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Fragment analysis

  • Analysis of molecular markers:
    • Qualitative: determination of the size of DNA fragments by means of capilar electrophoresis and fluorescence detection. Analysis of STRs, VNTRs, AFLPs, etc.
    • Semiquantitative: analysis of MLPAs (Múltiplex Ligation-dependent Probe Amplification).
  • Molecular identification of individuals: obtaining of genetic profile of human DNA samples with different origins (medical examiners, tissue banks, etc).
  • Other analyses: SNPs, SNaPshot, etc.

Many DNA fragments simultaneously amplified can be visualised by means of genotyping techniques and thus obtain banding patterns or fingerprints which can be used in many applications, such as the identification of specific DNA samples. 

Preparation of samples for fragment analysis

  • To send at least 10 μl of the PCR reaction in which the marking has been carried out or that of the corresponding dilution. 
  • To indicate the expected size range for the fragments. 
  • To indicate the fluorophores used in the marking of the primers.
Fluorophores which can be used for the fragment marking
Fluorophor Colour Max A (nm) Max E (nm)
6-FAM ™ Blue 494 522
JOE ™ Green 528 554
HEX ™ Green 535 553
VIC ® Green 538 554
NED ™ Yellow 546 575
Cy3C Yellow 550 550
PET ® Red 558 595
ROX ™ Red 587 607
LIZ ® Orange 638 655
  • Fluorophores combinations compatible with the existing equipments in the service are the following::
Emission Blue Green Yellow Red Orange Application
D 6-FAM ™ HEX ™ NED ™
Cy3C
ROX ™   Genotyping and microsatellites
F 6-FAM ™ JOE ™ NED ™ ROX ™   Genetic profiles
G5 6-FAM ™ VIC ® NED ™ PET ® LIZ ® Gentoyping and microsatellites
E5 dR110 dR6G dTAMRA ™ dROX ™ LIZ ® SNaPshot
  • Size markers available on the service are:
    • ROX-500 for fragments from up to 500 pb.
    • ROX-400 HD for fragments from up to 500 pb..
    • ROX-2500 for fragments from up to 2500 pb.
    • LIZ-500 for fragments from up to 500 pb. 

The markers with the ROX fluorophore can be used with the D and F filters and the marker with the LIZ fluorophore with the G5 and E5 filters.

Results delivery

  • The deadline for the analysis result of fragments is 5 days after the receipt day.
  • The results can be electronically transferred.
  • The software can interpret the results in GeneMapper by Applied Biosystems which is installed in a computer of the Central Service for Experimental Research or Peak Scanner, available on Applied Biosystems webpage.

Return of materials

The remaining PCR products will be preserved a maximum of four weeks to availability of the user and will be returned upon express request of the user. They will be deleted after this period. 

Molecular markers projects

  • Determination and analysis of genetic variation of molecular markers applied to the study of determined species.

Development of specific projects proposed by the user which require the determination and analysis of genetic variation by means of the use of molecular markers. The evaluation of the markers will be previously carried out (microsatellites, nuclear and organellar genes, etc.) and more suitable methods to the project (sequencing, AFLPs, VNTRs, etc.). Regarding the variation study, methods related to DNA or RNA extraction of the different celullar and tissue types which are required, electrophoresis, PCR amplification techniques, cloning, preparation of sequencing reactions, design of primers, editing and correction of obtained sequences, alignment and phylogenetic analysis will be applicated.
Applications will be carried out by the online platform LIMS. In the application you should make a brief description of the project stating necessarily the following: study institution, type of susceptible sample of the genetic analysis and conservation status of the sample (fresh, alcohol, acetone, etc.), type of required molecular marker (if known) and/or specific problem to be solved, as well as any other information that the user considers relevant for carrying out the project.

Identification of GMOs

  • Characterisation and identification by means of PCR techniques of Genetically-Modified Organisms.

Identification of transgenic organisms by means of PCR techniques:

  • Qualitative analysis: determination of transgenic material in any type of sample.
  • Quantitative analysis: determination of the transgenic material percentage contained in the sample.

The identification can be carried out from DNA obtained by the user or can be made in our DNA extraction laboratory on the basis of tissue samples.

Ribotyping

  • Automatic characterisation and identification of bacteria by means of RFLPs studies.

The identification and characterisation of bacteria and other microorganisms play a very important role for a great number of industries such as the food and agriculture field and health fields. In many cases, the use of a characterisation system such as RiboPrinter®, has allowed the analysis and resolution of problems which can be very tedious and expensive by using other more conventional methods.
Bacterial contamination in processed food products may originate from the ingredients, environment or personnel involved in the transformation process of the products. Ribotyping allows the establishment of unequivocal relationships between the isolated bacterial recovered from any of these sources and the processed product.
Another interesting application field of the ribotyping technique is the epidemiology. The system's ability to identify quickly the sources of infection opens a great potential for the development of more effective preventive methods in the hospital field.
In addition, the characterisation system RiboPrinter® has a wide application in the field of basic research.

Preparation and shipment of samples

  1. The bacteria should be plated in the appropriate medium. We must make a culture that allows to obtain isolated colonies. The optimal incubation conditions depend on the type of microorganism, but in any case we will work with young cultures.
  2. Since ribotyping procedure is slightly different for Gram + and Gram - bacteria, you should have this information for the sample you want to analyse. Also you must indicate if it is a lactic acid microorganism.
  3. If you have information about the identification of the microorganism you want to ribotype by means of other tests, you should indicate it in the application form of analysis.
  4. Indicate the enzyme with which you want to perform the DNA restriction. The enzymes available in the service are EcoRI, PstI and PvuII. If you want to use other enzymes or double digestions, check with the section staff. 
  5. Because of the characteristics of the test, the samples will be analysed for a maximum of 24 hours after receipt. For this reason, get in touch with lab personnel prior to shipment.
  6. The samples must be shipped to the service address, indicated in the contact page.
 
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