DNA Sanger Sequencing

Il Servizio fornisce la possibilità di compilare un'apposita richiesta direttamente dalla nostra pagina web. Per conseguire l'accesso alla nostra richiesta elettronica, ciascun cliente dovrà registrarsi alla pagina web di Cogentech contattando sales[@]cogentech.it

I primers forniti al servizio devono avere le seguenti caratteristiche:

• 18-20 bp
• un'ancora G/C al 3' e se possibile al 5'
• un contenuto in GC del 30-70%
• Tm: 48-65 °C
• non devono avere regioni che possono formare strutture secondarie o promuovere la formazione di dimeri
• l'appaiamento deve avvenire almeno 20-30 bp a monte della regione di interesse

È sempre consigliato, prima di sottoporre una richiesta di sequenziamento con primers universali e non, effettuare una ricerca per similarità tra primers di interesse e sequenza target (vettore e inserto) per essere sicuri che l'appaiamento avvenga in modo specifico.

I campioni possono essere consegnati in qualsiasi momento al Servizio che si trova nell' Edificio11 al secondo piano e possono essere lasciati nel frigo dedicato. I campioni consegnati oltre le 09.00 am, saranno di regola processati il giorno successivo.

I campioni devono essere inviati al seguente indirizzo:
DNA Sanger Sequencing Unit
Cogentech SB Srl,
Via Adamello 16
20139 Milan
I campioni che arriveranno dopo le 09:00 am saranno di regola processati il giorno successivo. Non è possibile effettuare consegna di campioni durante il weekend. Se si inviano I campioni il venerdì, assicurarsi che siano consegnati entro le 4:00 p.m.

I risultati sono di norma spediti entro 1-2 giorni lavorativi. L'utente riceverà una email che avvisa della spedizione dei risultati che possono essere scaricati dal sito online.

Se una reazione di sequenziamento fallisce, verrà ricaricata, qualora si siano escluse altre motivazioni note per il fallimento. Se la ricarica risultasse ancora fallita, il cliente verrà contatatto per risolvere in modo mirato il problema.

Di seguito alcuni programmi che è possibile scaricare gratuitamente:

• 4 Peaks (Mac)
• Finch Tv (Mac e Windows)
• Chromas (Windows)

Sanger sequencing is a chain terminator sequencing method developed in the Seventies by Frederick Sanger. In this method the DNA strand analyzed is primed by an oligonucleotide and serves as a template for extension by a DNA polymerase. Chain termination is achieved through the addition of the nucleotide analog 2',3'-dideoxynucleotides (ddNTPs) to the nucleotide mix in the reaction. When a dideoxynucleotide is incorporated at the 3' end of the growing chain, chain elongation is terminated selectively at A, C, G, or T because the ddNTP lacks a 3'-hydroxyl group.

We use Dye terminator sequencing, in which four differently fluorescently labeled ddNTPs allow detection of the four bases due to different emission spectra. After separation of the sequencing reaction on capillary gels, the differently labeled fragments are detected and the 4 nucleotides in the sequence are displayed as differently colored peaks in a chromatograph.

If you have many samples that need to be sequenced with the same primer, it is more convenient to provide the samples in a 96-well plate format. This way you can use the dedicated request form, which is quicker to fill in, we are quicker in pipetting and as a consequence the price/sample is reduced.

There are some templates/regions that are difficult to sequence when using a standard DNA sequencing protocol:

• GC rich regions (> 60% GC)
• Di- and Tri-nucleotide repeats
• Hairpin structures (e.g. si/shRNA inserts and pDONR/pDEST vector series from Invitrogen)
• A/T homopolymers (e.g. polyA resulting from oligodT primed amplification of mRNAs).

The Facility has tested and optimized alternative chemistries to overcome most of these problems. Please let the facility know if you anticipate that your templates might be “difficult”.

For many cases single strand sequencing gives you enough information. In specific cases where you need to be a 100% sure about the result, a more accurate result can be obtained by sequencing both strands of the DNA. This double strand strategy is required for variant detections (e.g. SNP or mutations) both at diagnostic and at a research level. The double strand sequencing strategy is also used for publication grade data or for specific projects (e.g. primer walking). The DNA Sanger Sequencing Staff will help with primer design and with the set up of the PCR conditions. In addition, they will do the data assembly and analysis.

When you want to sequence an insert that is more than 1.2 Kb long, it is necessary to use the primer walking strategy. Starting by sequencing with primers that flank the insert, new primers are designed at the end of the sequence that is obtained until the entire sequence of the insert is covered. If the sequence is known, the primers can by designed a priori and are spaced every 500-600 bp. The DNA Sanger Sequencing Staff will help you in primer design and in data alignment/editing if needed.

Resequencing is the sequencing of part of an individual's genome in order to detect sequence differences between the individual and the reference genome. Usually it involves sequencing of exons, splice junctions and UTRs of a specific candidate disease gene using the double strand sequencing approach. The DNA Sanger Sequencing Staff will help with primer design, set up of PCR conditions and do the data assembly and analysis.

Fragment analysis is a general term used to describe experiments, which rely on the detection of changes in the length of a specific DNA sequence to indicate the presence or absence of a genetic marker. For this purpose the genomic region of interest is amplified using fluorescently labeled primers and the generated PCR fragments are resolved through a capillary sequencer. A resolution of size difference of up to 1 bp can be achieved with this technique. Fragment Analysis has a wide array of applications such as Microsatellite Analysis and Cell line verification as further explained below.

Microsatellites are di-, tri-, or tetra nucleotide tandem repeats in DNA sequences. The number of repeats is variable among different persons and within the alleles of an individual. If you flank a microsatellite with fluorescent PCR primers and amplify it, you will have a pair of fluorescent allelic products that will vary in size according to their repeat length. For Microsatellite studies a prior meeting with the Staff is required for primer design and experimental set-up.

For human cell line the GenePrint® 10 System by Promega is used. This kit allows co-amplification and three-color detection of ten human loci (STR regions). These loci collectively provide a genetic profile with a random match probability of 1 in 2.92 × 109. An internal lane standard (ILS) and allelic ladder are provided for sizing and genotyping of amplified fragments, and a control DNA is supplied as a positive control. An optimized protocol is supplied by the Staff to help you using the Promega kit. The Service offers, in addition to the run of the reactions, also the comparison between the results and specific DataBase informations, previously provided by the user.

Sanger Sequencing Tutorial:

• Sanger Sequencing of DNA (Part I)
• Sanger Sequencing of DNA (Part II)
• Isolating Plasmid DNA [great 10 min video on plasmids and miniprep (1^st year students). Start at 2.52 minutes if you want to go directly to the miniprep (uses Qiagen kit)].