Discovering the Transcriptome
What is a transcriptome?
When one approaches the study of a gene, some information is initially very useful. If a sequence or part of a sequence is known then probes can be designed to study the mRNA transcripts. Sequence information may be derived from a homologous set of genes from other organisms, from amino acid sequencing and reverse translation, or from direct DNA sequencing.
If no sequence information is avaliable for probe development, then identification must be done at the protein level. It is helpful to have an antibody that is reactive with the protein of interest. This allows for screening of expression cDNA libraries to isolate clones that produce proteins that react with the identifying antibody. In this way cDNA clones can be identified and then sequenced to get information to make a probe to study transcription.
If no sequence information nor antibody is available but only protein gel migration patterns, then using Hybrid Release Translation (HRT) or Hybrid Arrest Translation (HART) can be used to screen for clones that code for protein with matching gel migration properties.
HRT
HART
Northern Blot Analysis
Assuming that a labeled probe can be designed and synthesized, a Northern blot analysis can be performed. A Northern blot is made by isolating RNA from a cell or tissue and separating the RNA by size on an agarose gel. A copy of the separated RNA pattern is transferred to a membrane made of nylon and crosslinked to the membrane by exposure to UV light. The membane is then exposed to a labeled probe which allows identification of the RNA band(s) corresponding to the transcripts of the gene of interest.
Questions that can be answered with the Northern blot analysis are:
How many transcript sizes are made.
How much of the transcript is made. (Comparable between different tissues
or times)
How rapidly is transcript made or degraded.
real-Time-PCR
Northern blot analysis can be used to estimate the relative concentration of RNAs but a sensitive method that is capable of being automated is real time PCR The .pdf file below contains a nice description of the Taqman and SYBR qantitative methods. There are other methods
Microarrays
Microarrays allow for the simultaneous comparison of thousands of genes for quantity of expression products. The animation and .pdf files are from the Affymetrix and give a description of their current technology and use.
How to use GeneChip Microarrays to study gene expression.
Two color microarray technology
Gene array data analysis (3 pages)
S1 Nuclease Protection Assay
Nuclease protection assays expose hybrids of mRNA and DNA to the S1 nuclease. Regions of the DNA that are single-stranded are degraded.
Questions that can be answered using this technique:
Intron/exon location.
Transcript 5' or 3' end location.
(The 5' end of the transcript can also be found using a primer extension and run-off assay.)
Identification of proteins and DNA regulatory sites
Deletion Assays
When initially determining the DNA segments upstream of the transcription start site that are essential to transcription and its regulation.
A clone of the potential transcription regulatory region and the promoter are cloned adjacent to a reporter gene.
Reporter genes may be genes such as luciferase, beta-galactosidase, the green fluorescent protein or any assayable marker protein.
Deletions are then made in the putative regulatory region and the amount of reporter product is monitored. Regions of DNA deletion that correspond to low reporter expression are deemed potential regulatory sites and can be further assayed by DNase footprinting and gel shift assays.
Deletions/insertions to interrupt function can be made in various ways.
Deletions and interrupting insertions can be introduced by
- Deleting DNA between
two restriction enzyme sites.
- Deleting overhanging bases at restriction enzyme sites.
- Oligo scanning (insertion of a short oligo at restriction sites
- Bal 31 deletions followed by trimming and ligation.
DNase Footprinting
The location of protein binding sites can be determined by allowing the proteins to bind to the DNA followed by the addition of DNase I. The locations of protein binding will protect the DNA from nicking by DNase I and when fragments are separated on a gel, fragments will be absent in the protected areas.
Gel Shift Assays
Proteins that bind to DNA can be eluted and characterized by protein gel electrophoresis. The specificity of protein binding can be tested by gel-shift or gel-retardation assays. The migration of labeled DNA fragments through a gel will be retarded or shifted when a protein is tightly bound to the DNA. Competition assays combined with gel-shift assays to determine the binding affinity of the protein to the DNA.
Modification Interference Assay
Dimethyl sulphate modifies bases and is the first step in chemical sequencing of Gs and As. Methylation of DNA can sometime prevent proteins from binding to the DNA and typically DNA regions that bind to proteins are undermethylated. Gel shift assay bands that are not retarded in their migration are then isolated and exposed to piperidine which cleaves the DNA at the modified G or A. The size of the remaining band then can tell the location of the DNA binding site.
Site-directed Mutagenesis
Single base substitutions or short segment substitutions can be introduced into a DNA sequence by site directed mutagenesis. DNA cloned into a plasmid containing an F1 origin of replication is able to replicate single strands of plasmid DNA. These single-stranded DNAs are primed with an oligonucleotide that has the mismatched base(s) and extended with the Klenow fragment of DNA polymerase I. The plasmid is re-transformed into a bacterial host and allowed to replicate. Half of the progeny will contain the mutatation. A modification of this techniques uses two mutating primers, one to mutate the base of interest and a separate primer that mutates a selectable marker, such as a drug resistance gene, to be either functional or non-functional. With this approach, mutants can be idenfiied by selection instead of being screened by sequencing or restriction site analysis.
