Chapter 12 DNA Technology and Genomics (aka GENETIC ENGINEERING) ALIGNED WITH “Ch. 12 DNA Technology and Genomics Questions” Worksheet

1. What makes recombinant DNA technology different from just DNA technology? (12.1) DNA Technology - method for studying & manipulating genetic material Recombinant DNA – DNA in which two different sources are combined into the same DNA molecule

1. What are some examples of recombinant DNA technology? (12.1) Mass production of materials (cancer drugs, pesticides, insulin) Transfer of genes from one organism to another Production of pest-resistant plants, heartier crops, more muscular farm animals Piggies + Jellyfish Gene While these glowing pigs may be just “fun,” Asian researchers have created a glowing fish whose luminescence can be used as an indicator of the presence or absence of certain chemical pollutants

2. What are plasmids? What makes them such a good tool in gene cloning? (12.1) Plasmid – small, circular DNA not part of chromosome Plasmids can carry foreign genes (genes from other organisms) and replicate QUICKLY and CHEAPLY in the lab  good for making LOTS of copies of the foreign gene AND the protein product of the gene in mass quantities

3. Briefly describe the process of gene cloning. (12.2) Bacterial plasmid (vector) and gene of interest (already cut by RE) are mixed… complimentary base pairing assures that GOI (gene of interest) gets incorporated into plasmid (step 3) DNA ligase covalently bonds nucleotide backbone (step 4) Result is a recombinant plasmid which, when inserted into a bacterial cell, will multiply the new DNA (clone) (steps 5-6) Note: the plasmid vector usually also contains an antibiotic resistance gene that will allow scientists to isolate colonies that have the GOI. (Will grow bacteria on pates w/antibiotic – those with out the plasmid will be killed, those with plasmid can survive).

4. What is genetic engineering and what is its role in biotechnology? (12.1) direct manipulation of genes for practical purposes ex: DNA fingerprinting, genetically modified organisms and food, transplantation of genes, cloning Biotechnology uses GE to use organisms and their components to make useful products

5. What are restriction enzymes and how do they function in cloning genes? (12.2) Restriction Enzyme (RE) – molecular scissors which identify and “cut” certain nucleotide sequences, creating restriction fragments. Leave either blunt ends (not useful) or “sticky ends” which can be combined with other sticky ends (see next slide) In nature, these enzymes protect bacterial cells from “intruder” DNA from bacteriophages (will chop up foreign DNA); bacteria protect their own DNA from RE’s by chemical modification (methylation – adding methyl groups to their A’s and C’s) Use in gene cloning: Isolate the gene of interest using a restriction enzyme Cut the donor DNA & plasmid with the same RE to make complimentary sticky ends!!

6. What is meant by the term “sticky ends”? Why are they so important in genetic recombination? (12.2) Sticky ends: the single stranded ends of double stranded DNA fragments; the unpaired nucleotides can pair with complimentary bases from another molecule cut with the same RE!!!! Only sticky ends cut with same RE will be complimentary hill.com/olc/dl/120078/bio37.swf EcoRI – cleaves GAATTC 7. Why is it important to cut the two DNA sources with the same restriction enzyme? (12.2)

Sample RE’s and their restriction sites

8. What is a genomic library? What two types of vectors can be used to create these libraries? (12.4) When cutting with a RE, you get not only the gene of interest, but all possible fragments resulting from that cut!! Genomic Library – the entire collection of all the cloned DNA fragments from a genome Bacterial plasmids and bacteriophages can be used as vectors to create libraries

9. What is cDNA? Explain how reverse transcriptase works to create cDNA. (12.5) cDNA – DNA that is complimentary to processed mRNA; obtained by REVERSING TRANSCRIPTION from a mRNA sequence (catalyzed by reverse transcriptase) Single-stranded DNA molecule then creates a compliment using DNA polymerase

10. What is the advantage for using cDNA in the cloning process? (12.5) The resulting cDNA lacks all the introns…it only has the important genetic information because all the introns were spliced out during RNA processing Since bacteria don’t have introns, they can’t remove them from a foreign DNA insert when making the mRNA. cDNA is used to clone human genes This technique also helps us to see what part of the original gene is intron and what is exon. We can now compare original gene (introns and exons) vs. the cDNA (all exons) Also useful in studying particular cell functions cDNA animation

11. Describe the advantages for using bacteria, yeast, and mammalian cells for mass production of gene products. Give one example and its use for each. (12.6) BACTERIA Readily available & cheap Have plasmids Grow rapidly Produce large quantities of gene and protein QUICKLY YEAST Easy to grow Good at synthesizing eukaryotic proteins (can remove introns) Have plasmids MAMMALIAN CELLS Attach sugars correctly to make glycoproteins Can mass produce gene product by whole animals rather than cells (sheep produce CF treatment in milk) 1982: Humulin The first recombinant drug made by bacteria and approved by the FDA

12. Briefly describe how DNA technology is being used to create vaccines. (12.7) Vaccine – a harmless variant or derivative of a pathogen (usually bacteria or virus) that is used to prevent an infectious disease by stimulating an immune response in the vaccine recipient (human) Genetically engineered cells can be used to produce large amounts of the virus’ outer protein coat (hepatitis B vaccine) Mutant viruses can be made by altering one or more of the genes; immune response still triggered Replace some genes in harmless virus to provide immunity to several diseases simultaneously HepB vaccine animation

13. Explain how DNA probes tag a gene. (12.8) A radioactively labeled complement (the probe) to the gene of interest is created and introduced to single stranded DNA from a phage clone Can be used to screen an entire bacterial colony to discover which one(s) contain genes of interest

GENETICALLY MODIFIED (GM) ORGANISMS Use Ti plasmid of Agrobacterium tumefaciens as the vector  GM plant ex: soybeans and cotton crops receive bacterial genes to make them resistant to herbicides and pests ex: “golden rice” = rice with a few daffodil genes added. Rice plant can now make B-carotene, needed for vitamin A production. Vitamin A deficiency (and resulting blindness) is a serious problem for HALF of the world who depend on rice as their staple food. Ti plasmid animation