Primer walking of constructs (Sequencing of cloned inserts) is an effective strategy to sequence the entire cloned DNA insert. BDRC offers to read upto 5kb insert, bidirectionally using this technique. Initially, sequence data is obtained using a standard primer which hybridizes to the vector sequence upstream of the insert DNA. Based on this initial data a custom oligonucleotide is designed, synthesized and used to prime a second sequencing reaction. The data obtained from the second reaction overlaps with initial data and extends the sequence further downstream of the initial primer. By repeated cycles of custom oligonucleotide synthesis and DNA sequencing the cloned insert is sequenced completely in one direction. For bi-directional sequencing, a second standard primer is used which binds the vector in the other direction, the above process is then repeated and the complete sequence read for both strands. This service includes DNA extraction, purification, Primer designing, synthesis, desalting, sequencing and assembling of the final sequence. Information & sequence report will be provided to the customer at each stage of the sequencing by primer walking. In case of unforeseen problems due to GC/AT rich regions of template DNA, the customer will be informed for suggestions and billed according to the number of reaction/primer synthesized.
How To Process Sample & Primers
Send DNA samples along with primers. Please note that DNA and primer should be of high quality refer note no. 1 (Sample Processing) & note no. 2 (Primer Processing). The sample should be sent along with the Application for Automated DNA Sequencing Service. Agarose gel analysis and / or spectrophotometric check of template DNA will be done for all samples received free of cost. Sequencing will be performed, only if the quantity and quality of DNA are found satisfactory; if found not satisfactory the same would be informed.
Note no. 1: Sample Processing
BDRC recommends the use of protocol optimised by ABI for DNA preparation. Template DNA supplied should satisfy the following criteria :
1. Salt free
2. RNA free and
3. EDTA free
Template DNA must be finally resuspended in deionised water only. Quantity (and concentration) of template DNA required for SPA services:
ssDNA 50-100 500-600
dsDNA 250-500 600-800
PCR product 50-100 500-600
Note no. 2: Primer processing
Criteria for primers used in sequencing:
Primer sequence selection, synthesis and purification have significant effect on the quality of sequencing data. We recommend the following criteria :
1. High Purity (either HPLC or Desalted) Primers.
2. Primers 18-24 bases long usually give good specificity. Long Primer (>35mer) is not preferred
3. Preferably 50 to 55% GC content.
4. No mismatch is present and no alternative hybridization sites are present in the template.
5. RAPD primers (10 mer) cannot be used for cycle sequencing reaction due to low annealing temperature.
6. For cycle sequencing, primer with melting temperature between 50 to 60° C is recommended. Avoid low melting temperature (40 to 45°C.) Melting temperature (Tm) is calculated as 4 (G+C) + 2 (A+T).No secondary structure particularly at the 3′ end is present. (ng/μl)
Quantity (and concentration) of primers to be provided for sequencing :
Concentration: At least 10 picomoles/μl
Quantity: Minimum 20-30 picomoles
How To Send Samples
DNA must be transported in dry ice / coolant packs at sufficiently low temperature to prevent degradation. DNA in solution should not be sent at room temperature. DNA (especially PCR product) can be sent as lyophilised sample. Care should be taken to see that lyophilised DNA is not excessively dry as this leads to problem in reconstitution. Customers are requested to send the Application Form and purchase order duly filled in along with the email and contact number for sending samples for Sequence Analysis.
Result of DNA Sequencing Services, low cost genome sequencing, Next Generation DNA Sequencing Analysis
BDRC will provide data by email -of each sample in the form of
(1)sequence (2) Electropherogram
• SPA services: Approximately 1 week.
• Primer walking services: Approximately 2 weeks for every 1 kb.