Maxam-Gilbert reactions

Lyophilize the purified end-labeled DNA that contains chloroacetaldehyde-modified MAR sequences at equal quantity in two separate Eppendorf tubes one for the hydrazine reaction and the other for the formic acid reaction. The step-by-step procedures for hydrazine and formic acid reactions for Maxam-Gilbert reactions are described in Sambrook et al. (1989). We have made the following deviations (a) the temperature employed for chemical reactions is 15°C instead of 20°C (b) at each step of precipitation of DNA with ethanol, there is no need to chill at -70°C before centrifugation and DNA is centrifuged at 10,000 g for 10 min at 4°C after the piperidine reaction, the sample is transferred to a new tube that contains 100 fi of 0.6 M sodium acetate at pH 5 in TE (10 mAf Tris-HCl, pH 7.5, 1 mAf EDTA) and precipitated with three volumes of ethanol. Redissolve the DNA pellet in 200 fi of 0.3 M sodium acetate and reprecipitate with ethanol. Wash the DNA pellet once with 70% ethanol, and lyophilize. Resuspend the DNA samples in 90% formamide in 1 x TBE (89 mAf Tris-borate, 89 mAf boric acid, 2 mAf EDTA) loading buffer, heat at 95 C for 5 min followed by quick chilling on ice. Separate the DNA samples on a polyacrylamide gel in 8.3 M urea, 100 mAf Tris-borate, pH 8.3, and 2 mAf EDTA. For best visualization of approximately 100-200 base pairs from the labeled end, 6% polyacrylamide gel is recommended. For visualizing 30-100 base pairs, an 8-10% polyacrylamide gel is typically used. 

The base-specific chemical cleavage (or Maxam-Gilbert) method starts with a single-stranded DNA that is labeled at one end with radioactive (Double-stranded DNA can be used if only one strand is labeled at only one of its ends.) The DNA strand is then randomly cleaved by reactions that specifically fragment its sugar-phosphate backbone only where certain bases have been chemically removed. There is no unique reaction for each of the four bases. However,... 

Note that the key to Maxam-Gilbert sequencing is to modify a base chemically so that it is removed from its sugar. Then piperidine excises the sugar from its 5 - and 3 -links in a /3-elimination reaction. The conditions of chemical cleavage described in Figures 12.4 and 12.5 are generally adjusted so that,... 

Fig. 3 Autoradiograms of the denaturing sequencing gel for photoreactions of duplexes 9/10, 11/12, 13/14, and 15/16. Lanes 1-4, ODN 9 lane 5, ODN 11 lane 6, ODN 13 lanes 7 and 8, ODN 15 ODNs in lanes 3-7 were photoirradiated all ODNs except in lane 3 were heated with piperidine lanes 1 and 8, Maxam-Gilbert G+A sequencing reactions for ODNs 9 and 15, respectively. Partial base sequences of oligomers were shown on the side. dCNBPU was located opposite to the A (shown with a box)...

Fig. 8 Autoradiograms of a denaturing polyacrylamide gel for photooxidation of duplex 35/36 in the presence of BamH I. ODNs 35 (a) and 36 (b) were separately 5 -32P-end labeled and hybridized to a non-labeled complementary strand. Lane 1, Maxam-Gilbert G+A sequencing reactions lane 2, in the absence of BamH I lanes 3-5, BamH I. ODNs in lanes 2-4 were irradiated at 312 nm. All samples except in lane 2 were heated with piperidine. The BamH I site and dCNBPU (X) are shown in bold face. For clarity, the autoradiogram for ODN 36 is shown upside-down...

The DNA sequencing chemistry begins with a base-modification reaction, the extent of which determines the frequency of DNA cleavage in the subsequent phosphate-elimination reaction. The number of bases modified in each molecule depends on the concentration of dimethylsulphate (G and G A reactions) and hydrazine (C T reactions) as well as the temperature and duration of the reaction. For speed and convenience the Maxam-Gilbert procedure makes use of temperature shifts and dilution to control the rate and extent of these reactions. The reagents are mixed at 0°C, incubated at 20°C for the required time and the DNA precipitated with cold sodium acetate and ethanol to slow down or halt the reaction. A fixed concentration of the different reagents is usually used so the main factor determining the extent of reaction is the time of incubation at 20°C. 

DNA sequencing Maxam-Gilbert method restriction endonuclease restriction fragment palindrome Sanger dideoxy method DNA synthesis DMT ether phosphoramidite phosphite polymerase chain reaction (PCR)... 

Small DNA segments can be synthesized in the laboratory, and commercial instruments are available for automating the work. Sequencing of DNA can be carried out either by the Maxam-Gilbert method, which uses chemical techniques, or by the Sanger dideoxy method, which uses enzymatic techniques. Small amounts of DNA can be amplified by factors of 10 using the polymerase chain reaction (PCR). 

Figure 10.10. Autoradiogram of DNA sequencing gel obtained after Maxam-Gilbert sequencing reactions of 32P-ACTGTAGC.

Another sequencing technique is the Maxam-Gilbert method (developed by Alan Maxam and Walter Gilbert). In this method, a single DNA strand is labeled at the 5 end with radioactive phosphorus (P ). The radioactive DNA is divided into four portions and each one is exposed to different chemical reactions. Each reaction causes a 5 cleavage adjacent to either... 

Stop the reaction by addition of 5 p.1 Maxam-Gilbert load bufferb... 

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