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Sanger Sequencing: General

DNA sequencing is the determination of the base-pair sequence of a DNA fragment by the formation of extension products of various lengths. DNA sequencing on capillary electrophoresis platforms is the process of reading nucleotide bases in a DNA molecule. During Sanger sequencing, DNA polymerases copy single-stranded DNA templates by adding nucleotides to a growing chain (extension product). This method involves copying single-stranded DNA with chemically altered bases called dideoxynucleotides which, when incorporated at the 3' end of the growing chain, terminate the chain selectively at A, C, G, or T. The terminated chains are then resolved by capillary electrophoresis. 

Fragment analysis determines the relative size of a DNA fragment, typically a PCR product. One of the PCR primers is fluorescently labeled and the labeled PCR product is combined with a size standard to extrapolate the base-pair size of the sample. 

Our website provides many resources for obtaining information regarding sequencing applications. A few are listed below:

The Sanger sequencing reaction cannot be multiplexed, as the reaction should contain only one template and one primer. Multiple priming sites will result in sequencing noise and low quality data. 

The amount of template DNA is dependent on the template type. Please see our guidelines below:

DNA template

 

Quantity used with most DNA sequencing purification protocols 

 

Quantity if using BigDye™ XTerminator™ Purification Kit*

 

PCR product:

 

 

 

100–200 bp 

 

1–3 ng

 

0.5–3 ng

 

200–500 bp 

 

3–10 ng

 

1–10 ng

 

 500–1000 bp 

 

5–20 ng

 

2–20 ng

 

1000–2000 bp 

 

10–40 ng

 

5–40 ng

 

 >2000 bp 

 

20–50 ng

 

20–50 ng

 

Other template:

 

 

 

Single-stranded DNA 

 

25–50 ng

 

10–50 ng

 

Double-stranded DNA 

 

150–300 ng

 

50–300 ng

 

Cosmid, BAC 

 

0.5–1.0 μg

 

0.2–1.0 μg

 

Bacterial genomic DNA

 

 2–3 μg

 

 1–3 μg

 

* The BigDye™ XTerminator™ Purification Kit is intended to purify the DNA sequencing reaction after thermal cycling by sequestering cycle-sequencing reaction components such as salt ions, unincorporated dye terminators, and dNTPs, to prevent their co-injection with dye-labeled extension products into a capillary electrophoresis DNA analyzer.

RNA cannot be sequenced directly; instead we recommend transcribing the RNA to cDNA. The cDNA can then be used as a template for sequencing. For more information on recommended kits to convert RNA to cDNA go here.

Template quality impacts the DNA sequencing reaction. We recommended a spectrophotometer to determine DNA quality. Optimum absorbance ratios (A260/280) are between 1.8 and 2.0.

  • After the sequencing reaction, post thermal cycling, the samples can be stored at 4 degrees C overnight or at –15 degrees C or –25 degrees C for long-term storage.
  • After purifying with Centri-Sep™ or ethanol precipitation, dry down the samples, seal the plate with MicroAmp™ Clear Adhesive Film, and store, protected from light, at 4 degrees C for capillary electrophoresis (CE) preparation or at –20 degrees C until use.
  • After BigDye™ Xterminator purification, to store for up to 10 days, seal the plate with MicroAmp™ Clear Adhesive Film, and store at 4 degrees C for CE preparation or at –20 degrees C. BDX plates can be stored at room temperature for up to 48 hours inclusive of time on the CE instrument.

For other DNA sequencing purification methods, please refer to the reagent-specific protocol. 

For long-term storage, the purified sequencing reaction should be dried and stored at –20 degrees C, protected from light. 

The sequencing standard is intended to be used after resuspension in Hi‑Di™ Formamide. It can be injected multiple times; the number of times will vary depending on the instrument-specific standard used. This information can be found in the product insert. The sequencing standard should be stable for 24 hours on the instrument. 

Once the sample has been resuspended in Hi‑Di™ Formamide, we recommend that you load it immediately on the instrument. It should be stable for 24 hours on the instrument. 

We recommend using Hi‑Di™ Formamide due to the sample stability in the solution. 0.1 mM EDTA, pH 8, can also be used as an injection solution but is more susceptible to evaporation. The use of water as an injection solution causes highly variable quantities of DNA to be injected, because there is no competition for the charged DNA/salts. 

The sequencing standard has a shelf life of one year from the date of receipt, unless otherwise listed on the CofA. Our Terms and Conditions of sale can be found here.

We recommend a primer amount of 3.2 pmoles in the sequencing reaction. 

The pGEM control and M13 primer provided in the kit should be used for troubleshooting purposes. The pGEM is a control template that can be used to isolate issues with sample quality, thermal cycler, kit or sequencing reaction purification. 

We have the Primer Designer™ Tool, which is a free online tool to search for the appropriate PCR/Sanger primer pair from a database of >650,000 pre-designed primer pairs for resequencing the human exome. Go here for more information, including a direct link to purchase the designed primers online.

Primer Designer™ Tool is a free online tool to search for the appropriate PCR/Sanger primer pair from a database of >650,000 pre-designed primer pairs for resequencing the human exome. For more information, including a direct link to purchase the designed primers online go here

The M13-tailed primers are used to simplify the workflow when sequencing PCR products and they reduce the loss of the 5’ unresolvable bases. When the PCR primers contain M13 tails on their 5’ ends, the M13 sequence is incorporated into the amplicons. This enables the use of sequencing master mixes containing either the universal M13 forward or M13 reverse primers. The sequence for the M13 forward and reverse primers are as follows:

  • M13 forward primer sequence: 5′ TGTAAAACGACGGCCAGT 3′
  • M13 reverse primer sequence: 5′ CAGGAAACAGCTATGACC 3′

Below is an illustration of the location of the M13 primers on the 5’ end of the target specific PCR primer: 

Offscale data refers to data from samples that have signal intensities resulting in saturation of the CCD camera, on the capillary electrophoresis instruments. The maximum signal thresholds for raw data are 32,000 rfu for the Applied Biosystems™ 3730/3730xl DNA Analyzers and 3500/3500xL Genetic Analyzers, and 8,000 rfu for the Applied Biosystems™ 310 Genetic Analyzer and Applied Biosystems™ 3130/3130xl Genetic Analyzers. 

The Sanger Sequencing Kit is designed for our complete sequencing workflow on the SeqStudio, 3500 series, and 3730 series genetic analyzers. The new kit provides all the components needed to clean up a PCR product for sequencing, perform cycle sequencing, and then purify the sequencing product. This simple, fast workflow enables completion of Sanger sequencing, from sample to answer, in less than a workday.

Yes, all the components in the Sanger Sequencing Kit are available separately. The individual components can be found on the following product pages:

- For the ExoSAP-IT Express PCR Product Cleanup Reagent please go here. We recommend replacing the strip septa after each run. Bleach, detergents, or solvents should not be used to clean the septa. Use of these reagents can cause them to dry out faster and risk contaminating the system.

- For the BigDye Terminator v3.1 Cycle Sequencing Kit, please go here.

- For the BigDye XTerminator Purification Kit, please go here.

The Sanger Sequencing Kit includes the following components:

- ExoSAP-IT Express PCR Product Cleanup Reagent, which removes excess primers and unincorporated nucleotides from a PCR reaction

- BigDye Terminator v3.1 Cycle Sequencing Kit, which provides premixed reagents for Sanger sequencing reactions

- BigDye XTerminator Purification Kit, a fast, simple purification method for DNA sequencing reactions, that removes unincorporated BigDye terminators and salts

For the sequencing reaction, we recommend using 3 µL of purified PCR product (purified using ExoSAP-IT Express PCR Product Cleanup Reagent from the kit).

When using the Sanger Sequencing Kit, we recommend using a sequencing reaction volume of 10 µL.

BigDye™ Terminator v1.1 and v3.1 Cycle Sequencing Kits

The BigDye™ Terminator kit versions have different fluorescent dyes attached to the dideoxy terminators, and the dNTP/ddNTP ratio differs between the kits. The BigDye™ Terminator v1.1 Cycle Sequencing Kit is optimized for shorter fragments and for obtaining sequence close to the primer. The BigDye™ Terminator v3.1 Cycle Sequencing Kit is more suitable for larger templates and for obtaining longer read lengths. 

If DNA sequence immediately after the primer is needed, the BigDye™ Terminator v1.1 Cycle Sequencing Kit has been designed to deliver optimal 5' resolution and basecalling in shorter fragments when used in combination with POP-6™ polymer and a 50 cm array. 

If longer read lengths are needed, the BigDye™ Terminator v3.1 Cycle Sequencing kit has been formulated to deliver robust performance across a wide variety of DNA sequences while maximizing read lengths.

The composition of the BigDye™ Terminator 5X buffer v1.1 & v3.1 and BigDye™ Terminator 5X buffer v1.0 & v3.0 chemistries is different and therefore should only be used with the matching chemistry. Use of the incorrect BigDye™ Terminator 5X buffer can negatively impact sequencing results.

We recommend using HPLC-purified primers for DNA sequencing. This will ensure the presence of full-length, highly purified primers that will minimize cycle sequencing noise and provides longer sequencing reads.

The primer amount in the final reaction should be 3.2 pmoles. If the sequencing reaction is being diluted, the primer concentration may need additional optimization.

The BigDye™ Terminator v1.1 & v3.1 5X Buffer is used to maintain the optimal buffer concentration in the sequencing reaction when less than the recommended volume of BigDye™ Terminator Ready Reaction Mix is used. The BigDye™ Terminator v1.1 & v3.1 5X Buffer amount varies depending on the volume of the BigDye™Terminator Ready Reaction Mix. To determine the amount of BigDye™ Terminator v1.1 & v3.1 5X Buffer, please use the Applied Biosystems™ BigDye™ Terminator v1.1 & v3.1 5X Sequencing Buffer Calculator

We provide the BigDye™ Terminator v1.1 & v3.1 5X Buffer to enable dilution of the BigDye™ Terminator Ready Reaction Mix. However, without optimization, diluting the BigDye™ Terminator Ready Reaction Mix may cause deterioration of sequencing quality. We cannot guarantee the performance of BigDye™ chemistry when it is diluted. 

The calculation can be done manually or by using our Applied Biosystems™ BigDye™ Terminator v1.1 & v3.1 5X Sequencing Buffer Calculator.

The formula is provided below:

Volume of BigDye™ Terminator v1.1 & v3.1 5X Buffer (µL) = 0.5 x [(total reaction volume/2.5) - volume of BigDye™ Terminator Ready Reaction Mix]. 

For example, if you are using 2 µL of the BigDye™ Terminator Ready Reaction Mix and a 15 µL reaction volume,

Volume of BigDye™ Terminator v1.1 & v3.1 5X Buffer (µL) = 0.5 x [(15/2.5) – 2] = 2 µL

PCR products may contain excess dNTPs and primers. Excess dNTPs will alter the optimized dNTP/ddNTP ratio in the sequencing reaction and may alter signal uniformity. The excess primers may have different priming sites, resulting in sequencing noise. If the PCR primers and sequencing primers are the same, the primer concentration will be higher in the sequencing reaction and may impact signal uniformity of the sample. If the PCR reaction is optimized to exhaust the primers and dNTPs, a PCR purification may not be necessary depending on the application. Please view a list of PCR clean-up kits here.

Purification of the sequencing reaction gets rid of salt ions, unincorporated dye terminators, and dNTPs. These components can compete with the sample for injection into the capillary and obscure data in the early part of the sequence and can interfere with basecalling.

For sequencing reactions, it is only necessary to remove the fluorescently labeled ddNTPs as they can interfere with the fluorescent sequencing detection. For sequencing, the extension products are single stranded and their lengths will vary, starting with primer +1, +2, +3 etc. Depending on the method, PCR cleanup methods are intended to remove primers or products below a certain basepair length, and loss of the smaller sequencing extension products can occur if a PCR purification method is used to purify the sequencing reaction. 

Sequencing reaction purification can be performed with BigDye™ XTerminator, Centri-Sep™ System or ethanol/EDTA precipitation. The protocols can be found in the BigDye™ Xterminator Purification Kit manual, Chapter 4 of the BigDye™ Terminator v1.1 Cycle Sequencing Kit manual,  BigDye™ Terminator v3.1 Cycle Sequencing Kit manual or the DNA Sequencing by Capillary Electrophoresis Guide.

The control is pGEM™-3Zf(+) , plasmid DNA which serves as a double-stranded control.

The pGEM control is 200 ng/µL and the M13 primer 0.8 pmol/µL

We do not sell the pGEM control separately from the DNA sequencing kit. 

We do not sell the –21 M13 forward control primer separately, however we provide the primer sequence for your convenience: TGTAAAACGACGGCCAGT

Please feel free to use our oligo services to order this primer.

We also offer an –21M13 forward primer that is one basepair shorter than that contained in the kit (Cat. No. N52002). 

Component

 

Quantity per reaction

 

BigDye™ Terminator 3.1 Ready Reaction Mix

 

8 μL

 

–21 M13 Control Primer (0.8 pmol/μL)

 

4 μL

 

pGEM (200 ng/μL)

 

1-2 μL

 

Water

 

Adjust based on

pGEM volume. i.e., 7 or 6 μL to bring final reaction volume to 20 μL 

 

Total volume

 

20 μL

 

The recommended annealing temperature is generally 5-10 degrees C lower than the primer melting temperature (Tm). 

Please refer to the COA for the expiration date. When stored at -20 degrees C in a non-frost free–freezer, the kit is guaranteed stable until the expiration date. 

The kit can undergo up to 10 freeze/thaw cycles. 

BigDye™ Direct Cycle Sequencing Kit

The BigDye™ Direct Cycle Sequencing Kit streamlines the workflow when sequencing PCR products. The kit combines the components for the PCR reaction, PCR purification and cycle sequencing, which results in substantial time savings. 

Components of the BigDye™ Direct Cycle Sequencing Kit are specially formulated and optimized to work together and they cannot be combined with other PCR master mixes. Other PCR master mixes are not recommended as they can introduce variability into the workflow and yield suboptimal results.

The BigDye™ Direct Cycle Sequencing Kit can be used on any organism. However, we only supply Human CEPH-1347-02 control DNA in the kit. A control for your organism should be obtained from an appropriate vendor. 

No. The gene-specific PCR primers used in the BigDye™ Direct Cycle Sequencing Kit must be modified with the M13 universal primers on the 5’ end. 

No, the M13 universal primers must be used, and are included in the BigDye™ Direct Cycle Sequencing Kit. 

The PCR guidelines in the BigDye™ Direct Cycle Sequencing Kit manual are general guidelines designed to work for a wide variety of samples. However, there will be cases where the PCR conditions may need to be modified (e.g. Tm of primer is very high, GC rich products, etc.) to best work with whatever you are trying to amplify and sequence. The primers must have the M13 tail on the 5’ end. For further suggestions, see Page 38 in the DNA Sequencing by Capillary Electrophoresis Guide. Some of those suggestions include:

  • Primers should be at least 18 bases long (not including the M13 tail which is not part of the PCR reaction)
  • Tm of the primers should be 45 degrees C or higher
  • Avoid runs of identical nucleotides, especially runs of 4 or more "Gs"

No, the BigDye™ Direct Cycle Sequencing Kit formulation has been optimized to work at full strength and the PCR and sequencing chemistry is optimized to work together for a simpler workflow. Diluting the product may lead to poor results.

The M13 tails are necessary as the BigDye™ Direct Cycle Sequencing Kit contains the universal M13 forward and universal M13 reverse primer, without the M13 tails the sequencing reaction will fail. The use of M13 sequencing primers also reduces the loss of valuable 5’ unresolvable bases.

  • M13 forward primer sequence: 5′ TGTAAAACGACGGCCAGT 3′
  • M13 reverse primer sequence: 5′ CAGGAAACAGCTATGACC 3′

It is an enzymatic cleanup and, due to the proprietary nature of the kit, we cannot disclose additional information. 

The BigDye™ Direct samples have specific mobility and basecaller files depending on the instrument. 

  • KB_3130_POP7_BDTv3direct.mob and KB_3130_POP7_BDTv3direct.bcc
  • KB_3500_POP7_BDTv3direct.mob and KB_3500_POP7_BDTv3direct.bcc
  • KB_3730_POP7_BDTv3direct.mob and KB_3730_POP7_BDTv3direct.bcc

The control that would be used for the sequencing reaction is the same control for the PCR reaction. For instance, in the kit, we provide the Control DNA CEPH 1347-02; this control should be taken into the PCR reaction and sequenced. Please see here for setting up the PCR reaction and the sequencing reaction. 

dGTP BigDye™ Terminator Cycle Sequencing Kit v1.0 and v3.0

The dGTP BigDye™ Terminator Cycle Sequencing Kits are kits that can be used for sequencing difficult templates where early signal loss may occur. The kit is not recommended for routine sequencing as the kit uses dGTP instead of dITP and compressions can occur with the data. 

The dGTP BigDye™ Terminator Cycle Sequencing Kit should be used for difficult templates, such as templates that are GT-rich, G-rich templates or are difficult to sequence with other kits. They should be used only when you cannot obtain good data using standard BigDye™ Terminator kits.

The dGTP BigDye™ Terminator Cycle Sequencing Kit can be run on all of our capillary electrophoresis platforms:

  • Applied Biosystems™ 3500/3500xL Genetic Analyzers
  • Applied Biosystems™ 3730/3730xl DNA Analyzers
  • Applied Biosystems™ 310 Genetic Analyzer
  • Applied Biosystems™ 3130/3130xl DNA Analyzers

However, due to the nature of the chemistry, compressions in the data are expected.

BigDye™ XTerminator™ Purification Kit

The BigDye™ XTerminator™ Purification Kit is intended to purify the DNA sequencing reaction after thermal cycling. 

The BigDye™ XTerminator™ Purification Kit sequesters cycle-sequencing reaction components such as salt ions, unincorporated dye terminators, and dNTPs, to prevent their co-injection with dye-labeled extension products into a capillary electrophoresis DNA analyzer. The SAM™ Solution also stabilizes the sample after purification. 

There are many advantages to using the BigDye™ XTerminator™ Purification Kit including:

  • Simplifies workflow:
    • No need for spin column or ethanol precipitation
    • Fast
    • Little hands-on time needed
  • Flexible throughput with purification in single tubes, 96-well or 384-well formats
  • No bead removal prior to injection (depending upon the instrument used for sequencing)
  • Efficient removal of  “blobs” 
  • No danger of removing sample pellet (as can happen with ethanol precipitation)
  • Higher signal intensity for the purified sample decreases quantity of DNA required

We recommend that you use wide-bore pipet tips (>1.0 mm) for pipetting the BigDye™ XTerminator™ Solution. 

The wide-bore tips are necessary when using the BigDye™ XTerminator™ Purification Kit to ensure that the correct amount of BigDye™ XTerminator™ Solution is used in the purification. The wide-bore tips are not necessary for pipetting the SAM™ Solution. If wide-bore tips are not used, an inadequate amount of BigDye™ Xterminator™ Solution may result and lead to uneven distribution of the BigDye™ Xterminator™ Solution from well to well. This may result in the liquid level of the BigDye™ Xterminator™ Solution to be consumed faster and result in a viscous solution that is difficult to pipet. 

Because of the inconsistency that may occur with manually cutting pipet tips, we do not recommend it. However, if it is needed in the short term, while waiting for your order of wide-bore pipet tips, the tip orifice should be >1.0 mm. 

The recommended vortexers for the BigDye™ XTerminator™ Purification Kit are the following:

  • Digital Vortex-Genie™ 2 
  • IKA MS3 Digital 
  • IKA Vortex 3 
  • Taitec MicroMixer E-36 
  • Union Scientific Vertical Shaker 

No. BigDye™ XTerminator™ Solution is heat-sensitive and should never be heated. 

For a 384-well plate, a minimum 5 µL reaction volume should be used. For a 96-well plate, a minimum 10 µL reaction volume should be used. 

The BigDye™ XTerminator™ run modules can be downloaded using the following link

The BigDye™ XTerminator™ Solution is expected to perform to specification if frozen no more than once.

The BigDye™ XTerminator™ Solution is expected to perform to specification if left out for no more than one 24-hour period. We cannot guarantee performance for reagent left out for a longer period of time.

Yes. The precipitate should be resuspended by warming the SAM solution (remember, never heat the BigDye™ XTerminator™ Solution) to 30-37 degrees C and gently vortexing. Bubbles will form on the surface of the SAM™ Solution when vortexed. These will dissipate within a few minutes. Allow the SAM™ Solution to cool to ambient temperature before use. Do not use it while warm as using warm SAM™ Solution will produce poor results. 

The precise ratio of liquid phase and insoluble phase in the BigDye™ XTerminator™ Solution is critical to achieving specified performance. We strongly recommend against resuspending this material and cannot guarantee the specified performance of the material if you do so. It is recommended that you use wide-bore pipette tips (>1.0 mm) when pipetting the BigDye™ XTerminator™ Solution to ensure that adequate amounts of the liquid and insoluble phase are aspirated during pipetting.

The pre-mixed SAM™ Solution and BigDye™ XTerminator™ Solution can be stored at 4 degrees C for up to 5 days. Vortex the pre-mix prior to use. 

Methylation Analysis

 We offer the Cells-to-CpG™ Bisulfite Conversion Kit, which converts unmethylated cytosines to uracil but does not change methylated cytosines. The Cells-to-CpG™ Bisulfite Conversion Kit is used during sample preparation and the sample can then be PCR amplified for use in the DNA sequencing reaction. Any of the BigDye™ Terminator kits are able to sequence bisulfite-converted DNA. Additional information regarding methylation analysis can be found on this page: Methylation Analysis by Sanger Sequencing.

The primer design is different for bisulfite-converted DNA. Primer design is critical as the converted DNA is difficult to amplify due to the less complex, 3-base genome, different Tm calculation and increased non-specific priming. We offer freeware to assist with the primer design called Methyl Primer Express™ Software v1.0.

Methyl Primer Express™ Software v1.0 is compatible with Windows™ 2000 (Service Pack 4), Windows™ XP (Service Pack 2). 

Purified gDNA, cultured cells, and blood can be used.

The optimal amounts for the specific sample types are the following:

  • Purified gDNA - 100 ng to 1 µg 
  • Cultured cells - 5000 cells to 105 cells 
  • Blood - 2.5 µL 

Yes, there are two controls: Unconverted Human Male gDNA and Converted Human Male gDNA. They can be ordered using Cat. No. 4445552.

The Cells-to-CpG™ Bisulfite Conversion Kit should be stored at room temperature.

Store the converted DNA at 4 degrees C for up to 6 months. For long-term storage, store at -20 degrees C or -70 degrees C. Store in aliquots to avoid multiple freeze-thaw cycles.

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