Separation of RNA according to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde.

Samples of RNA may be denatured by treatment with formamide and separated by electrophoresis through agarose gels containing formaldehyde. In this method, RNA is fractionated by electrophoresis through an agarose gel containing 2.2 m formaldehyde.

Transfer and Fixation of Denatured RNA in Agarose Gels to Membranes by Capillary Transfer.

In most cases, fractionation of RNA by agarose gel electrophoresis is but a prelude to hybridization of the fractionated population to specific labeled probes that detect particular target mRNAs. RNA is first transferred from an agarose gel to a 2D support, usually a nylon membrane. This protocol presents the steps involved in the transfer of RNA from an agarose gel to a membranous support, facilitated by the upward flow of buffer, followed by various methods for fixation of the RNA to the membrane in preparation for hybridization. An alternative method for transfer by downward capillary flow is also given.

Dot and Slot Hybridization of Purified RNA.

Dot blotting and slot blotting are techniques for immobilizing several preparations of nucleic acids on the same solid support, usually a charged nylon membrane. The concentrations of the target sequence of interest can be estimated by hybridizing the immobilized samples to an appropriate probe. The amounts of target sequence are estimated by comparing the intensity of signals emitted by dots containing the test samples with standards containing known concentrations of the target sequence. This protocol describes the blotting and subsequent hybridization of RNA that has been purified from cells or tissues.

Analysis of RNA by Northern Blotting.

Northern hybridization is used to measure the amount and size of RNAs transcribed from eukaryotic genes and to estimate their abundance. No other method is capable of obtaining these pieces of information simultaneously from a large number of RNA preparations; northern analysis is therefore fundamental to studies of gene expression in eukaryotic cells. To prepare a northern blot for hybridization, RNA must be separated according to size through a denaturing agarose or polyacrylamide gel and transferred to a solid support in a way that preserves its topological distribution within the gel. These important steps in northern analysis are discussed here.

Hybridization of Oligonucleotide Probes in Aqueous Solutions: Washing in Buffers Containing Quaternary Ammonium Salts.

In this protocol, hybridization is first performed in conventional aqueous solvents at a temperature well below the melting temperature, and the hybrids are then washed at higher stringency in buffers containing quaternary alkylammonium salts. TMACl is used with probes that are 14-50 nt in length, whereas TEACl is used with oligonucleotides that are 50-200 nt in length.

Purification of Labeled Oligonucleotides by Precipitation with Ethanol.

If labeled oligonucleotides are to be used only as probes in hybridization experiments, complete removal of unincorporated label is generally not necessary. However, to reduce background to a minimum, the bulk of the unincorporated label should be separated from the labeled oligonucleotide. Most of the residual unincorporated precursors can be removed from the preparation by differential precipitation with ethanol, as described in this protocol, if the oligonucleotide is >18 nt in length.

Preparation of Labeled DNA, RNA, and Oligonucleotide Probes.

Labeled nucleic acids and oligonucleotides are typically generated by enzymatic methods such as end-labeling, random priming, nick translation, in vitro transcription, and variations of the polymerase chain reaction (PCR). Some of these methods place the label in specific locations within the nucleic acid (e.g., at the 5' or 3' terminus); others generate molecules that are labeled internally at multiple sites. Some methods yield labeled single-stranded products, whereas others generate double-stranded nucleic acids. Finally, some generate probes of defined length, whereas others yield a heterogeneous population of labeled molecules. Options available for generating and detecting labeled nucleic acids, as well as advice on designing oligonucleotides for use as probes, is included here.

Digoxigenin.

In molecular cloning, digoxigenin is used as a ligand that can be incorporated into DNA and RNA probes and detected after hybridization with an anti-digoxigenin-antibody enzyme conjugate. Methods to label nucleic acids with digoxigenin and to detect digoxigenin-labeled probes are introduced here.

Labeling of DNA Probes by Polymerase Chain Reaction.

The polymerase chain reaction (PCR) can be used to produce both nonradiolabeled DNA probes and radiolabeled DNA probes with high specific activity. In this protocol, PCR is used to generate double-stranded probes. Related methods, including the generation of asymmetric probes by PCR, are also discussed.

Total RNA Extraction from Saccharomyces cerevisiae Using Hot Acid Phenol.

Isolation of RNA from yeast is complicated by the need to first break the thick, rigid cell wall. The protocol provided here uses a cycle of heating and freezing of cells in the presence of phenol and the detergent sodium dodecyl sulfate (SDS). The extraction is performed in the presence of low salt so that, following separation of the aqueous and phenol phases by centrifugation, DNA can be collected from the interface while RNA remains in the aqueous phase. This protocol should yield ∼50-250 µg of RNA from 10 mL of culture. The RNA isolated using this approach is suitable for most follow-up applications such as northern blot hybridization, reverse transcriptase-polymerase chain reaction (RT-PCR), and cDNA construction.

Alkaline Agarose Gel Electrophoresis.

Alkaline agarose gels are run at high pH, which causes each thymine and guanine residue to lose a proton and thus prevents the formation of hydrogen bonds with their adenine and cytosine partners. The denatured DNA is maintained in a single-stranded state and migrates through an alkaline agarose gel as a function of its size. Other denaturants such as formamide and urea do not work well because they cause the agarose to become rubbery.

One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution.

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.

Cloning and Transformation with Plasmid Vectors.

Plasmids occupy a place of honor in molecular cloning: They were used in the first recombinant DNA experiments and, 40 or more years later, they remain as the carriage horses of molecular cloning. After almost half a century of sequential improvement in design, today's plasmid vectors are available in huge variety, are often optimized for specific purposes, and bear only passing resemblance to their forebears. Here, various features of plasmid vectors and methods for transforming E. coli cells are introduced.

Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I.

In this method, a short primer is hybridized to an oligonucleotide template whose sequence is the complement of the desired radiolabeled probe. The primer is then extended using the Klenow fragment to incorporate [α-32P]dNTPs in a template-directed manner. After the reaction, the template and product are separated by denaturation followed by electrophoresis through a polyacrylamide gel under denaturing conditions. With this method, it is possible to generate oligonucleotide probes that contain several radioactive atoms per molecule of oligonucleotide and to achieve specific activities as high as 2 × 1010 cpm/µg of probe. Because the end product of the reaction is dsDNA, whose strands must be separated and the labeled product isolated, this method is generally not used to prepare nonradiolabeled oligonucleotides.

Purification of Labeled Oligonucleotides by Size-Exclusion Chromatography.

When labeled oligonucleotides are to be used in enzymatic reactions such as primer extension, virtually all of the unincorporated label must be removed from the oligonucleotide. For this purpose, chromatographic methods or gel electrophoresis are superior to differential precipitation of the oligonucleotide with ethanol or cetylpyridinium bromide (CPB). This protocol describes a method to separate labeled oligonucleotides from unincorporated label that takes advantage of differences in mobility between oligonucleotides and mononucleotides during size-exclusion chromatography. Although size-exclusion chromatography can, in principle, be used to purify either radiolabeled or nonradiolabeled oligonucleotides, this protocol is geared toward purifying radiolabeled oligonucleotides, whose elution from the column is monitored using a minimonitor and whose separation from unincorporated nucleotides is monitored by liquid scintillation counting.

Biotin.

Biotin is a water-soluble vitamin that can be attached to a variety of proteins and nucleic acids, often without altering their properties. Its use in molecular biology is introduced here.

Cloning Polymerase Chain Reaction (PCR) Products: TOPO TA Cloning.

This protocol describes the use of TOPO-activated TA vectors for cloning. Manufacturers of cloning kits provide excellent manuals that explain in detail what to do and why to do it. This makes TOPO cloning easy, but not foolproof. When setting up TOPO cloning for the first time, set up a trial experiment as described here.

A Guide to Cloning the Products of Polymerase Chain Reactions.

This introduction outlines various methods to clone amplified DNAs and to facilitate the construction of complex multicomponent genetic units. Because of the ease with which the termini of amplified DNAs can be tailored by polymerase chain reaction (PCR), many of the methods outlined here use PCR not only to synthesize DNAs but also to link them together into purpose-designed constructs. The most recent refinements however have been the development of modular genetic units that can be harnessed to target DNAs not by PCR but by site-specific recombination enzymes.

Labeling the 5' Termini of DNA with Bacteriophage T4 Polynucleotide Kinase.

The removal of 5' phosphates from nucleic acids with phosphatases and their readdition in radiolabeled form by bacteriophage T4 polynucleotide kinase is a widely used technique for generating 32P-labeled probes. When the reaction is performed efficiently, 40%-50% of the protruding 5' termini in the reaction becomes radiolabeled. However, the specific activity of the resulting probes is not as high as that obtained by other radiolabeling methods because only one radioactive atom is introduced per DNA molecule. Nevertheless, the availability of [γ-32P]ATP with specific activities in the 3000-7000 Ci/mmol range allows the synthesis of probes suitable for many purposes. This protocol includes procedures for labeling the 5' ends of dsDNA and oligonucleotides.

Labeling the 3' Termini of Oligonucleotides Using Terminal Deoxynucleotidyl Transferase.

Terminal deoxynucleotidyl transferase (TdT, also simply called terminal transferase) is a template-independent polymerase that catalyzes the addition of deoxynucleotides and dideoxynucleotides to the 3'-hydroxyl terminus of a DNA molecule. Cobalt (Co2+) is a necessary cofactor for the activity of this enzyme. Incorporation at the 3' terminus can be limited to just 1 nt by using [α-32P]ddATP or biotin-, digoxigenin (DIG)-, or fluorescein-ddUTP. Because none of these molecules carries a 3'-hydroxyl group, no additional molecules can be incorporated. Alternatively, the enzyme is capable of adding several (2-100) nt to 3' ends in a so-called homopolymeric "tailing" reaction. A tailing reaction is performed in the presence of a mixture of labeled and unlabeled dNTPs. The rate of addition of dNTPs, and thus the length of the tail, is a function of the ratio of 3' DNA ends to dNTP concentration and, in addition, the specific dNTP that is used.