CHAPTER 20: DNA TECHNOLOGY AND GENOMICS
recombinant DNA–
genetic engineering–
biotechnology–
DNA CLONING
I.
DNA TECHNOLOGY MAKES IT POSSIBLE TO CLONE GENES FOR BASIC RESEARCH AND
COMMERCIAL APPLICATIONS: AN OVERVIEW See Figure 20.1 p. 376
II.
RESTRICTION ENZYMES ARE USED TO MAKE RECOMBINANT DNA
A. Recognition site–
B. Restriction fragments–
*methylation–
C. Sticky ends–
D. DNA ligase–
III.
GENES CAN BE CLONED IN RECOMBINANT DNA VECTORS: A CLOSER LOOK
A. Cloning Vector–
B. Procedure for Cloning a Eukaryotic Gene in a Bacterial Plasmid See Figure 20.3 p. 378
1.
2.
3.
4.
5.
a) nucleic acid hybridization–
b) nucleic acid probe–
c) denaturation–
C. Cloning and Expressing Eukaryotic Genes: Problems and Solutions
1. Expression vector
2. Complementary DNA, cDNA–
a) isolate a mRNA–
b) reverse transcriptase–
3. Yeast artificial chromosomes (YACs)–
4. Electroporation
IV.
CLONED GENES ARE STORED IN DNA LIBRARIES
A. Genomic Library–
B. cDNA Library–
V.
THE POLYMERASE CHAIN REACTION (PCR) CLONES DNA ENTIRELY IN VITRO
A. Methods–
B. Uses–
DNA ANALYSIS AND GENOMICS
genomics–
gel electrophoresis–
VI.
RESTRICTION FRAGMENT ANALYSIS DETECTS DNA DIFFERENCES THAT AFFECT RESTRICTION
SITES
A. Hybridization–
1. Southern blotting– (see Figure 20.10 p.385)
2. *Northern blotting–
B. RFLP Analysis–
restriction fragment length polymorphisms–
1. Methods–
genetic markers–
2. Uses–
VII.
ENTIRE GENOMES CAN BE MAPPED AT THE DNA LEVEL
A. Human Genome Project–
Analyzing the Genomes of Other Species
B. Genetic (Linkage) Mapping
C. Physical Mapping: Ordering DNA Fragments
1. Chromosome walking–
2. Bacterial artificial chromosome (BAC)–
D. DNA Sequencing– the Sanger Method– (see Figure 20.12 p. 388)
E. Alternative approaches to Whole-Genome Sequencing–
Celera’s Methods
VIII. GENOME SEQUENCES PROVIDE CLUES TO IMPORTANT BIOLOGICAL QUESTIONS
A. Analyzing DNA Sequences
1. Surprisingly few genes in the human genome
2. Studying and comparing genes
B. Studying Gene Expression–
DNA microarray assays–
C. Determining Gene Function
1. in vitro mutagenesis–
2. RNA interference–
D. Future Directions in Genomics
1. Proteomics–
2. Bioinformatics–
3. Single nucleotide polymorphisms–
PRACTICAL APPLICATIONS OF DNA TECHNOLOGY
IX.
DNA TECHNOLOGY IS RESHAPING MEDICINE AND THE PHARMACEUTICAL INDUSTRY
A. Diagnosis of Diseases
1. Detection of genetic diseases–
2. Identification of heterozygote carriers–
B. Human Gene Therapy
1. Principle behind human gene therapy–
stem cells–
2. Technical questions–
3. Social and ethical questions–
C. Pharmaceutical Products
1. Insulin–
Human growth hormone–
*Erythropoietin–
Tissue plasminogen activator
2. Vaccines–
X.
DNA TECHNOLOGY OFFERS FORENSIC, ENVIRONMENTAL, AND AGRICULTURAL APPLICATIONS
A. Forensic Use of DNA Technology
1. DNA fingerprinting–
simple tandem repeats (STRs)–
2. Reliability–
3. Ethical problems–
B. Environmental Uses of DNA Technology
C. Agricultural Uses of DNA Technology
1. Animals Husbandry “Pharm” Animals–
a) transgenic organisms–
b) farm animals producing human proteins–
2. Genetic engineering in plants–
Ti plasmid–
3. herbicide resistance–
4. higher vitamin concentrations–
5. the nitrogen fixation challenge–
6. “pharm” plants
XI.
DNA TECHNOLOGY RAISES IMPORTANT SAFETY AND ETHICAL QUESTIONS
A. Genetically Modified (GM) Organisms
B. Other Concerns–