Techniques Described in 7.28
Technique
Lecture 1
dNTP Incorporation Assay
Primer Extension Assay
Filter Binding Assay
Gel Electrophoresis
DNA Helicase Assay
Helicase polarity
Template Challenge
Lecture 2
Topoisomerase assay
EM Autoradiography
2-D Agarose Gels
Southern Blotting (Genomic)
Assay For
DNA Synthesis (Fast; Quantitative for Amount of Synth.).
DNA Synthesis (Slow; Quantitative for Length of Synth.).
Method to separate incorporated from unincorporated dNTPs.
Method to separate DNA on the basis of length.
In vitro DNA Helicase Activity.
Assay for directionality of helicase movement; used to
study function of replication termination
sequences.
Assay Assay for Processivity.
Assay for changes in DNA topology; distinguish
supercoiled, relaxed, nicked, linear, forms of
DNA and measure catenation/decatenation.
Assay for Presence of Origins of Replication (No ability
to identify origin DNA sequences).
Assay for Origin of Replication (Must have an idea of
where the origin is to test).
Identifies specific DNA molecules transferred to
membrane.
Lecture 3
Plasmid Replicator Assay
Mutational Mapping Assay
Biochemical Fractionation
Identifies region of DNA sufficient for Replicator activity
Identifies regions of DNA necessary for Replicator activity
Mechanism to purify biochemical activity that can be
assayed in vitro.
Genetic Screen for DNA Replication Mutants (self explanatory)
Lecture 4
Biochemical Complementation Combines mutant extracts with biochemical fractionation to
identify proteins required for assay (in this case
DNA replication).
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).
1
DNA Unwinding Assay
Template Association Assay
Assays for formation of ssDNA through the use of a
ssDNA specific nuclease.
Uses gel filtration to separate molecules bound to a
plasmid from molecules that are not
bound to a plasmid (see also Reading
Assignment One).
Lecture 5
Micrococcal Nuclease Digestion
Lecture 6
Heteroduplex DNA analysis
Assay for the assembly of nucleosomes
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.
Lecture 7
Ames Test
Nicking of circular DNA
Lesion bypass DNA
synthesis
Lecture 8
Strand Exchange Assay
Lecture 9
ATPgamma-S
DMS footprinting
Cooperative DNA binding
Lecture 10
Concensus sequences
Native-Denaturing 2D
Gel
Lecture 11
DNA bending assay
Assay for chemicals and other factors that increase the
frequency of mutations.
Assay for excision-type mechanism of DNA repair.
Assay for ability of replication/repair proteins to synthesize
DNA off of a damaged template.
Assay for homologous DNA pairing & branch migration.
Used in determining whether ATP binding or
ATP-hydrolysis is required for a reaction.
Assay for protection of the bases in DNA, rather than the
phosphodiester backbone.
Gel-shift assay to measure cooperative vs.
non-cooperative DNA binding.
Identification of important elements by conservation.
Detection of nicks vs DSB’s in DNA
Assay for whether a protein bends DNA upon binding.
and position of bend
2
Lecture 12
Genomic southern
Plasmid mating
Lecture 13
S1 Protection Assay
Northern Blot
Incorporation Assay
DNA Unwinding Assay
Identifies specific DNA molecules transferred to
membrane (here used for transposition).
Assay for transposition in vivo by determining movement
Of drug resistance to a new plasmid.
Assay for Transcription. Maps Start Site
Assay for Transcription. Maps steady state RNA in vivo.
Assay for transcription in vitro.
Assay for Open Complex Formation
Lecture 14
Genetic Screens to identify and characterize activators and repressors.
Promoter Fusions
Assay to determine promoter activity indirectly
Lecture 15
Run off transcription assay
Antibody Supershift Assay
DNA Array Hybridization
In vitro incorporation assay that creates transcripts of
known size without termination. Important
for incorporation assays using eukaryotic
RNA polymerases.
Assay to determine the components of protein-DNA
complex
Assay to determine levels of transcription across many
genes simultaneously.
Lecture 16
Transcription Factor DNA Binding Domain Mapping
Transcriptional Factor Activation Domain Mapping
Cross-linking Assay for Protein Multimerization
Lecture 17
R-loop formation
Gel electrophoresis assay
Assay for regions of mRNA that anneal to different DNA
fragments.
For intermediates and products formed during splicing in
vitro (done using labeled RNA or by
Northern hybridization).
Lecture 18
Calculation and
Rnase digestion
cDNA cloning
Exon trapping
Determination of RNA secondary structures.
Determine mRNA sequence and structure, protein
expression.
Find exons in DNA fragments of regions thought to
contain genes.
3
Lecture 19
None
Lecture 20
None
Lecture 21
Incorporation Assay for Translation
Gel Filtration Analysis of
Protein Binding
Measures Binding of Small Molecules to
Larger Molecules (e.g. Binding to
Ribosome)
Measures Binding of RNA to Protein
Identifies regions of RNA protected
by Protein or RNA Binding.
Filter Binding Assay
Modification Interference Assay
Lecture 22
None
Lecture 23
Protein Phosphorylation Assay
Protein Processing Assay
4