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

hosts for

c. cDNA inserted into vector 7. Describe two [sic] advantages of using yeast cells instead of bacteria as 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 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 b. Allowed reconstruction of the sequences between the widely

to detect

sequences scattered genes

each

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 contributed to the genome mapping project. a. Linkage mapping = ordering the known "genes" (about 5000) b. Physical mapping = isolating a complete set of overlapping c. DNA sequencing = determining the base order on each segment 17. Describe the alternate approach to whole-genome sequencing pursued by

segments 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 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 of other function microarray d. Then, post-translational modification further diversifies the products 20. Describe what we have learned by comparing the human genome to that organisms. a. New human gene may be homologous to a gene with a known b. Confirms evolutionary connections/relevance of model systems 21. Explain the purposes of gene expression studies. Describe the use of DNA 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 ii. Hybridized with fluorescently labeled cDNA iii. Rapidly determines patterns of expression 22. Explain how in vitro mutagenesis and RNA interference help to discover 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 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 ii. Fundamental; represents the raw material for phenotype 24. Explain the significance of single nucleotide polymorphisms in the study of the human genome.

greater

DNAs

the functions

introduced

interactions

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 PRACTICAL 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