Additional Handouts
Practice problems on Homologous Recombination (PDF)
Laboratory Techniques (PDF)
The following techniques are described over the course of the semester in 7.28.
| LEC # | TOPICS | TECHNIQUES | ASSAYS FOR |
|---|---|---|---|
| 1 | Replication (Replication Enzymes) | dNTP Incorporation Assay | DNA Synthesis (Fast; Quantitative for Amount of Synth.). |
| Filter Binding Assay | Method to separate incorporated from unincorporated dNTPs. | ||
| Gel Electrophoresis | Method to separate DNA on the basis of length. | ||
| Primer Extension Assay | DNA Synthesis (Slow; Quantitative for Length of Synth.). | ||
| Template Challenge Assay | Assay for Processivity. | ||
| 2 | Replication (Replication Fork) | DNA Helicase Assay | In vitro DNA Helicase Activity. |
| Helicase Polarity Assay | Assay for directionality of helicase movement; used to study function of replication termination sequences. | ||
| Topoisomerase Assay | Assay for changes in DNA topology; distinguish supercoiled, relaxed, nicked, linear, forms of DNA and measure catenation/decatenation. | ||
| 3 | Replication (Prok. System) | 2-D Agarose Gels | Assay for Origin of Replication (Must have an idea of where the origin is to test). |
| Southern Blotting | Identifies specific DNA molecules transferred to membrane. | ||
| DNA Microarray Assay | Assays the relative amount of replicated DNA sequences during a synchronized round of DNA replication to identify sites of replication initiation. | ||
| Plasmid Replicator Assay | Identifies region of DNA sufficient for Replicator activity. | ||
| Mutational Mapping Assay | Identifies regions of DNA necessary for Replicator activity. | ||
| Biochemical Fractionation | Mechanism to purify biochemical activity that can be assayed in vitro. | ||
| Biochemical Complementation | Combines mutant extracts with biochemical fractionation to identify proteins required for assay (in this case DNA replication). | ||
| 4 | Replication (Euk. Systems) | Genetic Screen for DNA Replication Mutants | |
| DNase I Protection Assay | Assay for Sequence Specific DNA Binding (Slower and not quantitative; Gives Information about bound DNA sequence). | ||
| Gel Mobility Shift Assay | Assay for Sequence Specific DNA Binding (Fast and Quantitative but less information about bound sequence). | ||
| DNA Unwinding Assay | Assays for formation of ssDNA through the use of a ssDNA specific nuclease. | ||
| 5 | Chromatin Assembly | Template Association Assay | Uses gel filtration to separate molecules bound to a plasmid from molecules that are not bound to a plasmid. |
| 6 | DNA Mismatch Repair | Heteroduplex DNA analysis | Assay for DNA repair in vivo. |
| Restriction Analysis | Assay for methylation state of DNA. | ||
| Restriction site repair | Assay of DNA repair in vitro; also used incorporation assay. | ||
| 7 | DNA Repair II | Ames Test | Assay for chemicals and other factors that increase the frequency of mutations. |
| Nicking of Circular DNA | Assay for excision-type mechanism of DNA repair. | ||
| Lesion bypass DNA synthesis | Assay for ability of replication/repair proteins to synthesize DNA off of a damaged template. | ||
| 8 | DS Break Repair, Homologous Recombination | Strand Exchange Assay | Assay for homologous DNA pairing and branch migration. |
| 9 | Homologous Recombination Enzymes | ATPγS | Used in determining whether ATP binding or ATP-hydrolysis is required for a reaction. |
| DMS Footprinting | Assay for protection of the bases in DNA, rather than the phosphodiester backbone. | ||
| Cooperative DNA Binding | Gel-shift assay to measure cooperative vs. non-cooperative DNA binding. | ||
| 10 | Meiotic Recombination | Consensus Sequences | Identification of important elements by conservation. |
| Native-Denaturing 2D Gel | Detection of nicks vs DSBs in DNA. | ||
| 11 | Site-specific Recombination | DNA Bending Assay | Assay for whether a protein bends DNA upon binding and position of bend. |
| 12 | Transposition | Genomic Southern | Identifies specific DNA molecules transferred to membrane (here used for transposition). |
| Plasmid Mating | Assay for transposition in vivo by determining movement of drug resistance to a new plasmid. | ||
| 13 | Transcription: Prok. Machinery and Promoters | Incorporation Assay | Assay for Transcription in vitro. |
| S1 Protection Assay | Assay for Transcription. Maps start site of transcription. | ||
| Northern Blot | Assay for Transcription. Maps steady state RNA in vivo. | ||
| Microarray Analysis of RNA | Assay for relative levels of gene expression. Can assay many genes at once. | ||
| Pulse Labeling of in vivo RNA | Assay for actively transcribing genes. | ||
| Promoter Fusion Assay | Assay to indirectly measure promoter activity. | ||
| 14 | Transcription: Euk. Machinery and Promoters | DNA Unwinding Assay | Assay for open complex formation. |
| Indirect end-labeling | Method to extend a labeled DNA primer to measure breaks or modification of DNA. | ||
| 15 | Transcription: Regulation I | Antibody Supershift Assay | Assay to determine the components of protein-DNA complex in a gel shift assay. |
| 16 | Transcription: Regulation II | Transcription Factor DNA Binding Domain Mapping | |
| Transcriptional Factor Activation Domain Mapping | |||
| Chromatin Immunoprecipitation | Assay to determine the in vivo binding site of proteins. | ||
| 17 | RNA Processing I | R-Loop Formation | Assay for regions of mRNA that anneal to different DNA fragments. |
| Gel Electrophoresis Assay | For intermediates and products formed during splicing in vitro (done using labeled RNA). | ||
| 18 | RNA processing II | Calculation of "Consensus Fold" and Rnase Digestion | Determination of RNA secondary structures. |
| 19 | RNA Processing: Regulation | cDNA Cloning | Determine mRNA sequence and structure, protein expression. |
| RT-PCR | Generate segments of amplified DNA starting with specific RNA template. | ||
| Exon Trapping | Find exons in DNA fragments of regions thought to contain genes. | ||
| 20 | RNA Editing, RNAi and miRNAs | ||
| 21 | Translation: Basic Machinery I | ||
| 22 | Translation: Basic Machinery II | ||
| 23 | Translation: Regulation I | Incorporation Assay for Translation Polysome Assay | Identifies mRNAs that are being translated in vivo. |
| Gel Filtration Analysis of Protein Binding | Measures binding of small molecules to larger molecules (e.g., binding to ribosome). | ||
| Filter Binding Assay | Measures binding of RNA to protein. |