Services | DNA Sequencing | PCR

Amplification of nucleic acid sequences by polymerase chain reaction (PCR) is now a standard process that is indispensible for molecular biology. By using PCR one can amplify products of various sizes, typically a few hundred to a few thousand bases in length. It can be adventageous to sequence such PCR products without first cloning them into a vector. We term this "direct" PCR product sequencing. It is important to realise that direct sequencing of PCR products has its advantages and disadvantages. Customers must decide whether they wish to try directly sequencing a PCR product or cloning it into a vector and then sequencing that (using our standard plasmid sequencing service).

There is no universal rule that will determine which is the "best" approach. However, below are some points that may help customers decide which approach to take. In order to attempt direct PCR product sequencing:

  1. One really needs a very clean PCR product that contains a single amplified product
  2. The product should be at least 200-300bp in size
  3. The PCR reaction should give a high yield of product

There is more information about direct sequencing of PCR products in our Resources section.

Should your PCR product not meet the above requirements (either before or after purification), then direct sequencing is not likely to be very successful.

Please also note that whilst we can sequence virtually any length of PCR product, the amount of material that is needed will change according to the length of the product. Details can be found below.

Please review the information below prior to submission of your samples so that you get the best out of our direct PCR product sequencing service.

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Materials Required Per Reaction

Please send PCR product that has been diluted or purified such that the final diluent is water or a buffer that does not contain EDTA (TE buffer is therefore not appropriate). EDTA can inhibit the sequencing reaction by chelating out magnesium (required for the DNA polymerase). Like plasmids, we also need specific amounts of material. However, the amount of PCR product required varies according to the size of the PCR product as indicated below:

PCR Product Length

PCR Product Required*

(per reaction)

Primer Quantity Required**

(per reaction)

100-200 bp

2-6 ng

3.2 pmole

200-500 bp

6-20 ng

3.2 pmole

500-1000 bp

10-40 ng

3.2 pmole

1000-2000 bp

20-80 ng

3.2 pmole

>2000 bp

80-200 ng

3.2 pmole

Please note that it is critical that quantitation of PCR products is not done with a spectrophotometer even if the product has been purified. Rather, evaluation on an agarose gel provides a more accurate estimate. This is because background "smears" which may not be obvious on a gel can seriously affect spectrophotometric values. Additional details about how to quantitate PCR product template is provided in our FAQs.

*This material should be supplied so that the amount stated above is in a total volume of 30µl, which allows us to retain some material in case a reaction needs to be repeated.  Hence, taking a 500-1000bp PCR product as an example, we need 10-40ng of that product in 30ul per reaction to be performed (final concentration of material you supply should be 0.3ng/ul to 1.3ng/ul).

**Note: This applies if you are submitting the primer and template together in a single sequencing reaction. 3.2 picomoles is 1µl of a 3.2 uM stock solution.

When sending primer separately, please send a minimum of 10µl of 3.2 uM primer stock to avoid issues associated with evaporation.

Primers

Special Considerations

PCR Template Purity

In order for direct sequencing of PCR templates to work well, it is critical that customers provide “cleaned” single band PCR products. These are PCR products free from "off-target" PCR products and from which the PCR primers and dNTPs have been removed or diluted to a point where they do not interfere with the sequencing reaction. This can be accomplished in various ways (e.g. optimisation of the PCR conditions and post-PCR clean-up using propriety kits, Exo/SAP treatment or simple dilution in water). The choice of which post-PCR clean-up strategy to use is dependent on various factors and we do not promote or recommend a specific method. Failure to clean up your PCR product at all will result in poor sequence in most cases. However, we should say that sometimes the act of purifying PCR products (especially if gel purifying or using column kits) can result in the introduction of contaminants that inhibit the sequencing reaction. The best approach is to optimise your PCR conditions so that you amplify just one product and reach an end-point where most primers and dNTPs are consumed. Often all that is needed with such PCR products is to dilute them in water.

Additional details can be found below in the section entitled "Key Points about PCR Product Sequencing that Affect Results".

PCR Template Quantitation

Please note that when using Exo/SAP treatment, you must quantitate your PCR product on a gel and then dilute it appropriately. A spectrophotometer will not give an accurate reading of the PCR product concentration due to the presence of degraded primers and residual dNTPs. Even if you have column purified your product, it is still not a good idea to use a spectrophotometer to quantify your product because background "smears" that often occur during PCR can interfere badly with spectrophotometric analysis.

PCR Template Length for Sequencing Reactions

Customers should bear in mind that PCR products often need some optimisation in order to get the best conditions for a particular product. This is because PCR products tend to vary more than plasmids in their abilities to act as good sequencing templates. If the product is ≥ 200 bp, then direct sequencing should be fairly straightforward (although care is still needed, especially to ensure that the product is clean and at the right concentration).

If the product is less than 200 bp, then generally direct sequencing of the product is going to be tricky. In this case, cloning is the better solution. When cloning the product, it is recommended that customers sequence at least 3 clones to be sure that you catch any PCR generated mutations.

Additional details about read lengths that customers can expect to obtain can be found in our FAQs.  

Read Length

There are many factors that can affect the read length one obtains. We will endeavour to provide customers with the longest read possible depending on service requested. 

With a template of appropriate quality, customers can expect read lengths of up to 1000 bases of sequence from a single primer. However, read lengths will decrease with templates of low quality and/or quantity. If you have problems achieving good read lengths with what you believe are high quality templates, please take a look at our resources section. If you still need more help, please feel free to contact us.

Pricing

Reactions

Services Requested Pricing (per Reaction)
Sequencing  £5.00
Sequencing + Quant-It* NA
Sequencing + Augmented Protocol^ £5.50
Sequencing + Quant-It* + Augmented Protocol^ NA

 

  • *Quant-It: Due to difficulties associated with getting accurate concentrations for PCR products using spectrophotometric analysis (which is the only method we can employ to determine concentrations), we are unable to offer a Quant-It service for PCR products.
  • ^Augmented Protocol: If your template has known secondary structure or a high GC content, £0.50 will be applied per reaction to facilitate the sequencing reaction using an 'Augmented Protocol'

Do you routinely sequence a large number of PCR Reactions?

Why not contact our staff to learn about savings options?

Our pre-payment option will help you save more per reaction!

Primer

  • Standard primers offered by MRC PPU DNA Sequencing & Services are free!
  • Custom primer synthesis prices will reflect the size of the primer – the cost to the customer is £0.50 per base

Learn more about all our pricing and turnaround.

Please note that prices are exclusive of VAT, which customers outside The University of Dundee will be charged at the appropriate rate (currently 20%) on all products and that all sales are subject to MRC PPU DNA Sequencing and Services' Terms and Conditions.

Turnaround Time

Results are routinely supplied to customers within 24 hours upon receipt of primer and template. Our standard practice is to perform the cycle sequencing reactions on the day the samples are supplied to us, followed by evaluation of productions on our sequencers. 

Shipping

Shipping Details

  • Labels
    • Please ensure that samples are clearly labelled so that we can identify them.
  • Packing Materials
    • Eppendorf Tubes
      • Please also ensure that your samples are well protected and packaged to meet Post-Office or courier regulations
        • A "Jiffy" style padded envelope for DNA samples in plastic tubes works well
        • We discourage use of regular envelopes as in the past, we have received envelopes with tears and/or crushed tubes in them

 

Postage Costs

  • Customers are responsible for paying all postage / carriage charges
    • For DNA samples or agar plates, normal first class post should be sufficient
      • As this does not always arrive as "next day post", we recommend that customers who have deadlines to meet send their samples by a guaranteed next day delivery service
  • Insufficient postage provided on a package will cause a delay in use receiving the package
    • If MRC PPU DNA Sequencing and Services is then charged upon receipt, please note that we will need to pass this cost onto the customer

 

Postal Address:

DNA Sequencing and Services
Medical Sciences Institute
School of Life Sciences
University of Dundee
Dundee
DD1 5EH
UK

Telephone:

(01382) 388019

Fax:

(01382) 388729

Email:

info@dnaseq.co.uk

Key Points about PCR Product Sequencing that Affect Results

How PCR Product Mutations Affect Sequencing Results

One of the main advantages of sequencing a PCR product directly (as opposed to cloning it first) is that you will not see PCR-generated mutations. The reason for this is that you have a large population of templates in your initial PCR reaction and even a mutation introduced in one PCR product in the first round of amplification will only be present in that one product out of thousands produced by the reaction. This very low frequency means that this error is swamped out by the majority (correct) sequence and so is not seen. As the reaction proceeds, the error is propogated, but so is the proportion of correct sequence at the same place in other templates. Hence, the error is still only present at a very low frequency and is not seen.

Contrast the above scenario with what happens when you clone the PCR product. Each clone is obtained originally from a single PCR product being inserted into a single vector. Hence, if that product contains an error, all the cloned plasmids derived from it will contain the same error. For this reason, one must sequence at least three independent clones in order to be sure that you are seeing the actual sequence and not a PCR-generated error.

PCR Primers vs Sequencing Primers

If your PCR primers are very long (over 30 bases), then it will probably be best to make shorter primers for performing the sequencing. This is because the very long primers might give a high background.

You may also wish to use "nested primers". These are primers that will bind slightly further into the PCR product than the primers used for PCR. This only needs to be a few bases. What this does is prevent any non-specific PCR products from taking part in the sequencing reaction and can, therefore, produce a much cleaner sequence. Customers are strongly advised to consider the use of nested primers.

Purification of PCR products

In order for direct sequencing of PCR templates to work well, it is critical that customers provide “cleaned” PCR products to remove unwanted PCR products (if present) and remove (or dilute) excess PCR primers and remaining dNTPs from the PCR reaction. Failure to do this is very likely to result in a messy sequence result. 

There are various options available for performing PCR product purification. What will work the best in each case depends on various factors.

EXO-SAP

  • If you have a very clean and strong single band, you might want to try EXO-SAP. This is a very simple method that removes residual primers and dNTPs (these are the things that cause problems for sequencing as noted above).
  • The benefit is that you don't lose any product (it just gets diluted a little).
  • However, this will not remove extraneous PCR products, so only works if you have a clean single band. It also will not remove primer dimers.

Column Purification

  • Any of the various column purification kits available will also work to remove primers and dNTPs.
    • Yield is likely to be less than EXO-SAP, but it might remove primer dimers since these may not "stick" to the column.
  • An alternative is to use a size cut-off membrane centrifugation approach to remove small molecules (dNTPs, primers, buffer components).

Gel Purification

  • Gel purification is also an option and needed if you do not have a single pure PCR product.
    • The trouble with this approach is that yield can be low and you run a real risk of introducing extra contaminants into the prep in the form of components from the agarose gel.
  •  It is vital to use a high quality agarose if doing gel purification.

Dilution of PCR Productions

  • A simple option is to dilute your PCR product in water with no further cleanup.
  • If you have a well optimised PCR reaction, the residual primers and dNTPs will be low and diluting may be all that is necessary.
  • For this approach to work, you need to have a single strong PCR product present.

Methods for Quantitating PCR Products

Please note that it is critical that quantitation of PCR products is not done with a spectrophotometer (such as a nanodrop). Rather, evaluation on an agarose gel provides a more accurate estimate.

Most of the absorbance can often be from free dNTPs and primers and not from the PCR product. Hence, customers often over-estimate the yield and consequently send far too little PCR product.

This is especially important if you clean up your product by EXO-SAP or simply diluting it. What is needed is a ball-park concentration for the PCR product – it does not need to be picogram accurate.  

For this reason, estimation on an agarose gel is best:

  1. Run a known volume of PCR product on a gel and also run a size marker (a couple of different loadings is good).
  2. Look at it and pick a marker band that is close in intensity and size to the PCR band.
  3. The marker product sheet will tell you that if you load Xµl, band Y will have Z nanograms of DNA in it.
  4. Hence, you can calculate your nanogram amount of PCR product and can work out the concentration.
  5. You can also use software to interpret the gel image and work this out for you, but that is not really needed once you are familiar with estimating by eye.

It is important that any estimation of yield is done right at the end of the cleanup process, in case the product is lost during purification.  

Do you need any help? Please get in touch and we’ll be happy to lend a hand.