Protocol:DNA sequencing

DNA sequencing 

Principle of Sanger or dideoxy method
DNA sequencing allows the analysis of the exact nucleotide sequence of the gene, or area of gene, under study. The method most commonly used is based on that first described by Sanger et al, 1977 (52). The method depends on the in vitro synthesis of DNA strands copied from a DNA template (usually generated using PCR nowadays), during which the synthesis of each nascent DNA chain is stopped at every point along the strand, following the random incorporation of a dideoxynucleotide triphosphate (ddNTP). ddNTP’s can be incorporated by DNA polymerase into the growing chain, but the absence of a hydroxyl residues at the 3’ position prevents the extension of the DNA chain.

The basic reaction includes the template DNA (single-stranded), of which the nascent sequencing strand will be a complementary copy, a synthetic oligonucleotide or ‘primer’ about 20bp long which is complementary to a small region of the template DNA close to the target area for sequencing, a specialized DNA polymerase enzyme (T7, or a thermostable DNA polymerase) and free nucleotides, most of which are deoxynucleotide triphosphates (dNTP’s), with a small fraction of dideoxynucleotide triphosphates (ddNTP’s).

There are variations of the Sanger protocol, which differ relative to the DNA polymerase employed for strand synthesis and/or the detection system used to detect the newly synthesised DNA strands. For the so-called manual sequencing method the product of the sequencing reaction is usually radioactively labelled and is separated on a vertical acrylamide gel, which is then exposed to produce an autoradiograph for manual reading. More recent modifications to the Sanger protocol include cycle-sequencing (exploiting the use of a thermal stable DNA polymerase) and detection of fluorescently labelled nascent DNA using an automatic sequencer (53). Following the cycling reactions, the products are separated according to size on a polyacrylamide gel (or capillary), which is monitored by a laser to detect the fluorescently labelled nascent strands. There are two main fluorescent dye chemistries to label the nascent strand, involving either a fluorescent primer or fluorescent dNTP’s.

In this chapter the basic steps of 2 general sequencing protocols are outlined:


 * 1) Manual Sequencing using single-stranded DNA template, T7 polymerase and radioactive label.
 * 2) Cycle sequencing with use of fluorescent labels and analysis on automatic sequencer

Preparation of single-stranded DNA template by asymmetric PCR (Manual sequencing) :
This method involves a first amplification to produce double-stranded DNA. For a 50μl reaction, add the following to an eppendorf tube, on ice:


 * 1) 0.1-0.5 μg genomic DNA
 * 2) 20 pmol of each primer (forward and reverse for relevant fragment)
 * 3) H2O up to 50 μl final volume
 * 4) 5 μl of 10x reaction buffer (usually provided with the Taq polymerase by the manufacturer, containing 500 mM KCl, 100 mM Tris-HCL, pH 8.0, 25 mM MgCl2, 2 mg/ml BSA)
 * 5) 2 mM dNTP’s
 * 6) 1 unit of Taq polymerase
 * 7) Overlay with 50 μl paraffin oil.

Method for PCR (Manual sequencing) :

 * 1) With 30 cycles of: denaturation at 95 oC for 60 sec, annealing at a temperature appropriate for Tm of primer pair, for 60 sec, and extension at 72 oC for 90 secs.
 * 2) A second PCR reaction follows using 2 μl of the first PCR and only ONE of the 2 primers in a100 μl reaction volume.
 * 3) Following the second PCR, the product is cleaned to remove excess dNTP’s etc:
 * 4) Add 1 volume of chloroform, mix and spin in microcentrifuge at 10,000 rpm for 5 minutes.
 * 5) Remove supernatant to a clean eppendorf tube and add 100 μl of 5 M ammonium acetate and 200 μl isopropanol.
 * 6) Leave to stand for 10 min, then spin at 10,000 rpm for 10 minutes.
 * 7) Discard supernatant, wash pellet with 70% ethanol and dry. Dissolve single-stranded DNA pellet in 15 μl of distilled water.
 * 8) Check 3-5 μl of product on a 1% agarose gel in TBE, stain with ethidium bromide and view under UV light. (Single-stranded DNA product usually runs slower than equivalent double-stranded product).

Method for sequencing reaction (manual sequencing) :
==== This forsees the use of one of the many available kits which provide reaction buffers, labelling mixes (including dNTP’s), termination mixes (including ddNTP’s), T7 DNA polymerase and enzyme dilution buffer. The user has to purchase separately the radioactively labelled dNTP (usually either [a-35-S]-dATP or [a-35-S]-dCTP. ====
 * 1) Take 10 μl single-stranded DNA product, and add 2 μl sequencing primer.
 * 2) Incubate at 37 oC for 10-20 minutes and then leave at room temperature for a further 10-20 minutes to ensure primer-template annealing.
 * 3) To the annealed primer-template mix add 3 μl labeling mix, 1μl radioactive dNTP (10 μCi) and 2 μl T7 polymerase (usually diluted just prior to use with enzyme dilution buffer).
 * 4) Mix gently and leave at 37 oC for 5 minutes
 * 5) During this time label 4 eppendorf tubes (1 each for A,C,G and T), add 2 μl of the appropriate ddNTP termination mix to each and warm at 37 oC for a few minutes.
 * 6) Distribute 4.5 μl of completed labelling reaction to each of the 4 ddNTP termination mixes and incubate for a further 5 μl.
 * 7) Add 5 μl stop solution and place on ice.
 * 8) Immediately before loading, heat samples to 80 oC for 2 minutes and place on ice.

Preparation of denaturing polyacrylamide gel, electrophoresis and autoradiography (manual sequencing):
There are many types of electrophoresis equipment available commercially, and the arrangement of glass plates and spacers may vary. Standard sequencing gels are usually about 40 cm long with spacers and combs that are 0.4 mm thick. All sequencing electrophoresis apparatus includes a heatable “plate” to warm the surface of the gel during the run.

The concentration of acrylamide in the gel depends upon the size of the DNA fragments to be analysed. Sequences between 20-250 nucleotides from the primer can be read from 40 cm length gels and 6% acrylamide with a single loading.

Reagents (Manual sequencing):
The following solution containing 6 or 8% acrylamide can be prepared as follows:


 * 1) 6% acrylamide

Acrylamide 17.1 g

Bis-acrylamide 0.9 g

Urea 150 g

10x TBE* 30 ml

Distilled H2O to 300 ml


 * 1) 8% acrylamide

Acrylamide 22.8 g

Bis-acrylamide 1.2 g

Urea 150 g

10x TBE* 30 ml

Distilled H2O to 300 ml

(*10x TBE: 0.89 M Tris, 0.89 M boric acid, 0.02 M EDTA).

The solution is stable at 4 oC for several months.

Method for acrylamide electrrophoresis (Manual sequencing) :

 * 1) Sequencing gels should be made at least 2 hours before loading.
 * 2) For plates 40 cm long, 20 cm wide and 0.4 mm wide, about 40 ml of gel is required, as follows: 40 ml of 6% or 7% acrylamide/8M urea, 300 μl 10% ammonium persulphate and 30 μl TEMED. The mixture will polymerise within 1 hour.
 * 3) Once polymerised, remove the well-former from the top, clean away excess acrylamide and place in electrophoresis apparatus. Fill upper and lower buffer reservoirs with 1x TBE and pre-run gel for about 30-60 minutes to reach a temperature of about 50 oC.
 * 4) Place a sharks-tooth comb on top of gel. Flush out wells and load 2-3 μl of each dNTP from each sample per lane (ie one sample is 4 lanes, A,C,G and T).
 * 5) Electrophoresis is performed at 1500-2000 volts for 2-3 hours depending upon the distance from the primer to be analysed.
 * 6) After electrophoresis, turn off the power, remove one glass plate and place a piece of Whatman paper (35x42 cm) to stick over the entire surface of the gel, taking care to avoid formation of bubbles and not to split the gel.
 * 7) Carefully cover the gel side with Saran-wrap (or equivalent) and place paper side down on a gel dryer and dry under vacuum for about 90 mins at 80 oC.
 * 8) Remove wrap from gel and in a dark room place a 35x42 cm X-ray film (Kodak XAR-2 or equivalent) and place in an X-ray cassette. Perform autoradiography overnight, and read the sequence of nucleotides according to size (Figure 1).

Cycle sequencing
Since the details of the method for an automatic sequencer depends upon the particular system used, a general protocol will be outlined. Overall, most of the preparation steps are supported by a wide range of commercially available template preparation and sequencing kits. Some machines are able to detect only a single fluorescent label, and others have the ability to detect multiple fluorescent labels. The level of automation relative to gel (or capillary) preparation and sample loading may vary considerably between machines. Additionally each system will have particular software for controlling running conditions and analysing data. The quality of template and precision of the sequencing reactions are paramount to the generation of good data for base calling.

Preparation of double-stranded DNA template and purification by commercially available columns (cycle sequencing).

For a 50 μl PCR reaction to produce double-stranded DNA, add the following to an eppendorf tube, on ice:


 * 1) 0.1-0.5 μg genomic DNA
 * 2) 20 pmol of each primer (forward and reverse for relevant fragment)
 * 3) H2O up to 50 μl final volume
 * 4) 5 μl of 10x reaction buffer (usually provided with the Taq polymerase by the manufacturer, containing 500 mM KCl, 100 mM Tris-HCL, pH8.0, 25 mM MgCl2, 2 mg/ml BSA)
 * 5) 2 mM dNTP’s
 * 6) 1 unit of Taq polymerase
 * 7) Overlay with 50 μl paraffin oil.
 * 8) With 30 cycles of: denaturation at 95 oC for 60 sec, annealing at a temperature appropriate for Tm of primer pair, for 60 sec, and extension at 72 oC for 90 secs.
 * 9) Following the amplification reaction, a 5 μl aliquot can be checked on a 1% agarose gel in 1x TBE. The template may then be purified by using a commercially available PCR-product purification kit eg by Qiagen.

PCR reaction (cycle sequencing).

There are several commercially available cycle-sequencing kits and most manufacturers will recommend that (or those) most suited to the automatic sequencer to be used. Additionally there are two main fluorescent dye chemistries used to label the nascent strand, involving either a fluorescent primer or fluorescent dNTP’s.

Essentially cycle sequencing is a PCR reaction which involves making a template-primer mix which is then distributed amongst 4 tubes containing dNTP’s and very small quantities of either ddATP or ddCTP or ddGTP or ddTTP, an appropriate buffer and a thermostable polymerase.

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