Introduction to Automated DNA Sequencing Sanger Dideoxy DNA polymerases copy single-stranded DNA templates, by adding nucleotides to a Sequencing growing chain (extension product). Chain elongation occurs at the 3´ end of a primer, an oligonucleotide that anneals to the template. The deoxynucleotide added to the extension product is selected by base-pair matching to the template. The extension product grows by the formation of a phosphodiester bridge between the 3´-hydroxyl group at the growing end of the primer and the 5´-phosphate group of the incoming deoxynucleotide (Watson et al., 1987). The growth is in the 5´Æ3´ direction (Figure 1-1). DNA polymerases can also incorporate analogues of nucleotide bases. The dideoxy method of DNA sequencing developed by Sanger et al. (1977) takes advantage of this ability by using 2´,3´-dideoxynucleotides as substrates. When a dideoxynucleotide is incorporated at the 3´ end of the growing chain, chain elongation is terminated selectively at A, C, G, or T because the chain lacks a 3´-hydroxyl group (Figure 1-1). Extension product

Template

3´ hydroxyl group

no 3´ hydroxyl group Figure 1-1 DNA strand synthesis by formation of phosphodiester bonds. The chain is terminated by the use of dideoxycytidine triphosphate (ddC) in place of deoxycytidine triphosphate (dCTP). The inset shows a schematic representation of the process.

1-2 Introduction

Fluorescent In the Applied Biosystems strategy for automated fluorescent sequencing, fluorescent Sequencing dye labels are incorporated into DNA extension products using 5´-dye labeled primers (dye primers) or 3´-dye labeled dideoxynucleotide triphosphates (dye terminators). The most appropriate labeling method to use depends on your sequencing objectives, the performance characteristics of each method, and on personal preference. Applied Biosystems DNA sequencers detect fluorescence from four different dyes that are used to identify the A, C, G, and T extension reactions. Each dye emits light at a different wavelength when excited by an argon ion laser. All four colors and therefore all four bases can be detected and distinguished in a single gel lane or capillary injection (Figure 1-2).

Figure 1-2 Four-color/one-lane fluorescent sequencing vs. one-color/four-lane method such as radioactive sequencing

Introduction 1-3

Cycle Sequencing Cycle sequencing is a simple method in which successive rounds of denaturation, annealing, and extension in a thermal cycler result in linear amplification of extension products (Figure 1-3). The products are then loaded onto a gel or injected into a capillary. All current ABI PRISM DNA sequencing kits use cycle sequencing protocols. See Chapter 3 for information on cycle sequencing protocols.

Figure 1-3 Cycle sequencing

Advantages of Cycle ♦ Sequencing ♦

1-4 Introduction

Protocols are robust and easy to perform. Cycle sequencing requires much less template DNA than single-temperature extension methods.

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Cycle sequencing is more convenient than traditional single-temperature labeling methods that require a chemical denaturation step for double-stranded templates.

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High temperatures reduce secondary structure, allowing for more complete extension.

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High temperatures reduce secondary primer-to-template annealing.

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The same protocol is used for double- and single-stranded DNA.

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The protocols work well for direct sequencing of PCR products (see page 3-14).

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Difficult templates, such as bacterial artificial chromosomes (BACs), can be sequenced.

ABI PRISM Sequencing Chemistries AmpliTaq DNA AmpliTaq® DNA Polymerase, FS is the sequencing enzyme used in ABI PRISM cycle Polymerase, FS sequencing kits. It is a mutant form of Thermus aquaticus (Taq) DNA polymerase and contains a point mutation in the active site, replacing phenylalanine with tyrosine at residue 667 (F667Y). This mutation results in less discrimination against dideoxynucleotides, and leads to a much more even peak intensity pattern (Tabor and Richardson, 1995). AmpliTaq DNA Polymerase, FS also contains a point mutation in the amino terminal domain, replacing glycine with aspartate at residue 46 (G46D), which removes almost all of the 5´Æ3´ nuclease activity. This eliminates artifacts that arise from the exonuclease activity. The enzyme has been formulated with a thermally stable inorganic pyrophosphatase that cleaves the inorganic pyrophosphate (PPi) byproduct of the extension reaction and prevents its accumulation in the sequencing reaction. In the presence of high concentrations of PPi the polymerization reaction can be reversed (Kornberg and Baker, 1992), a reaction called pyrophosphorolysis. In this reaction, a nucleoside monophosphate is removed from the extension product with the addition of PPi to form the nucleoside triphsphate. In a sequencing reaction, if a dideoxynucleotide is frequently removed at a particular position and replaced by a deoxynucleotide, eventually there is little or no chain termination at that location. This results in a weak or missing peak in the sequence data (Tabor and Richardson, 1990).

Dye-Labeled With dye terminator labeling, each of the four dideoxy terminators (ddNTPs) is tagged Terminators with a different fluorescent dye. The growing chain is simultaneously terminated and labeled with the dye that corresponds to that base (Figure 1-4).

Figure 1-4 One cycle of dye terminator cycle sequencing

Features of Dye-labeled Terminator Reactions ♦ An unlabeled primer can be used. ♦

Dye terminator reactions are performed in a single tube. They require fewer pipetting steps than dye primer reactions.

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Four-color dye labeled reactions are loaded in a single gel lane or capillary injection.

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False stops, i.e., fragments that are not terminated by a dideoxynucleotide (see page 7-30), go undetected because no dye is attached.

See Chapter 2 for information on ABI PRISM™ DNA sequencing kits. Introduction 1-5

Dye-Labeled With dye primer labeling, primers are tagged with four different fluorescent dyes. Primers Labeled products are generated in four separate base-specific reactions. The products from these four reactions are then combined and loaded into a single gel lane or capillary injection (Figure 1-5).

Figure 1-5 One cycle of dye primer cycle sequencing

Features of Dye-labeled Primer Reactions ♦

Dye primer chemistries generally produce more even signal intensities than dye terminator chemistries.

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Labeled primers are available for common priming sites. Custom primers can also be labeled.

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Four-color dye-labeled reactions are loaded onto a single lane or capillary injection.

See Chapter 2 for information on ABI PRISM™ DNA sequencing kits.

1-6 Introduction