FESTIVE CLOSURE

Festive Closure

The service will close down over the festive period. We aim to process any sequencing samples arriving before or on the first post of Friday 22nd of December. Any sequencing samples arriving after that will be stored and processed on our return (3rd January). For other sample types please contact us.

Services | Fragment Analysis

Fragment Analysis, a type of genetic marker analysis, is a technique that facilitates identifying the presence or absence of a DNA sequence that is linked to an allele of interest. It does not provide the sequence information of a particular gene; instead changes in the length of specific DNA sequences provide key information to distinguish changes in markers. Often, analyses such as these are used to examine inheritance patterns of alleles of interest in families (for instance, those associated with a disease) and even populations – including humans, animals and plants.

We provide a range of fragment analysis options to customers and are happy to assist customers with their data analysis needs as well. We encourage you to review the information below prior to submission so that your reaction has the potential to yield the most robust information for your studies.

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Materials Required

All our processing and handling of fragment analysis samples is undertaken in 96-well format so we require customers to submit samples in a 96-well plate (the exception is if we undertake initial optimisation work where smaller numbers of samples are diluted to varying amounts). Please see below (under "special considerations") for information about types of 96 well plate you can send. Your fluorescently labeled PCR products do not need to be cleaned up in most cases, but you should take steps to optimise your reactions (please see below under "special considerations"). Please refer to the ‘Fluorescent Dye’ section below if you need help deciding on dyes to use.

Once we receive your samples, we will process them in the following manner:

  • Dilute these into an appropriate size marker mix
  • Run them on our genetic analyser
  • Provide the data to you

Fluorescent Dyes

There are several dyes (indicated below) that are supported by Applied Biosystems. As some ABI supported dyes are proprietary, as well as expensive, we have suggested alternative dyes in the table below which are available from various manufacturers (e.g. Eurofins MWG) that customers may wish to consider. Customers should note that several of the suggested dyes are not officially supported by Applied Biosystems on our instruments and so ABI will not offer technical support. 

Dye Colour Absorbance/Emission Alternative Dyes Description Considerations
6-FAM Blue 495 nm/
520 nm
  Fluorescein, derivatized as NHS ester via a carboxyl at position 6. ABI-supported dye.
HEX Green 535 nm/
555 nm
Alternative to VIC when used with LIZ size standard Hexachlorofluorescein, NHS ester. Can only be used on 5' end of oligo. 

ABI-supported dye when used with ROX size marker.

Please note that this is not an ABI-supported dye when used instead of VIC (which uses a LIZ size marker). If HEX is used instead of VIC, the spectral calibration on the instrument takes away the amount of signal that it would expect to see from VIC, but this does not remove all the HEX signal from the NED (or TAMRA) channel and so you see peaks corresponding to NED (or TAMRA) under the HEX peaks. There is nothing we can do about this and so customers who choose not to use the recommended dyes will have to take account of this.

VIC Green 538 nm/
554 nm
  ABI proprietary "green".  

ABI-supported dye.

This is the supported and recommended green channel dye when using the 5-dye setup (LIZ size marker). This is used because it has a tighter peak profile than HEX and this helps to prevent fluorescence cross-talk with NED (or TAMRA).

NED Yellow 553 nm/
575 nm
TAMRA or ATTO 550 (when used with either LIZ or ROX size standard) ABI proprietary "yellow". ABI-supported dye.
TAMRA Yellow 559 nm/
583 nm
Can be used with either LIZ or ROX size standard, in lieu of NED Carboxy tetramethyl rhodamine, avail. as NHS ester, or direct linked.  ABI-supported dye.
ATTO 550 Yellow 554 nm/
576 nm
Can be used with either LIZ or ROX size standard, in lieu of NED ATTO 550 is a novel fluorescent label produced by ATTO-TEC GmbH related to the dyes Rhodamine 6G and Rhodamine B.

NOT an ABI-supported dye.

ATTO 550 does not exbhibit bleed-through when used in conjunction with HEX.  

PET Red 558 nm/
595 nm
ATTO 565  (when used with LIZ size standard)   ABI-supported dye.
ATTO 565 Red 563 nm/
592 nm
Can be used with  LIZ size standard, in lieu of PET ATTO 565 is a novel fluorescent label produced by ATTO-TEC GmbH belonging to the class of Rhodamine dyes. NOT an ABI-supported dye.

Customers should also be aware that not all dyes have equal fluorescent intensity, especially when not excited at the Amax. On our instruments, customers will find that the following intensity trend is seen:

Blue > Green > Yellow > Red

This means that the same molar amount of a fragment will appear brighter when labeled with a blue dye than when labeled with a red dye. Please take this into consideration when deciding on dilutions of material.

Size Markers

We have a number of size markers that we can use, which cover the most popular dye combinations and fragment sizes:

HOW THE DYES CAN BE USED IN CONJUNCTION WITH ONE ANOTHER DYE SET 
Blue Green Yellow Red Orange  
6-FAM HEX NED ROX 500 - ABI Dye Set D, DS-31
6-FAM HEX NED ROX 400HD -  
6-FAM VIC NED PET LIZ 500 ABI Dye Set G5, DS-33

 

Dye Combinations we utilize are shown in the table below:

Marker Name Colour Size Range Absorbance/Emission Description
ROX 500 Red 50 to 500 base pairs 588 nm / 608 nm Carboxy X-rhodamine, NHS ester.
ROX 400HD Red 50 to 400 base pairs 588 nm / 608 nm Carboxy X-rhodamine, NHS ester. This marker has a "High Density" of peaks and so should provide an even greater accuracy than the standard "500" markers. 
LIZ 500 Orange 50 to 500 base pairs 638 nm / 655 nm ABI proprietary "orange".

 

Please note that only the dye combinations shown can be used (e.g. you cannot use a VIC-labeled fragment with a ROX size standard).

Should you still have questions upon revewing the information above, please feel free to contact us

Special Considerations

PCR Reactions

Due to the individual conditions required for optimal PCR of fluorescently labeled fragments (especially if multiplexing is being used), we are unable to perform your PCR reactions for you. We are also unable to tell you what dilution of your products will "work" for you. There is no simple correlation between a dilution ratio and how your fragments will appear on the run. Our experience does, however, tell us that it is almost always better to perform some form of dilution of the PCR reactions prior to mixing with the size marker and running on the analyser. This may only be 10-20x or it may be 200-400x. The range is quite large.

To assist with optimisation of dilutions, we are more than happy to run a test plate for you. We will take a selection (e.g. 8) of your samples and run these at serial two fold dilution, usually starting at 1:10. This means you will get back 48 results with dilutions ranging from 1:10 to 1:320. We can provide an "overview" of how these results look, but, for obvious reasons, we are not able to tell you exactly which dilution is best for you. The test plates are charged at the same rate as normal plates even though it is considerably more work for us. Please contact us should you wish to discuss this further.

Analysis of Results

We will undertake limited quality control analysis of all runs to include checking that the marker bands are present and well defined and that sample injection has occurred in all capillaries. This is included in the basic cost of the work.

In addition, we can offer to undertake a limited analysis of the results for you. This is limited to analysing the samples to the stage of generating a tabulated output of fragment sizes for each capillary. For this work we charge an additional £10 per plate (48 or 96 samples). The tabulated output will be included with your .fsa files if you pay for this additional analysis. We are afraid that we cannot offer to undertake a full analysis of your results and produce allele information.

Please note that customers can make use of free software available from Applied Biosystems called PeakScanner for their analysis and display of the data.

96-well Plate Recommendations

You are free to send us your samples in any standard 96 well plate of your choice. However, please note that it must be a standard 96 well plate containing 12 columns of 8 wells. Please ensure it is clear which corner is the "A1" well and note that we always work in columns of 8 and NOT rows of 12! When sending 48 or less samples, also please note that our 48 capillary machine is set up to take samples from the odd columns (1,3,5,7,9,11) and so your samples should be put into those columns.

When sealing the top of the plate, there are various methods you can use such as heat sealing, adhesive film or a silicon sealing mat. We recommend only using a silicon mat as in our experience adhesive film does not stick well and can result in leakage out of wells. Also, heat sealing is difficult for us to remove in order to access the samples. For customers wishing to use silicon mats, we recommend "Axymats" from Axygen. The one we use has product code "AM-96-PCR-RD". We will return mats in batches to customers for re-use. Whichever sealing method you choose, please ensure it is effective. We don't like having to tell customers that their sealing did not work and all the wells have been mixed/emptied of contents....

For those customers who only require a small number of plates and do not wish to buy a whole box, please contact us and we will supply you with the number of plates you need at £10 per plate (including postage & packing) along with a protective base plate and a silicon sealing mat to keep your samples in the wells when you return the plate together with your samples.  

Organization of Samples on Plate

  • You may send either 48 or 96 samples per plate. 
  • Please note that even if you send less than 48 or 96 samples, we will need to charge you for the full 48 or 96 samples. 
  • Please organize the samples into the wells in the format given below. 
    • Failure to do this will result in the samples not being processed. 
  • Please ensure that you know where each of your samples is on the plate, since result files will be numbered as illustrated below (i.e. consecutively from 1...48 or 1...96).

Organising 48 and 96 Well Plates

  • Please note the cut corner on the plate (upper right) and the reference numbers/letters that will also appear on the microtitre plate. 
  • Please ensure that your samples are placed exactly as per the illustrations above or you will not receive your samples back in the correct order.  

Once all your samples are ready, seal the plate and send to us. 

Pricing

Fragment Analysis Alone

  • 48-sample Plate: £48
  • 96-sample Plate: £96

Fragment Analysis + Data Analysis

  • 48-sample Plate: £58
  • 96-sample Plate: £106

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

Upon receipt of 96-well plates, samples are routinely processed, results analysed, and returned within 24 hours.

Shipping

Shipping Details

  • Labels
    • When sending multiple plates, please ensure that they are clearly labeled so that we can identify them.
  • Packing Materials
    • 96-well Plates
      • When sealing the top of the plate, there are various methods you can use such as heat sealing, adhesive film or a silicon sealing mat.
        • We recommend only a silicon mat
          • For customers wishing to use silicon mats, we recommend "Axymats" from Axygen.
            • The one we use has product code "AM-96-PCR-RD".
          • We will return the mat to you for you to re-use.
        • We have found that adhesive film does not stick well and can result in leakage out of wells and heat sealing deforms the tops of the plates and causes us lots of problems when it comes to performing the sequencing.

 

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

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