The importance of chemical sequencing has always been demonstrated by obtaining sequences of oligonucleotides, functional analysis of transcriptional regulatory signals (methylation interference analysis, Carey and Smale 2000), and identification of these signals in living cells (genomic imprinting, Church and Gilbert 1984). This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Chemical sequencing assay experiment
Materials and Instruments
Acetic Acid Anhydrous Ethanol Dimethyl Sulfate DMS DMS Buffer DMS Termination Solution EDTA Formamide Formamide Sampling Buffer Hydrazine Hydrazine Termination Solution NaCl NaOH-EDTA Solution Piperidine Aqueous Solution Piperidine Carboxylic Acid Sodium Acetate Polyacrylamide Sequencing Gel Salmon Sperm DNA Radiolabeled Target DNA Yeast tRNA Move makings For more product details, please visit Aladdin Scientific website.
Dry ice-ethanol baths Cerenkov counters Ice-water baths Small centrifuge tubes Rotary vacuum dryers Recirculating water baths with tight-fitting lids Gel sequencing units and water baths with adjustable power supply at 37°C and 90°C
Solutions and buffers
Refer to Appendix 1 for formulations of storage solutions, buffers and reagents.
Acetic acid (1 mol/L): Prepared from glacial acetic acid (17.4 mol/L).
Anhydrous ethanol: -20°C
Dimethyl Sulfate (DMS): Concentration 99% (Aldrich; Gold Label 99%)
DMS (10% V/V) Dissolved in Ethanol
DMS Buffer
50 mmol/L sodium dimethylarsenate (pH 7.0)
1 mmol/I.EDTA (pH8.0)
WARNING: Use gloves and mask when weighing sodium dimethylarsonate and gloves when using DMS buffer.
DMS Termination Solution
1.5mol/L sodium acetate (pH 7.0)
1mol/L β-mercaptoethanol
250ug/ml yeast tRNA
EDTA(0.5mol/L,pH8.0)
Formamide
Formamide Sampling Buffer
Hydrazine (95%)
Hydrazine (EastmanKodak), dispense in small portions in tightly closed microcentrifuge tubes and store dry at -20°C.
Hydrazine Termination Solution
0.3mol/L sodium acetate (pH 7.0)
0.1 mmol/L EDTA (pH 8.0)
100ug/ml yeast tRNA
NaCl(5mol/L)
NaOH-EDTA solution: 1.2 mol/L NaOH with 1 mmol/LEDTA
1mol/L piperidine solution
This solution should be prepared fresh by mixing 9 times the volume of water with 1 times the volume of piperidine (10 mol/L; Fisher) in a graduated glass tube.
1mol/L Piperidine Carboxylic Acid
A 4% aqueous solution of formic acid can be made up to pH 2.0 with 10mol/L piperidine.
3mol/L sodium acetate (pH 5.2)
Gel
Polyacrylamide sequencing gel
Maxam-Gilben sequencing reaction products are typically analyzed on 6% or 8% denaturing polyacrylamide gels (see protocol 8).
Clostridial and Oligoclostridial Acids
Salmon Sperm DNA (lmg/ml) Aqueous Solution
Trimmed salmon sperm DNA is commonly used as a carrier for chemical sequencing. In fact, almost any DNA will do. Trimmed salmon DNA can be purchased as a commercial product and is also available in the laboratory (see Chapter 6, Scheme 10).
Radiolabeled target DNA
The labeled fragment must consist of a minimum of 5x105Ci/min of 32P, dissolved in water up to 5000Ci/min/ul (see Supplementary Methods: Preparation of End-Labeled DNA for Chemical Sequencing for details). the DNA solution must be salt-free. If the amount of radiolabeled DNA is limited. Sequence results can sometimes be obtained by performing only 4 of the 5 reactions (C, C+T, A+G, G).
Yeast tRNA (1 mg/ml aqueous solution)
Specialized devices
Dry ice-ethanol bath
Cerenkov counter (see Appendix 8)
Ice-water bath
Small centrifuge tubes
Cutting reactions are usually performed in standard 1.5 ml small centrifuge tubes. When different experiments are performed at the same time, different colored tubes may be used to distinguish them. Some researchers use silicone-treated tubes. This is not necessary, as it is sufficient to dissolve the DNA thoroughly after each precipitation step. For example, the walls of the tubes can be rinsed thoroughly with buffer aspirated from a Tip, or the vortexing time can be extended.
Rotary Vacuum Dryer
such as the SavantSpeedVac.
Circulating water bath units with tight-fitting lids
e.g. ResearchProductsInternational products.
Optional, see step 4.
Gel Sequencing Unit and Power Supply
See Option 11.
Adjustable 37°C and 90°C water baths
Methods
1. Prepare radiolabeled DNA and follow the flow chart in Table 12-16.
Usually, only four reactions (C, C+T, A+G, G) are performed to obtain the sequence results.
2. Add 100 ul of 1 mol/L piperidine to the above 4 or 5 lyophilized DNA samples containing the modified bases and resuspend with a vortex shaker.
Piperidine is used to break the sugar-phosphate bond at the chemically modified site on the DNA strand.
3. Cover the tube tightly and mix with a vortex shaker. If needed, centrifuge slightly (2000r/min) to bring the mixture together at the bottom of the tube.
4. Incubate the tubes at 90°C for 30 min. To prevent the caps from popping off, place a weight on the tubes, seal the mouths of the tubes with plastic tape, or react in a circulating water bath with a tight-fitting cap.
5. When the tubes have cooled to room temperature, open the lids, seal the open tubes with paraffin film (Parafilm), and puncture several holes in the paraffin film with a 21-gauge needle. Evacuate it on a rotary vacuum dryer.
To avoid blurring of the bands on the final sequencing gel, the piperidine from the base-holding cleavage reaction must be thoroughly removed. It is best to use a rotary vacuum dryer in the case of lyophilization. Depending on the efficiency of the dryer, this step often takes 1-4 hours. Some researchers prefer to freeze the samples with dry ice-ethanol prior to drying.
6. Remove the tubes. Remove paraffin film and add 20ul of water. Cap the tube and vortex and oscillate to dissolve the DNA for 30 s. Centrifuge slightly to allow the solution to collect at the bottom of the tube. Use a handheld micro-counter to confirm that the labeled DNA on the wall of the tube has been completely dissolved in the aqueous solution.
7. Repeat the process and dry the tubes on a rotary vacuum dryer, which often takes 15-30 min depending on the efficiency of the dryer.
(Refer to step 5). 
8. Repeat steps 6 and 7.
To ensure that the piperidine is completely removed. Some researchers prefer to dissolve the DNA in 10ul of water again and drain. However, this step is generally only necessary if the sample is oily or requires a long drying time.
9. Estimate the radioactivity in the tube using a Cerenkov counter, and place the modified and cut reactants in sequencing gel buffer. For overnight exposure on Kodak XAR-5 film, cut reactants after one base require about 25,000 cpm (C and G reactions), and cut reactants after two bases require about 50,000 cpm (C+T, A+G, A>G). Therefore, the C and G reactions dissolved in sequencing buffer should reach 25,000 cpm/3ul; and the C+T, A+G and A>G reactions should reach 50,000 cpm/3ul. Vortex the mixture to completely dissolve the DNA, and centrifuge briefly to bring the mixture together at the bottom of the tube. Samples can be stored at -20°C for several hours.
10. Denature the DNA by heating at 90°C for lmin and quench on ice. Analyze the results by denaturing polyacrylamide gel electrophoresis (see protocols 8, 9, 10, 11 and 12). 
