Classic Protocol

Pulsed electrical fields can be used to introduce DNA into a wide variety of animal cells^1,2. Electroporation works well with cell lines that are refractive to other techniques, such as calcium phosphate–DNA coprecipitation. But as with other transfection methods, the optimal conditions for electroporation of untested cell lines must be determined experimentally.

Northern blotting and hybridization are used to study gene expression by detecting RNA species of interest, and to identify alternate RNA splicing patterns. It is analogous to Southern blotting, which is used to analyze DNA. This protocol describes the transfer of RNA from agarose gels to nylon membranes and the fixation of the RNA to the membrane^1,2,3.

In 1992, investigators working on mixing and fusing various domains of different DNA polymerases showed that, when combined with the robust reliability of Taq DNA polymerase, these enzymes produced longer amplicons. Long and accurate polymerase chain reaction (LA PCR) refers to the production of amplified product longer than ∼3 kilobases (kb) with high fidelity. Long PCR mixtures typically yield PCR products with some tenfold fewer mutations¹ than those observed in products resulting from conventional PCR. The mixture of DNA polymerases typically included either Taq or Klentaq1 (which have no 3′-exonuclease proofreading activity) as the major component and, as the minor component, an archaebacterial DNA polymerase (with proofreading activity) such as Deep Vent, Vent or Pfu¹. Among other factors that improve LA PCR are the enzyme deoxyuridine triphosphatase (dUTPase)², which prevents the incorporation of dUTP, the deaminated form of deoxycytosine triphosphate (dCTP) into DNA, and the chemical betaine. Although introduced for amplification of high G-C content targets³, betaine, when included at surprisingly high concentrations, usually helps to promote long PCR up to at least 20 kb. Since the introduction of mixtures of DNA polymerases, most PCRs of any length have improved in reliability and in yield of product. The following protocol is based on the use of ten various primer pairs for the amplification of a genomic template DNA of ∼5 kb in a reaction volume of 50 μl.

The immunoblotting method has evolved from early stages when antibodies were used to 'stain' polyacrylamide gels directly^1,2 to more versatile methods using replica techniques, in which the separated polypeptides are transferred to nitrocellulose membranes, chemically activated paper or nylon sheets. Although there are several variations on this basic theme, the most common and effective is electrophoretic transfer to nitrocellulose sheets^3,4. The separated proteins can then be probed with antibodies; this variation of the technique is known as immunoblotting (or western blotting). The membrane can also be probed with specific ligands, such as DNA, protein, small molecules (for example, heparin or GTP) or even whole cells. Electrophoretic transfer can be achieved either in a tank³ or in a semidry apparatus⁵, in which the buffer volume is reduced to filter paper pads. The following protocol presents the original method used for tank blotting.

Micrococcal nuclease (MNase) is unique among nucleases in its ability to induce double-strand breaks within nucleosome linker regions, but only single-strand nicks within the nucleosome itself. Because of this property, MNase can be used to determine whether a DNA fragment of interest is within a nucleosome^1,2. In addition, MNase can be used to determine the approximate positions of nucleosomes in a region of DNA, if the nucleosomes are consistently positioned. In brief, cell nuclei are isolated and limiting concentrations of MNase are added to the nuclei, resulting in cleavage at nucleosome linker regions. The genomic DNA is purified and the fragments are separated by agarose gel electrophoresis; the resulting ladder of stained bands corresponds in size to multiples of the nucleosome core plus the linker (∼200 base pairs (bp)). To determine whether a DNA fragment of interest is within a nucleosome, the genomic DNA is subjected to Southern blot analysis. If a probe derived from the DNA fragment hybridizes to the ladder of nucleosomal bands, the fragment may indeed be assembled into nucleosomes. To determine nucleosome positioning, the purified genomic DNA must be cleaved with a restriction enzyme before gel electrophoresis and Southern blot analysis.

This method is used to extend partial cDNA clones by amplifying the 5′ sequences of the corresponding mRNAs^1,2,3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target—in the case of 5′ RACE, to a homopolymeric tail added (via terminal transferase) to the 3′ termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5′ sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.