Automated sequencing provides the nucleotide sequence of a DNA sample, which can be cloned into a vector or directly amplified by PCR.

There are many fields of application for these techniques. Among the most relevant are the following:

• Basic research in molecular biology: e.g. phylogenetic and evolutionary studies of organisms.
• Biomedicine: molecular characterisation of clinically relevant genes, detection of genetic susceptibility to certain types of cancer, detection of mutations in oncogenes, etc.
• Human genetics: diagnosis of hereditary diseases. Genetic counselling.
• Forensic medicine: identification of individuals, paternity tests.
• Microbiology: identification of bacterial spices.
• Other: conservation of endangered spices, identification of plant varieties or virology.

Services offered

800+ bp reads from good quality DNA templates in a single sequencing reaction. The length of the DNA sequence is directly dependent on the quality and accurate quantification of the template DNA.

• Sequencing of PCR products, plasmids, phages, etc. Sequences are obtained using user primers or standard primers. Sequencing reactions are performed at 50ºC, unless the user indicates a specific temperature for their primers.
• Users of the DNA sequencing service have the option of carrying out the marking of samples for sequencing in their laboratory with prior agreement with the laboratory staff. In this case, only capillary electrophoresis will be performed in the laboratory.
• Visualisation, editing and evaluation of the quality of the results and sending of the sequence files by e-mail or via FTP.
• Determination of the concentration of the samples if not carried out by the user.
• Results within a maximum of 5 days from the reception of the sample.

In the Guide for automatic DNA sequencing you will find information on how to carry out the DNA sequencing process.

Sample preparation and requirements for DNA sequencing

• Cloned inserts: Plasmids/Lambda/Phages: There are a number of minimum quality requirements for the DNA to be sequenced. Different protocols can be used to obtain the DNA, but all of them have to produce DNA free of RNA, phenol, salts, ethanol, EDTA, proteins, detergents, etc. Each user can choose his own DNA purification protocol, according to the needs of his sample and his laboratory.
• The template DNA can be lyophilised or in WATER.
• A quantity of 200 ng per reaction is required, at a minimum concentration of 50 ng/µl.
• Indicate the type of vector, insert size and sample concentration on the test request form. Ensure that the name of the reaction (sample + primer) is less than 8 characters. Do not use punctuation marks or Greek letters.
• If the sample concentration is not indicated, send at least 10 µl for each sequencing reaction.

• PCR products:
  • In the case of direct sequencing PCR products, these must be free of by-products, excess primers, dNTPs and salts. Clean-up protocols can be followed, either by filtration or column purification (Quiaquick, Quiagen, Microcon, Millipore, ExoSAP, Amersham USB). Ammonium acetate precipitation can also be used when the total concentration of primers in the PCR reaction does not exceed 5 pmol.
  • DNA can be lyophilised or in WATER.
  • An amount of 20 ng per 100 bases in the fragment length is required, at a minimum concentration of 10 ng/µl.
  • Indicate the fragment length and concentration.
  • If the concentration of the sample is not indicated, send at least 10 µl per sequencing reaction.
• Primers:
  • Sequencing can be performed with gene-specific primers or with standard primers. Universal primers for vectors such as M13FW, M13Rev, T3, T7 Promoter, T7 Terminator and SP6 are available from the service.
  • The specific primers must be in ultrapure water at a concentration of 5 µM. The hybridisation temperature with the template DNA must be indicated so that the Unit technicians can determine the temperature at which DNA labelling will be performed.
  • Submit a volume of at least 2 µl for each sample to be sequenced.

Sequences of the primers available in the laboratory

Sequencing of marked samples

Users of the DNA sequencing service have the option of carrying out the marking of samples for sequencing in their laboratory, subject to prior agreement with the laboratory staff. In this case, our laboratory will only carry out the electrophoresis of the plates and, if necessary, the previous step of eliminating the excess of labelled terminators.

Delivery of results

• The deadline for delivery of the sequencing result is 5 days from the date of receipt.
• The sequence data will be transferred electronically, both the text and the electrophoregrams.
• If you do not have the software to interpret the electropherogram files, there is a version of the Sequence Scanner programme that you can download from the ThermoFisher website or the Chromas programme that you can download from the Technelysium website.

Return of materials

Remaining moulds will be kept for a maximum of four weeks at the user's disposal and will be returned at the user's express request. After this period, they will be disposed of.

Failed sequences

The procedures used are designed to minimise variability and the frequency of failed sequences. However, failures can occur due to three types of causes:

• Characteristics of the samples, either of the mould or of the initiators.
• Insufficient information from the user.
• Operational error in the internal operation of the service.

Failed sequences are invoiced when the causes of error are estimated to be of type a or b. Those caused by type c errors are repeated. If the repetition fails again, it shall be assumed that it was not an operational error.

Causes of error

The most common causes of error associated with the starting DNA are as follows:

• For samples in cloning vectors, the bacterial strain where the template is propagated can affect its quality. Reliable results are obtained with DH1, DH5a, DH10B or XL1-blue. MV1190, HB101 and JM101 are not recommended.

• Insufficient amount of template DNA. It is recommended to quantify the DNA by electrophoresis, using dilutions and comparing with markers of known mass and check the concentration and impurities by absorbance at 260/280 nm.
• Quality of the template. Even if commercial kits have been used for its preparation, impurities that interfere with sequencing may remain. For PCR products the most common impurities are fragments that co-purify with the template such as primer-dimer, which contain binding sites for sequencing primers.
• Problems with template-specific primers. The sample may contain different binding sites, or the primer does not bind to the template or insufficient amount of primer or error in the Tm calculation, etc.