RNA Technology RNA Analysis DNA Technology Analysis and Cloning Production
RNA Extraction and Purification
Purified, full-length RNA is required
for cDNA cloning. It is also used in some methods for analyzing gene expression
and for microarrays utilizing cDNA. RNA is prepared from bacteria, tissue
culture cells, tissues and plants. Preparations can be purified messenger
(mRNA) or total RNA that includes mRNA, ribosomal (rRNA) and transfer
(tRNA) RNA. The scale is from 1 microgram to1 milligram.
Northern blot analysis involves size
separation of the RNA species by electrophoresis followed by identification
with labeled gene-specific oligonucleotides or DNA probes. The size and
abundance of the desired RNA is determined. The predominant rRNA in a
total RNA preparation can also be detected by UV absorbance as a measure
Dot Blot and Slot Blot
Specific RNA species in an unfractionated
preparation can be measured by immobilizing a sample in a spot (Dot Blot)
or in a manifold slot (Slot Blot). Detection is by a labeled DNA probe
that hybridizes to the immobilized RNA. For the Dot Blot, quantitation
is usually visual whereas the Slot Blot format is more easily quantitated
by scanning with a densitometer.
Polymerase Chain Reaction
The usual PCR technique copies a
piece of DNA and greatly amplifies the copy number for analysis or storage.
RT-PCR starts with mRNA or total RNA, makes a cDNA complimentary strand
using reverse transcriptase, and then amplifies the product. The product
is used to determine the presence of the mRNA and for size determination,
sequencing or for quantitation by Quantitative-PCR.
In situ Hybridization
In situ hybridization localizes
the expression of a particular mRNA among different cells in a tissue
or cell preparation. A cell preparation is fixed on a slide and the RNA
contents probed with radiolabeled or fluorescent probe.
DNA Extraction and Purification
Purified DNA is required to prepare
DNA libraries or microarrays, to make probes, and to transfect genes.
DNA is prepared from bacteria, yeast, tissue culture cells, tissues, or
plants. Preparations can be genomic DNA or plasmids. The scale is from
1 microgram to 1 milligram.
Gene activation or gene expression
can be conveniently monitored by a DNA construct that links the regulatory
components of the gene to a reporter gene, such as for a fluorescent protein
or beta galactosidase.
cDNA Purification for Microarrays
DNA complementary (cDNA) to mRNA
is prepared for preparing microarrays for screening in different cell
sources. For screening, the total RNA is extracted from tissue or cells
and is hybridized to the cDNA immobilized onto a solid support. A detection
molecule is applied and is visualized.
Analysis and Cloning
DNA Restriction Fragment Mapping
Restriction endonucleases recognize
and cut DNA at unique nucleotide sequences. Delineation of these cleavage
sites provides a map of the DNA, produces characteristic sizes of the
fragments, and allows for the preparation of pieces of DNA to be used
for making new vector or plasmid constructs.
Subcloning is used to amplify a desired
DNA molecule. After a piece of DNA or a gene is obtained, by synthetic
synthesis, restriction digestion, joining to another DNA by a ligase,
etc., it is introduced into competent bacteria and transformants expressing
the new gene are identified. The bacteria are cloned on selection plates
whereby only those cells that contain the desired DNA survive and propagate
into colonies. The colonies are "picked" and expanded in liquid cultures.
cDNA and Genomic Libraries
A library is an amplified collection
of DNA representing the genes of an organism (genomic library) or of DNA
complementary to a preparation of mRNA (cDNA library). Either library
can be probed to pull out a specific gene of interest. cDNA libraries
are used to clone the expressed version of a gene comprised of the exons.
Genomic libraries contain both the exons, introns and regulatory portions
of the gene. Bacteriophage lambda and cosmid vectors are used in library
Library Screening and Gene Cloning
A DNA library is screened to determine
the presence of a gene and to clone out either a full-length or smaller
version of the gene. There are many strategies for cloning from a library.
Typically, the library is introduced into bacteria, the bacteria are plated
under cloning conditions, replicate plates of the bacterial colonies are
made, and the clones are probed for the existence of the desired gene
either by hybridization or through the expression of the specific protein.
For a positive clone, the DNA is extracted from original plate, recloned
To unequivocally identify a gene
or cDNA, the DNA is submitted for sequencing. Using overlapping portions
of the DNA, a contiguous sequence is generated which can be used for data
Transfection, Transient and Stable
Genes can be introduced to cells
by suitable molecular biology methods. A gene can be transfected into
cells simply with its own regulatory elements, or after making a construct
to achieve high, overexpression. Cells can also be transfected to conditionally
express a gene of interest. Transfections in mammalian cells can be transient
or permanent. Transient expression lasts only a few days. Permanent expression
requires cotransfection with a dominent selectable marker and several
rounds of selection for the few cells that stably integrate the gene in
the cell. This takes approximately three to four weeks .
Recombinant Protein Expression
Recombinant proteins are expressed
in bacteria, yeast, mammalian or in the baculovirus system. The gene of
interest is transfected into the appropriate host together with regulatory
elements that allow very high levels of production. Protein production
may be intracellular, or may accumulate in the bacterial periplasmic space,
or be secreted into the medium of cells.
The scale may be a few mL for analytical
purposes, or in batches of up to 20 L in bacterial or yeast fermentation,
or up to 50 L in mammalian suspension, hollow fiber or porous bead cultures.