The objective is to introduce the principles, methods, and applications of modern methods of gene modification.
 | Isolate and fragment of source DNA |
| Join source DNA to cloning vector |
| Incorporate DNA into host |
| Detect and purify desired clone |
| Replicate clone to high numbers |
3.3.1. Restriction Enzymes
| definition: enzymes that cut DNA in specific places |
| function: inactivate foreign DNA which can derange metabolism |
| breaks only palindrom sequences, i.e. those exhibiting two-fold symmetry |
| mechanism (9-1) |
| examples (9-2) |
| Important in DNA research, i.e. sequencing, hybridization |
| Companies purify and market restriction enzymes |
3.3.2. Modification Enzymes
| protect destruction of native DNA by native restriction enzymes |
| mechanism (9-3) |
| A nucleotide can be a substrate for a particular restriction enzyme or modification enzyme but not both |
3.4.1. Objective: to make multiple copies of a specific gene
4.4.2. Steps
| a (9-4) |
| b (9-5) |
| c (9-6) |
3.4.3. Method
| DNA is removed and purified |
| DNA is heated to denature (separate strands) |
| Specific primer are added to locate desired sequences along with DNA polymerase; primer initiates replication of desired fragments during cooling cycle |
| Cycle is repeated resulting in logarithmic increase in desired fragments |
| Amplified DNA is separated using electrophoresis. |
| Amplified DNA may now be sequenced or used for other purposes |
3.5.1. Plasmids
| example of plasmid (9-7) |
| cutting plasmid with restriction enzymes (a)(9-8) (b)(9-9) (c)(9-10) |
| DNA modification using plasmids (9-11) |
| advantages (9-12) |
3.5.2. Phage
| DNA modification using phage (9-13) |
| Steps in cloning with phage (9-14) |
3.5.3. Detection of Cloned Gene (9-15)
3.6.1. Gene Gun
| diagram (9-16) |
| photograph (9-17) |
| DNA-coated pellets are injected into recipient cells |
3.6.2. Microinjection
| method (9-18) injection into mouse sperm pronucleus |
| example (9-19) salmon eggs injected with gene for growth hormone (14 month-old fish) |
4. Other Tools and Methods
| Gels (9-20) |
| Micropipettes (9-21) |
| DNA Sequencing Gel (9-22) |
| DNA Fingerprints (9-23) |
| DNA Synthesis (9-24) |
| Reverse Transcription (9-25) |
| Gene Probes (9-26) |
| humans have about 30,000 genes (3.2 billion base pairs) in 23 pairs of chromosomes |
| 99.9% of the nucleotide sequence is the same in different (race, sex, etc.) humans |
| will be useful in detecting genetic diseases and gene therapy |
| Protein Sequencing |
| DNA Sequencing |
| DNA Synthesis |
| Protoplast Fusion |
| Electron Microscopy |
| Flow Cytometry |
| Antibody Probes |
| Hybridoma |
| Glycobiology |
| Biological Computing |
| Education and Training |
7.1. What is the general objective of genetic "engineering"?
7.2. What is the function and mechanism of restriction and modification enzymes?
7.3. How are plasmids viruses used as vectors?
7.4. What are two methods of detecting genetically modified organisms?
7.5. What is the objective and steps of PCR? What do you think discovery of PCR warranted a Nobel Prize?
7.6. How could one determine that blood on a defendant's shirt came from a murdered victim?
7.7. Why would one want to sequence some isolated DNA?
7.8. Why would one want to synthesize a DNA piece with a know base sequence?
| DNA synthesis | protoplast fusion | antibody probe |
| gene probe | electron microscopy | hybridoma |
| flow cytometry | gene chromatography | fluorescence |
| protein sequencing | DNA sequencing | dendragram |
| RNA probe |
| conjugation | transformation | transduction |
| cDNA | cloned DNA | DNA library |
| Dna ligase | eugenic engineering | gene therapy |
| genetic fingerprint | plasmid | RFLP |
| polymerase chain reaction | recombinant DNA technology | mutation |
| gene probe | restriction enzyme reverse transcription | mutagen |
The Genetic Code - S.D. Black, U. of Texas Health Center at Tyler Table relating nucleic acid triplet to the corresponding amino acid.
Molecular Biology - The Biology Project, Biology, University of Arizona Activities, Problems sets, and Tutorials: Molecular Genetics; Nucleic Acids; Recombinant DNA Technology; Eukaryotic Gene Expression
Prokaryotic Genetics and Gene Expression from the MIT Hypertextbook - M.I.T. Tools for studying prokaryotic genetics, biochemical genetics, Lac operation, etc.
The making of the nucleosome - D. Pruss Simplified images and a tour.
