Chef

CHEF Factors Affecting Resolution • uniformity of the two electric fields • lengths of the electric pulses • the ratio of the lengths of the pulses • the angles of the two electric fields • strengths of the electric fields

Factors effecting hybridization • temperature • ionic strength • chaotropic agents • probe length • probe mismatch • % GC Consistent with the above discussion, increasing the sodium concentration and percentage of GC base pairs will raise the Tm and increasing the formamide (a chaotropic agent) concentration and decreasing the amount of homology between the probe and target will lower the Tm. Generally, hybridization is discussed in terms of stringency and not the Tm. Stringency refers to the conditions of the hybridization. It is a relative term that is related to the Tm (Box) and reflects the homology between the probe and the target. For example, only a probe with a high degree of homology to the target DNA will hybridize under high stringency conditions, whereas low stingency will allow a less homologous probe to hybridize to the target DNA. Stringency vs. Tm high Tm - 15o moderate Tm - 25o low Tm - 35o The stringency is controlled by changing the hybridization conditions. For example, increasing the temperature, decreasing the salt concentration, or including formamide all increase the stringency. Likewise, the stringency can be decreased by lowering the temperature, increasing the salt concentration, or decreasing the formamide concentration. In practical terms it is often easier to vary the sodium or formamide concentrations rather than the temperature. A common buffer for hybridization is SSC (standard sodium citrate) which is a citrate buffered sodium solution. A stock solution of 20X SSC (= 3.3 M Na+) is diluted to achieve different levels of stringency. Typically ranges of 0.1-6X SSC are used with the lower SSC concentrations representing higher stringency. The formamide is typically used to lower the temperature and at the same time maintain a certain level of stringency. For example, hybridizations carried out at 60-65o in the absence of formamide or at 37-42o with 50% formamide are about equal in stringency. If incubations at high temperatures are inconvenient then formamide can be included in the buffers and the hybridizations carried out at lower temperatures. Stringency also applies to the wash steps. It is common to hybridize at low stringency and then to wash at a higher stringency. This insures that the probe will bind to the target DNA and any non-specific hybridization can be removed during the wash steps. The conditions for hybridization (i.e., stringency) need to be determined empirically in

conjunction with the above formula as guidelines. A single blot can be sequentially examined under different stringencies. Hybridization and washes are initially carried out at low stringency. The blot is wrapped in plastic and not allowed to completely dry. After exposure to x-ray film, the blot is washed under higher stringency and reexposed to X-ray film. Comparison of the different autoradiographs will allow one to determine how homologous the probe is to the target DNA and the degree of cross-hybridization to other DNA fragments. PREPARATION OF LABELED DNA PROBES The two major sources of probes are previously cloned genes and synthetic oligonucleotides. In both cases a label needs to be incorporated into the probe DNA. Radioactivity is a common label, but non-radioactive probes are also available. Four methods for incorporating label into DNA probes have been described (Box ). Nick translation is an older technique that has been replaced by random Stringency vs. Tm high Tm - 15o moderate Tm - 25o low Tm - 35o • • • •

Nick Translation Random Priming T4 Nucleotide Kinase Terminal Transferase

priming. Random priming is the method of choice for labeling cloned DNA fragments. Synthetic oligonucleotides are labeled using T4 nucleotide kinase. Random Priming. In random priming (Figure) DNA is denatured by heating and mixed with hexamers of random sequence (i.e., random primers). The random primers are usually synthesized and included as part of a kit. They can also be prepared from genomic DNA. A few of primers will be complementary to the probe DNA and the duplex formed between the primer and the probe DNA will serve as an initiation point for the DNA polymerase. The DNA polymerase used is Klenow. Klenow is the large subunit of DNA polymerase I in which the 5'→3' exonuclease activity is removed. The four dNTPs including a nucleotide containing a radioactive phosphate in the α-position are also added to the mixture. Therefore, the Klenow will make radioactive copies of the template DNA. The probe DNA is boiled immediately before use in the hybridization assay to convert the dsDNA to ssDNA. T4 Polynucleotide Kinase. T4 polynucleotide kinase transfers the γ-PO4 from ATP to the 5'-hydroxyl of polynucleotides. It is therefore necessary to dephosphorylate the DNA with alkaline phosphatase (AP) before carrying out the phosphorylation. A disadvantage of this technique is that only one radioactive atom is incorporated per DNA strand. However, 5'-terminal phosphorylation is widely used to label oligonucleotide probes that have been prepared synthetically. Synthetic oligonucleotides lack the 5’-phosphate and are too short for random priming. T4 kinase is also used in Maxim and Gilbert DNA sequencing and to phosphorylate (non-radioactive) synthetic linkers. Terminal Transferase. Terminal deoxynucleotide transferase (TdT) adds dNTPs to the 3-OH of either ssDNA or to 3' overhang. In the presence of Co2+ TdT will add dNTPs to the 3'-OH of either dsDNA or 5' overhangs. TdT can be used to radiolabel 3' ends if radioactive nucleotides are used. A more common use, however, is to generate homopolymer tails for molecular cloning. Non-radioactive Probes.

Several procedures have been devised for the detection of hybridization using non-radioactive probes (Box). All are based upon enzyme-linked systems using either alkaline phosphatase (AP) or horse-radish peroxidase (HRP). Biotinylated dNTPs can be incorporated into the probe DNA by random priming. The probe is then be detected with an enzyme-linked streptavidin. Another approach is to incorporate digoxigenin-11-(d)UTP into the DNA probe and then subsequently detected with enzymelinked antibody against the digoxigenin. A third method is to directly cross-link HRP to the DNA probe. Radioactive probes are generally more sensitive and reliable. However, non-radioactive probes can be adapted to many applications and eliminate some of the problems associated with the use of radioactivity such as waste disposal and safety issues. In addition, the use of non-radioactive probes is are particularly advantages in situation where the same probe is going to be used over a long period of time. The short half-life of 32P (14 days) necessitates that the probe be prepared on a monthly basis, whereas large amounts of a nonradioactive probe can be prepared and stored for long periods of time. Insoluble substrates, as described for Western blots, or chemiluminescent substrates can be used in association with Northern and Southern blots. Chemiluminescent substrates produce light when cleaved by the appropriate enzyme and this light is detected by autoradiography. Substrates for both alkaline phosphatase (1,2 dioxetane) and peroxidase (luminol) are available. The use of chemiluminescence allows the blot to be striped and reprobed.