Biol 335

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Basic cloning experiment

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Goal: isolate and maintain a piece of DNA
Techniques: Southern blotting, restriction endonuclease and ligation, restriction mapping, transformation and selection, colony lift hybridization
Procedure: insert the desired piece of DNA into a plasmid vector, library generation, and use selection markers and hybridization techniques to identify desired clones.
Example application: finding a gene responsible for conferring antibiotic resistance.

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Sanger dideoxy sequencing

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Goal: obtain sequence of a short stretch of DNA
Techniques: thermocycling with chain-terminating ddNTPs
Procedure: to purified template DNA, add 1 sequencing primer, ddNTPs, polymerase and reaction buffer. Thermocycle and run on sequencing gel/capillary.
Example application: finding the sequence of your favourite gene.

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Next Generation Sequencing

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Goal: obtain a large amount of DNA sequence using techniques that no longer relies on normal chain termination methods to obtain the reads, but rapidly obtains multiple reads by simultaneously reading multiple short DNA segments attached to a solid support.
Example application: re-sequencing the human genome, personalized medicine based on individual genetic makeup, sequencing the Neanderthal genome

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RNA Interference (RNAi)

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the use of small interfering RNA (siRNA) molecules or micro RNAs (miRNA) that prevent the translation of a particular mRNA by binding to the 3’UTR of that mRNA. This technique can be used to knock down the expression of any gene without mutating it (Loss-of-function experiments).
Example application: to study the effects of loss-of-function of a gene for which there is no known mutation.

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Genome-Wide Association (GWA)

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an examination of genetic variation across a given genome, designed to identify genetic associations with observable traits. In human studies, this might include traits such as blood pressure or weight, or why some people get a disease or condition.
Example application: determine the susceptibility to colorectal cancer given a particular genotype.

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Chromatin Immunoprecipitation (ChIP)

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an experimental method used to determine where proteins, such as certain transcription factors, are associated with a specific genomic region in living cells or tissues. This cell-based technique is often used together with non-cell-based assays to characterize protein:DNA interactions. The method is based on the principle that formaldehyde reacts with primary amines located on
amino acids and the bases on DNA or RNA molecules, forming a covalent crosslink between the specific protein to the DNA on which they are situated. Following crosslinking, the cells are lysed and the crude cell extracts are sonicated to shear the DNA to a smaller size. The protein:DNA complex is immunoprecipitated using an antibody against the protein of interest. The DNA protein cross-links are reversed by heating and the proteins removed by treatment with proteinase K. The DNA portion of the complex is then purified and identified by PCR using specific primers to the suspected binding region.
Example application: Finding the binding targets (i.e. enhancers) of a specific transcription factor.

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Electrophoretic Mobility Shift Assays (EMSA)

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a common affinity electrophoresis technique used to study protein-DNA or protein-RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex. A gene sequence (DNA) of interest is run on agarose gel in the presence of nothing else or various proteins. If the proteins bind or interact with the DNA sequence, the total size of the complex will increase and the DNA will move more slowly through the gel. This is then seen as an upward shift in the position of the DNA band on the gel.
Example application: Does TFIIC bind to DNA?

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Chromosome conformation capture (C3)

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Goal: to determine if two discontinuous stretches of DNA are in fact close to each other in vivo due to protein binding.
Procedure: crosslink DNA to bound proteins. Fragment DNA and add DNA ligase. DNA bits in close proximity (crosslinked to proteins) will likely be joined together. You may then PCR the fragments up, sequence them… etc.
Example application: determining if two boundary elements function in the ‘looping model’ to prevent enhancer function.

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Affinity chromatography

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Goal: to determine if two different molecules bind to each other at a set salt concentration. You can test DNA-protein binding or protein-protein binding.
Procedure: manufacture sepharose beads joined to your DNA sequence or protein of interest. Wash the cell protein extract through. Proteins from the extract may bind to the DNA/protein immobilized on the beads, or be washed through. Elute these bound proteins with low-salt buffer.
Example application: finding a protein that binds to an enhancer (DNA), finding the protein binding partner of a transcription factor (protein)

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Gene prediction

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Goal: Given the genomic sequence of an organism, define the transcriptional units.
Techniques: ORF scanning, consensus sequence scanning (TATA- or CAAT- boxes, AATAAA polyadenylation signal), splice site prediction (GT-AG introns), codon bias… etc.
Example application: annotating the genome of a newly sequenced species.

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RNA (Northern) Blot

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Used to study gene expression by detecting the amount of particular mRNA in a sample (ie. cell, tissue). A sample is homogenized and the mRNA is isolated. The RNA is then separated by gel electrophoresis. The RNA is then transferred from the gel to a nylon membrane. The membrane is washed with a labeled DNA probe for a specific RNA sequence of interest. Unbound probe is washed away and the resulting hybrid signals (seen as bands on the membrane) are used to quantify the amount of transcript of interest.
Advantage: Provides transcript size and abundance. Disadvantage: Time-consuming
Example application: is your favourite gene transcribed at embryonic day 9 in the mouse brain?

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Reverse Transcription Polymerase Chain Reaction (RT-PCR)

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Used to study gene expression by detecting the amount of particular mRNA in a sample (ie. cell, tissue). A sample is homogenized and the mRNA is isolated. The mRNA is reverse-transcribed into cDNA. PCR is done using primers specific for a particular gene, so only that gene is amplified. The product is run on a gel to visualize amount and size of transcript.
Advantage: Fast. Disadvantage: Crudely quantitative (prone to artifacts/contamination) and transcript size determination also crude.
Example application: is your favourite gene transcribed at embryonic day 9 in the mouse brain?

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In situ Hybridization

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Technique for determining the distribution of a transcript in an organism. A fixed (immobilized) set of cells is incubated with a gene- specific cDNA or RNA probe. The cells containing the gene of interest that hybridized to the probe will be visible based on the nature of the probe (ie. fluorescence, autoradiography).
Advantage: Provides specific information on the spatial distribution of the gene transcript in an organism. Disadvantage: Time-consuming, laborious, and provides little information on amount of transcript.
Example application: is your favourite gene transcribed at embryonic day 9 in the mouse brain?

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Microarray Hybridization

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Technique to determine the amount of many individual transcripts (hundreds/thousands) in a particular tissue of an organism. Complementary gene-specific oligonucleotides are synthesized and attached to a solid surface (hybridization membrane/chip). mRNA from a tissue is isolated, reverse-transcribed into cDNA, and then hybridized to the microarray. The gene-specific oligonucleotides fluoresce based on the amount of the complementary cDNA present. The level of gene expression for hundreds/thousands of specific transcripts is determined simultaneously this way.
Advantage: Provides information on the amount of RNA transcript for every gene included in the array. Disadvantage: Expensive, prone to artifacts, and results must be verified by a different method.
Example application: Of the ~20,000 genes in the mouse genome, which are the ones that are transcribed in the mouse brain?

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Promoter Traps

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a way to find promoter regions of genes. Consists of a reporter gene on a plasmid that is transformed into a host organism and then randomly inserts itself into the host genome. In order to be expressed, the reporter gene will need to insert downstream or near the appropriate regulatory element (promoter or enhancer) that it is lacking. Transformants are screened for reporter expression, and those expressing the reporter are isolated for the DNA surrounding the reporter gene to be cloned and further studied.
Example: where and what is the enhancer sequence that directs the expression of genes in the eye?

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Enhancer Trap

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Goal: Find an enhancer with desired expression pattern by observing which tissues turn X-gal blue.

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positional cloning

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Using the mutant phenotype as a genetic marker, the gene of interest can be mapped relative to well-defined genetic markers to establish a genetic position within the genome (eg. position on a specific chromosome).

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insertional tagging

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ii) isolate a mutant from a population mutagenized by insertion by a known element (eg transposon insertion). ii) use a probe for the transposon to identify a cloned DNA fragment from the mutant that carries the gene of interest (the insert should reside in the gene of interest).iii) Make a library and probe with the insertion sequences.