Hybridization / Single Base Extension
Single base-extensions (SBE) provide the basis for genotyping, haplotyping, and exon-typing with polonies (the latter two are essentially just multiplexed versions of the first). Multi-base sequencing (at least in its current implementation) uses a different set of nucleotides and a different protocol. SBEs can essentially be performed in two steps. A related protocol is hybridization with a labeled probe oligonucleotide. The protocol for this is essentially identical (the probe takes the place of the sequencing primer in the below protocol, and you can skip the extension step).
At the conclusion of the PCR reaction, amplicons are present in double-stranded form, with one strand anchored to the gel via the acrydite modification. Denaturation will permit us to remove the un-anchored strand, such that the remaining strand can be free to serve as a template for hybridization and sequencing reactions.
1. Prepare the following 70% formamide denaturation solution directly in a plastic Coplin jar (we generally keep a dedicated Coplin jar for use with formamide only)
2 mL 20x SSC
28 mL Formamide
10 mL dH20
Mix by stirring with one of the pipets.
2. Heat (without lid) to 70'C in a microwave (use a thermometer to check). Typically only 30 seconds or so is required to heat formamide to this temperature. Be careful not to boil the formamide over.
3. Deposit slides into the heated formamide solution, and incubate the Coplin jar for 15 minutes with shaking in a 70 degree incubator.
4. Remove jar from incubator and transfer slides to a seperate plastic Coplin jar.
5. Wash slides 1 x 3' in dH20 on shaker.
6. Wash slides 2 x 4' in Wash 1E on shaker.
In this step, a sequencing primer (or labeled probe) is annealed to a specific location within the now-single-stranded PCR. Although it is possible to use the unmodified amplification primer itself as the sequencing primer, it is generally advisable to use a sequencing primer that is internal to the PCR product (largely to avoid incidental sequencing of spurious PCR products, primer-dimer, etc.)
1. Prepare annealing mix:
1194 uL 6x SSPE w. Triton X-100
6 uL Sequencing primer (100 uM)
We usually prepare annealing mix fresh, but have found that it seems to last alright at room tempearture. Storing at -20 C is possible but can result in precipitation of salts out of solution.
2. Remove slides from Wash 1E-filled Coplin jar and dry edges with ChemWipe without touching the gel.
3. Apply blue FrameSeal chamber base to the slides.
4. Use blunt end of tweezers to seal down chamber, remove plastic surface covering sticky area.
5. Apply 125 uL of annealing mix to the center of each gel.
6. Cover chamber with plastic seal. Liquid should spread evenly over surface of gel.
7. Use blunt end of tweezers to seal down chamber and cover.
8. Place slides in thermal cycler (with labels facing out)
9. Run annealing program:
94'C for 6 minutes.
10. After the 56'C step has run for 15 to 20 minutes, remove slides individually from slide-block, and QUICKLY pull of the FrameSeal chambers and dunk the slide into a Coplin jar filled with Wash 1E. The goal here is to dilute away excess sequencing primer before the temperature can drop (to limit the amount of non-specific binding).
11. Wash slides 2 x 4' with shaking in Wash 1E.
12. If this is a labeled hybridization probe, proceed to scanning. If not, proceed to single base extension.
Obviously the selection of a specific Cy-label and dNTP combinations will depend on exactly what you are trying to sequence in a specific experiment. Cy-labeled dNTPs are NOT terminators, and experiments should be designed accordingly. More than one labeled or unlabeled base can be included in an extension reaction. A typical genotyping experiment, for example, might include 2 dNTPs (one Cy3 labeled and one Cy5 labeled). Note that in our hands, the signal-to-noise ratios with Cy5-labeled bases is considerably better than with Cy3. So another option is to extend with a single Cy5-labeled base, scan the gel, and then perform a second extension reaction with the other base. The genotypes can be determined by image processing after the fact. Even when we are trying to probe, rather than sequence over a SNP, we generally avoid using Cy-labeled hybridization probes and instead perform an SBE over a known base (just because labeled primers are so expensive).
1. Prepare the following extension mix at room temperature. One needs ~45 uL of extension mix per slide. The formula for 100 uL of extension mix is as follows:
1 uL Klenow (50 U / uL)
1 uL SSB
0.5 uL Cy5-dNTP or Cy3-dNTP (100 uM) [this is more than enough base; lower concentrations can even give better signal-to-noise]
98.5 uL 1x Klenow Buffer
2. Equilibrate slides by replacing buffer in Coplin jar with 1x Klenow Buffer for 2 to 3 minutes.
3. Remove slides and dry edges with Chemwipe without touching gel. This keeps the mix from being diluted or running off the gel.
4. Add 45 uL of extension mix to each slide.
5. Allow reaction to go for 2 minutes (tilt slides around a bit to get extension mix to cover the surface of the gel)
6. Wash slides 2 x 4' with shaking in Wash 1E.
7. Slides are ready for scanning.
To date, we only have experience with Perkin-Elmer and Axon instruments. We encourage you to take advantage of whatever equipment you have access to, and would be happy to send you test-slides (e.g. gels that are already amplified and labeled) if you wish to test the suitability of your scanning equipment.
Gels can be scanned dry or wet (the latter under a cover-slip). If you are planning to reuse the amplified gels (e.g. to denature and reanneal for a second round of sequencing, for example), we suggest that the slides be scanned wet so that they do not dry out. One important hint for keeping the gels stable over repeated use is to always dip the slides in liquid (e.g. Wash 1E) immedietely before removing the coverslip. We have found that gel damage over repeated uses of a given slide is primarily a consequence of removing the coverslip from a dry-gel after scanning.
We have generally found good signal-to-noise ratios with modest power and PMT settings (i.e. 50:1 signal-to-noise with Cy5 dye SBE scanned at 80/80). Also note that the focus can often differ from that which is required for scanning microarrays, so this is an important parameter to play around with.