Autoradiography

Autoradiography is a simple technique which can he carried out on a sample which has been rendered radioactive by the introduction of P. The sample is pressed into close contact with a photographic plate, which after a short time can be developed. The blacker areas on the plate correspond 

Examples of sequencing gels prepared as described are shown in Plates I-IV. The procedure used in reading the gel is described in Section 4.2.9. Hints on reading the sequence and trouble-shooting are given in Section 4.7. 

Plate I shows part of a sequencing gel which gives the sequence of an 80-nucleotide Alul fragment derived from the yeast 2 /x plasmid (Elleipan and Hindley, 1980). The sequences underlined correspond to the M13mp2 sequences around the site of insertion 

Plate II. Autoradiograph of a sequencing gel showing an increased background of all possible chain lengths. For discussion see text. 

Plate III. A sequencing gel showing the patterns obtained from eight M13mp2 recombinant phage DNAs picked at random. Chain extension was carried out in the presence of ddT only to identify recombinants containing identical cloned 

Diagram showing origin of sequences from cloned Alul fragment (cf Plate 1) 

Fogh and Fogh (1968) give details of a method using orcein to detect mycoplasma. 

Autoradiography of contaminated cultures labelled with tritiated thymidine shows grains over the whole cell rather than localised to the nucleus. This is a result of degradation of the thymidine to thymine followed by incorporation into mycoplasma DNA and other cell constituents. 

Quantification of the composition of the grain boundary from the Auger spectrum is not always trivial. For a homogeneous material the composition is 

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Also present in the first test tube is a synthetic analog of ATP in which both the 2 and 3 hydroxyl groups have been replaced by hydrogens This compound is called 2 3 dideoxyadenosme triphosphate (ddATP) Similarly ddTTP is added to the second tube ddGTP to the third and ddCTP to the fourth Each tube also contains a primer The primer is a short section of the complementary DNA strand which has been labeled with a radioactive isotope of phosphorus ( P) When the electrophoresis gel is examined at the end of the experiment the positions of the DNAs formed by chain extension of the primer are located by a technique called autoradiography which detects the particles emitted by the P isotope... 

The contents of each tube are then subjected to electrophoresis m separate lanes on the same sheet of polyacrylamide gel and the DNAs located by autoradiography A typical electrophoresis gel of a DNA fragment containing 50 nucleotides will exhibit a pattern of 50 bands distributed among the four lanes with no overlaps Each band cor responds to a polynucleotide that is one nucleotide longer than the one that precedes it (which may be m a different lane) One then simply reads the nucleotide sequence according to the lane m which each succeeding band appears... 

The nonquantitative detection of radioactive emission often is required for special experimental conditions. Autoradiography, which is the exposure of photographic film to radioactive emissions, is a commonly used technique for locating radiotracers on thin-layer chromatographs, electrophoresis gels, tissue mounted on sHdes, whole-body animal sHces, and specialized membranes (13). After exposure to the radiolabeled emitters, dark or black spots or bands appear as the film develops. This technique is especially useful for tritium detection but is also widely used for P, P, and 1. 

A. Rogers, Techniques of Autoradiography, Elsevier Scientific Publishing Co., Amsterdam, the Netherlands, 1979. 

The distribution of impurities over a flat sihcon surface can be measured by autoradiography or by scanning the surface using any of the methods appropriate for trace impurity detection (see Trace and residue analysis). Depth measurements can be made by combining any of the above measurements with the repeated removal of thin layers of sihcon, either by wet etching, plasma etching, or sputtering. Care must be taken, however, to ensure that the material removal method does not contaminate the sihcon surface. 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

Autoradiography Detecting radioactivity in a sample, such as a cell or gel, by placing it in contact with a photographic film. 

TLC Analysis. Samples were examined by TLC using standard procedures. Rf values were determined and compared with those of authentic reference materials. Radioactive components were located by scanning (Vanguard Instrument Corp., North Haven, Conn., Model 885) or by autoradiography (Eastman Kodak, Rochester, N. Y., type AA film). The relative Rf value of DCDD on silica gel plates (Brinkmann Instruments, Inc., Westbury, N. Y., type For,4) when developed with n-hexane dioxane acetic acid, 90 10 4, V/V/V, was 0.90. The observed impurity had a relative Rf value of 0.40. On Brinkmann alumina plates, developed with n-hexane, DCDD had a relative Rf of 0.32. Neither system separated the chlorinated dibenzodioxin isomers. 

The difference in rates of release of BCNU from wafers produced by the trituration or solution methods is also seen in vivo (11,14), as is shown in Fig. 6. Wafers of PCPP-SA 20 80 were prepared by either the solution or trituration methods, as described above, and were implanted into the brains of rabbits. The animals were sacrificed at various times after implantation and the brains were removed, fixed, and processed for quantitative autoradiography. To quantitate the percentage of the brain exposed to BCNU released from these wafers, the following calculation was performed. The percentage of the brain in which the radioactivity from the tritiated BCNU released from the wafers exceeded the background counts by at least two standard deviation units was plotted as a function of time following implantation in Fig. 6. A control set of rabbits had a solution of BCNU injected directly into the same location in the... 

FIGURE 6 Distribution of [ HlBCNU-associated radioactivity time in rabbit brain. The distribution of radioactivity was measured using quantitative autoradiography techniques following implantation of discs described for Fig. 5 into the brains of rabbits. Details of the experimental procedure are described in the text. 

Autoradiography The detection of radioactive molecules (eg, DNA, RNA, protein) by visualization of their effects on photographic film. Bacteriophage A virus that infects a bacterium. Blunt-ended DNA Two strands of a DNA duplex having ends that are flush with each other. cDNA A single-stranded DNA molecule that is complementary to an mRNA molecule and is synthesized from it by the action of reverse transcriptase. 

Signal The end product observed when a specific sequence of DNA or RNA is detected by autoradiography or some other method. Hybridization with a complementary radioactive polynucleotide (eg, by Southern or Northern blotting) is commonly used to generate the signal. 

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