AP Biology, Chapter 20
DNA Technology

Summary
Introduction

DNA CLONING
	Introduction
		1. Explain how advances in recombinant DNA technology have helped scientists study the 
		eukaryotic genome.
			a. Difficulties
				i. Long DNAs with many genes
				ii. Genes make up a small fraction of the genome
				iii. Genes are nearly identical to the noncoding sequences
			b. Solutions from DNA technology
				i. Work with small pieces
				ii. Many tools for dissecting and analyzing DNA
				iii. Can make many copies and express them
	DNA technology makes it possible to clone genes for basic research and commercial 
	applications: an overview
	Restriction enzymes are used to make recombinant DNA
		2. Describe the natural function of restriction enzymes.
			a. Made by bacteria to cut up invading DNA
			b. Host DNA protected my methylation at recognition sites
		3. Explain how the creation of sticky ends by restriction enzymes is useful in producing a 
		recombinant DNA molecule.
			a. Hundreds of restriction enzymes known
			b. Cut at specific sites leaving the same complementary ends
			c. Mixing DNAs cut with the same enzyme allows matching ends to bind
			d. DNA ligase heals backbones to make recombinant DNA	
	Genes can be cloned in recombinant DNA vectors: a closer look
		4. Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid.
			a. Cut plasmid and eukaryotic DNA with the same enzyme
			b. Mix fragments and allow them to match
			c. Ligate
			d. Transform into bacteria
			e. Identify desired clone
				i. Allow only plasmid-carrying bacteria to grow (AMP)
				ii. Add fragments to disrupt an easily screenable phenotype (GLO)
				iii. Use nucleic acid probe to ID clone with desired gene
		5. Describe the role of an expression vector.
			a. Goal: overcome differences between prokaryotes and eukaryotes 
			b. Expression vector has prokaryotic promoter next to restriction site
			c. Inserted eukaryotic gene now under prokaryotic host control
			b. Problem: introns
		6. Explain how eukaryotic genes are cloned to avoid the problems associated with 
		introns.
			a. mRNAs lack introns 
			b. mRNA  cDNA using reverse transcriptase
			c. cDNA inserted into vector
		7. Describe two [sic] advantages of using yeast cells instead of bacteria as hosts for 
		cloning or expressing eukaryotic genes.
			a. Easy to grow and have plasmids
			b. Process pre-mRNA
			c. Longer portions can be inserted in artificial chromosomes
			d. Perform some post-translational modification, can be engineered to do all
	Cloned genes are stored in DNA libraries
		8. Describe three techniques to aggressively introduce recombinant DNA into eukaryotic 
		cells.
			a. Electroporation opens holes in the membrane with electricity
			b. Micromanipulation with microinjection of single cells
			c. DNA-coated metal pellets fired into cells with a gun
		9. Define and distinguish between genomic libraries using plasmids, phages, and cDNA.
			a. Plasmid library: thousands of randomly cut pieces inserted into plasmids
			b. Phage: thousands of randomly cut pieces inserted into phages
			c. cDNA
				i. Only contains cloned copies of mRNAs
				ii. Isolated mRNA "transcribed" with reverse transcriptase
				iii. Then inserted in plasmids or phages
	The polymerase chain reaction (PCR) clones DNA entirely in vitro
		10. Describe the polymerase chain reaction (PCR) and explain the advantages and 
		limitations of this procedure.
			a. Advantages
				i. Quick and more specific and very sensitive
				ii. Amplifies only the sequence of interest
			b. Process
				i. Starting mix: Taq DNA polymerase, NTPs, single-stranded DNA primer
				ii. Process
					. Heat to separate target DNA strands
					. Cool to allow primers to bind to target DNA
					. Heat-resistant polymerase adds to primer
					. Heat again, separates new strands
					. Cool again, polymerase copies again
					. Repeat
				iii. Result: primer-specified copies double with each cycle
			c. Limitations
				i. Doesn't make large quantities of DNA
				ii. Errors in the chain reaction are amplified

ANALYSIS OF CLONED DNA
	Introduction
	Restriction fragment analysis detects DNA differences that affect restriction sites
		11. Explain how gel electrophoresis is used to analyze nucleic acids and proteins and to 
		distinguish between two alleles of a gene.
			a. Electrophoresis separates molecules by size, charge, shape
			b. Nucleic acid analysis
				i. Cut with a specific restriction enzyme  consistent set of pieces
				ii. Separate by electrophoresis  consistent pattern of bands
				iii. Each band is a specific piece of DNA
			c. Distinguishing alleles
				i. Alleles differ in base sequence
				ii. If one allele differs in a restriction site sequence it will not be cut
				iii. A different pattern of bands will result 
		12. Describe the process of nucleic acid hybridization.
			a. Mixed complementary nucleotide strands will bind
		13. Describe the Southern blotting procedure and explain how it can be used to detect 
		and analyze instances of restriction fragment length polymorphism (RFLP).
			a. Usefulness: identifies specific restriction fragments in complex mixtures
			b. Process of Southern blotting
				i. Cut with restriction enzymes, separate pieces in a gel
				ii. Blot fragments onto nitrocellulose paper
				iii. Soak paper in solution containing labeled probe sequence
				iv. Heat to separate strands
				v. Cool to allow hybridization
				vi. Detect radioactive label by overlaying X-ray film
		14. Explain how RFLP analysis facilitated the process of genomic mapping.
			a. There are many more differences in restriction sites in non-coding sequences
			b. Allowed reconstruction of the sequences between the widely scattered genes
			c. The differences are inherited allowing genetic identification
	Entire genomes can be mapped at the DNA level
		15. List the goals of the Human Genome Project.
			a. Map, sequence, and publish the entire human sequence
			b. Do the same for the major model systems: E. coli, yeast, C. elegans, 					Drosophila, and the mouse
			c. Develop sequencing technology = make it cheaper
		16. Explain how linkage mapping, physical mapping, and DNA sequencing each 
		contributed to the genome mapping project.
			a. Linkage mapping = ordering the known "genes" (about 5000)
			b. Physical mapping = isolating a complete set of overlapping segments
			c. DNA sequencing = determining the base order on each segment
		17. Describe the alternate approach to whole-genome sequencing pursued by J. Craig 
		Venter and the Celera Genomics company. Describe the advantages and disadvantages 
		of the private effort.
			a. Venter's approach: shotgun
				i. Skip the linkage and physical maps
				ii. Isolate random fragments
				iii. Determine their sequence
				iv. Have a computer identify overlaps and put together the map
			b. Advantage: it worked
				i. First to sequence a genome: Hemophilus influenzae
				ii. First to sequence Drosophila
				iii. Tied the government program to 90% of the human sequence
			c. Disadvantage: results not public, didn't develop technology
		18. Describe the surprising results of the human genome project.
			a. Only 20,000-25,000 genes
			b. A very small fraction of the total is genes
			c. Introns 10x longer than fly or worm introns
		19. Explain how the vertebrate genome, including that of humans, generates greater 
		diversity than the genomes of invertebrate organisms.
			a. More alternative pre-mRNA splicing
			b. Typical human gene makes 2-3 polypeptides
			c. Not more types of protein domains, put together in more combinations
			d. Then, post-translational modification further diversifies the products
		20. Describe what we have learned by comparing the human genome to that of other 
		organisms.
			a. New human gene may be homologous to a gene with a known function
			b. Confirms evolutionary connections/relevance of model systems
		21. Explain the purposes of gene expression studies. Describe the use of DNA microarray 
		assays and explain how they facilitate such studies.
			a. Purposes
				i. See what genes are active at different stages of development
				ii. … in different tissues
				iii. …in different states of health
			b. Microarrays
				i. = glass slides printed with dots of 1000s of single-strands DNAs
				ii. Hybridized with fluorescently labeled cDNA
				iii. Rapidly determines patterns of expression 
		22. Explain how in vitro mutagenesis and RNA interference help to discover the functions 
		of some genes.
			a. Especially useful for determining functions of unidentifiable genes
			b. in vitro mutagenesis
				i. Disable the cloned gene
				ii. Put it back in the organism, observe phenotype
			c. RNA interference
				i. Double-stranded RNA homologous to the unknown gene is introduced
				ii. Results in the breakdown of the mRNAs from the gene 
		23. Define and compare the fields of proteomics and genomics.
			a. Proteonomics
				i. = study of the whole set of proteins and their interactions
				ii. Essential; proteins determine phenotype
			b. Genomics
				i. = study of the whole set of DNA sequences and their interactions
				ii. Fundamental; represents the raw material for phenotype
		24. Explain the significance of single nucleotide polymorphisms in the study of the human 
		genome.
			a. Most human variation is due to single base differences
			b. = 1 in 1000 bp = 3 million differences
			c. Significance
				i. Allows reconstruction of human history
				ii. May determine health, susceptibility, treatment options

PRCATICAL APPLICATIONS OF DNA TECHNOLOGY
	Introduction
	DNA technology is reshaping medicine and the pharmaceutical industry
		25. Describe how DNA technology can have medical applications in such areas as the 
		diagnosis of genetic disease, the development of gene therapy, vaccine production, and 
		the development of pharmaceutical products.
			a. Diagnosis of disease
				i. PCR can find the DNA of pathogens in very small amounts
				ii. Diagnose hundreds of genetic disease
				iii. ID genetic diseases in embryos produced in vitro
				iv. Problem: some current RFLP markers are close to, but not at the 					abnormal gene
			b. Development of gene therapy; for example, using a retrovirus vector
				i. Insert RNA for the normal gene into retrovirus
				ii. Infect isolated bone marrow cells
				iii. Allow retrovirus to insert the normal gene into chromosome
				iv. Inject bone marrow cells back into patient
			c. Vaccine production
				i. Clone gene for antigenic protein from pathogen
				ii. Put cloned gene into bacteria
				iii. Bacteria make vaccine protein
			d. Development of pharmaceutical products
				i. Genes turned on abnormally in disease are targets for therapy
				ii. Cloned genes can be used to make proteins, like human growth 					hormone
	DNA technology offers forensic, environmental, and agricultural applications
		26. Explain how DNA technology is used in the forensic sciences.
			a. RFLP analysis can give a probable ID from 1000 cells = DNA fingerprint
			b. PCR of single tandem repeats is more powerful
				i. More variable  more certain ID
				ii. Requires only 20 cell sample
			c. Both require accompanying statistical arguments
		27. Describe how gene manipulation has practical applications for environmental and 
		agricultural work.
			a. Bioremediation
				i. Conversion of metals to recoverable forms
				ii. Break down harmful organic pollutants
			b. Transgenic organisms
				i. Goats with vaccine antigens cloned to appear in their milk
				ii. Corn and soybeans with bacterial insecticide genes
				iii. Enviropigs: have an added enzyme that gives them low P feces
				iv. Golden rice makes vitamin A
		28. Describe how plant genes can be manipulated using the Ti plasmid carried by 
		Agrobacterium as a vector.
			a. A segment of this plasmid is inserted into the host chromosome
			b. Desired new DNA is added to the Ti plasmid at that point
			c. Put back into Agrobacterium for infection or inserted directly
			d. Only works in dicots
	DNA technology raises important safety and ethical questions
		29. Describe the safety and ethical questions related to recombinant DNA studies and the 
		biotechnology industry.
			a. New genes in new combinations can pollute the gene pool
			b. Developing the technology to treat genetic disease may allow other 					manipulations