Study Materials

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.

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.