Chapter 10
Genetic Engineering: A Revolution in Molecular Biology
Chapter Outline
10.1.Basic Elements and Applications of Genetic Engineering
A. Genetics is considered basic science
B. Genetic engineering is an applied science
10.2.Tools and Techniques of Genetic Engineering
A. DNA: The raw material
1. Enzymes for dicing, splicing, and reversing nucleic acids
a. Restriction endonucleases
i. Palindromes
ii. EcoRI from E. coli and HindIII from Haemophilus influenzae
iii. Restriction fragments and restriction fragment length
polymorphisms (RFLPS)
b. Ligase
c. Reverse transcriptase
d. Complementary DNA (cDNA)
2. Analysis of DNA
a. Gel electrophoresis
3. Nucleic acid hybridization and probes
a. Gene probes
b. Southern blot method
c. FISH
4. Methods used to size, synthesize, and sequence DNA
- DNA sequencing: Determining the exact genetic code
i. Sanger method
5. Polymerase chain reaction: A molecular Xerox machine for DNA
a. Primers
b. DNA polymerases
i. Thermus aquaticus and Thermococcus litoralis
c. Steps in PCR cycle:
i. Denaturation
ii. Priming
iii. Extension
10.3.Methods in Recombination DNA Technology: How to Imitate Nature-Or to “One-Up” It
A. Introduction
1. Recombinant DNA technology
a. Clones
b. Vectors
c. Cloning hosts
B. Technical aspects of recombinant DNA and gene cloning
1. Characteristics of cloning vectors
a. Charon phage
b. Cosmids
c. Artificial chromosomes
2. Characteristics of cloning hosts
a. E. coli
b. Saccharomyces cerevesiae
c.Bacillus subtilis
C. Construction of a recombinant, insertion into a cloning host, and genetic expression
10.4.Biochemical Products of Recombinant DNA Technology
A. Human growth hormone
B. Insulin
C. Clotting factor VIII
10.5.Genetically Modified Organisms
A. Recombinant microbes: Modified bacteria and viruses
1. Pseudomonas syringae and Frostban
2. Pseudomonas fluorescens, Bacillus thuringiensis and insecticide genes
B. Transgenic plants: Improving crops and foods
1. Agrobacterium
C. Transgenic animals: Engineering embryos
1. Pharming
10.6.Genetic Treatments: Introducing DNA into the Body
A. Gene therapy
1. Adenosine deaminase (ADA) deficiency
2. SCID
B. DNA technology as genetic medicine
1. Antisense DNA: Targeting messenger RNA
10.7.Genome Analysis: Maps, Fingerprints, and Family Trees
A. Genome mapping and screening: An atlas of the genome
1. Locus and alleles
2. Linkage, physical and sequence maps
3. Genomics and bioinformatics
B. DNA fingerprinting: A unique picture of a genome
1. Single nucleotide polymorphism
2. Markers
a. Variable number of tandem repeats (VNTRs)
b. Microsatellite polymorphisms
3. Microarray analysis
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