastes Agricultural applications Insert pest-resistant or drought-resistant genes GMO (Genetically Modified Organisms) You eat GMO corn, soybeans, canola and cottonseed oil Probably at least weekly 46% of GMOs are grown in US Europe had 12 year moratorium on growing GE foods