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BAC/PAC clones as DNA sequencing templates
Previously there has been no means to directly sequence DNA inserts in large clones such as BACs or PACs. Currently the typical procedure for sequencing large genomic regions includes generation of minimally overlapping BAC libraries, random subloning of the large DNA fragments into smaller vectors, shotgun sequencing from these subclones and finally organizing the accumulated raw sequence data into final sequence.
So called BAC end sequencing, i.e. single read extending from a vector-binding sequencing primer, is a widely used method for selecting minimally overlapping BAC clones for the primary clone set in large scale sequencing projects.
The protocol steps in short:1. BAC/PAC DNA preparation for transposition reaction
2. In vitro transposition reaction
3. DNA preparation for sequencing
4. DNA sequencing reactions
1. BAC/PAC DNA preparation for transposition reaction
Purify the BAC/PAC target DNA for the TGS II transposition reaction using a DNA purification kit that is recommendend for large clones such as BACs and PACs (e.g. QIAGEN Large-Construct Kit) or alternatively using the alkaline lysis method that has been modified for large DNA purification . Make sure that the target DNA is pure enough for high-efficiency transformation using electroporation.
2. In vitro transposition reaction
Perform the in vitro transposition reaction as described in the Instruction Manual of theTGS™ II Kit (pdf). Use maximum of 2 µg of the target DNA per single standard 20 µl transposition reaction.
Dilute the reaction mixture ten-fold in deionized water and use 1-10 µl directly to transform electrocompetent E. coli cells with a high transformation efficiency (>109 cfu/ µg pUC19). We recommend that you use the <i>E. coli</i> strain DH10B or another strain that is commonly used with large DNA clones.
A typical result is more than thousand insertion clones (i.e. target DNA clones that contain an Entranceposon insertion at random location) per single transformation. It is also possible to increase the yield of transformants by precipitating the transposition reaction mixture prior to the transformation step and using more DNA per transformation reaction.
3. DNA preparation for sequencing
Pick transformant colonies in 5-10 ml of LB (or TB, depending on the DNA purification method) supplemented with the proper antiobiotics. Grow o/n at 37°C.
Prepare minipreps using a method that yields high quality BAC DNA.
The following commercial kit has been tested to give good results:
- Qiagen Large Construct Kit (using smaller Tip 20 columns)
4. DNA sequencing reactions
Only a small fraction of the BAC DNA serves as the actual template for the sequencing reaction. The lower molar concentration of the sequencing primer binding sites has to be compensated by using large amounts of template DNA (up to 2 µg per reaction) and higher concentrations of sequencing primers SeqA or SeqB.
The following commercial kit has been tested to give good results:
- ABI PRISM® BigDye Terminator Cycle Sequencing Kit (PE Biosystems)
Modifications to standard protocol
- BAC DNA 500 ng
- SeqA or SeqB primer 10 pmol
- 5 % DMSO added to the reactions
- Reaction volume 20 µl
| Modifications to standard protocol | ||
|---|---|---|
| 98°C | 2 min | 45-65 cycles |
| 95°C | 20 s | |
| 53°C | 10 s | |
| 60°C | 3 min | |
| 72°C | 5 min | |
Literature
Kelley, J. M., Field, C. E., Craven M. B., Bocskai D., Kim U. J., Rounsley S. D. & Adams M. D. (1999). High throughput direct end sequencing of BAC clones. Nucleic Acids Res. 27, 1539-46.
