The procedure which I used in the Fish 543 class to clone microsatellites is based on the paper by Hamilton et al. (1999) ("Universal linker and ligation procedures for construction of genomic libraries enriched for microsatellites" [Biotechniques Vol. 27, No. 3, pp 500-507]). We used some shortcuts and modifications suggested by Paul Bentzen, Pam Jensen and Ingrid Spies, so those are detailed here.
My aim is to produce a step by step account, complete with results for each stage, in case I ever have to do it again.
1. Extract DNA from tissue sample using the ammonium acetate extraction method (>>ammonium acetate protocol)
2. Run genomic DNA extraction on gel (>>gel 1)
1% agarose (TBE), 100v for 40 minutes, stain with Ethidium Bromide (EtBr). Loading: 4ml DNA + 2ml loading buffer. 3ml HiLo ladder (L)
Result is a band of lots of DNA at around the 10,000 bp + area. This is what we want to see.
3. Restriction digest with various enzymes to attempt to find one (or a combination) which cuts the genome into suitable sized fragments. 3 enzymes tested, Hin f1, Rsa1, Hae3. Buffer used in each case is the one supplied with the enzyme. Incubation is at 37oC for 60 minutes.
Volume (ml) H2O 5.9 10X buffer 1 BSA 0.1 DNA 2 (guessed concentration at 0.5mg/ml, we want 1mg) Enzyme 1 (enzyme is at 10units/ml) 10ml total reaction volume.
4. Run results of digest on another gel (>>gel 2)
1% agarose (TBE), 100v for 40 minutes, stain with Ethidium Bromide (EtBr). Loading 4ml DNA + 2ml loading buffer. 3ml HiLo ladder
Results: Hin f1 and Hae3 produce strong bands of short fragments (around 100 bp). Rsa1 gives a more diffuse smear in all size ranges (the apparent gap at 500 bp is caused by shadowing with the loading buffer dye). There is some undigested DNA left behind at the 10,000 + level. The decision at this stage is to continue using Rsa1.
5. Restriction digest with Rsa1 and Nhe1 in a higher volume to produce a lot of the fragments. We also include Nhe1 as this is required for the use of the SNX linkers. These fragments would be cut during ligation into the vector. Incubate at 37oC overnight.
Volume (ml) H2O 9.5 10X buffer (NEB 2) 5 BSA 0.5 DNA 30 Rsa1 3 (enzyme is at 10units/ml) Nhe1 2 (enzyme is at 10units/ml) 50ml total reaction volume.
6. Run results of digest on another gel (>>gel 3)
1% agarose (TBE), 100v for 40 minutes, stain with Ethidium Bromide (EtBr). Loading 4ml DNA + 2ml loading buffer. 3ml HiLo ladder
Results: Same as before, Rsa1 gives a diffuse smear in all size ranges. The Hamilton et al protocol calls for the use of Mung bean exonuclease at this stage to make the products blunt ended. I don't need this as Rsa1 is a blunt cutter.
7. Clean up the DNA fragments using a QiaQuick DNA purification column (50ml final elution).
8. Dephosphorylation of DNA fragments. Incubate with calf intestinal phosphatase at 37oC for 2 hours.
Volume (ml) H2O 3 10X buffer (NEB 2) 6 DNA 50 (from previous step) CIP 1 (enzyme is at 10units/ml) 60ml total reaction volume.
9. Clean up the DNA fragments again using a QiaQuick DNA purification column (30ml final elution). By now we should have blunt ended, dephosphorylated, DNA fragments of various lengths.
10. Ligation of double stranded SNX linker to blunt ended DNA fragments. This is done in the presence of the restriction enzyme Xmn 1, which cuts any SNX linker dimers
Volume (ml) 10X buffer (NEB 2) 3 100X BSA 0.3 DNA 10 DS SNX linker (5mM) 11.7 rATP (10 mM) 3 Xmn1 1 (enzyme is at 10units/ml) T4 DNA ligase 1 (enzyme is at 10units/ml) 50ml total reaction volume.
Incubate in thermocycler, 30 cycles of 30 minutes @ 16oC, 10 minutes @ 37oC. Follow this with 20 minutes at 65oC, to kill the restriction enzyme and the ligase.
This should give us many fragments that have the SNX linker ligated to each end. These SNX linkers can then be used as priming sites for PCR as we know their sequence.
11. First PCR. Protocol uses Vent polymerase, we used TAQ, because it seems to be more trustworthy.
Volume (ml) DNA 2 H2O 30.7 10X PCR buffer 5 dNTP (8 mM) 5 SNX F primer (10mM) 4 MgCl2 (25 mM) 3 TAQ polymerase 0.3 50ml total reaction volume.
For the controls, the DNA was replaced with 2ml of: DS SNX linker, digested genomic DNA (result of step 9.), and water.
Thermocycler profile: 1X 94oC for 5 minutes, 40X 94oC for 45 s, 62oC for 1 minute, 72oC for 1 minute.
12. Run results of PCR on another gel (>>gel 4)
2% agarose (TBE), 100v for approx 40 minutes, stain with Ethidium Bromide (EtBr). Loading 4ml DNA + 2ml loading buffer. 3ml HiLo ladder
Result is a very faint band in the 0-500 bp range for the sample. A clear band at <50 for the DS SNX linker, and nothing for the other 2 controls. This is as expected, but the band is faint. This is an indication of low success at producing ligated fragments.
13. Now the fancy bit. Repeat enrichment with biotin labeled oligos and streptavidin beads........
a) Hybridisation of DNA + linker fragments to biotinylated repeat oligonucleotide (16 bp GACA repeat in this case).
Volume (ml) Linker ligated DNA (approx 100 ng) 10 Biotinylated repeat oligo (1mM) 2 SSC (20X + SDS) 30 (final conc = 6X SSC .05% SDS) SNX F linker (10mM) 3 (30 picomoles to block dimerisation of fragments) Water 55 Chill out wax to prevent evaporation - 100 ml total reaction volume.
Incubate in heat block, 15 minutes @ 98oC to denature, then 30 minutes @ 48oC for hybridisation.
This should give us many SNX ligated fragments hybridised to GACA repeats.
b) While the DNA is cooking, prepare solutions:
For bead prep: 10X PBS + 0.1% BSA 250 ml For washes: 2X SSC 500 ml 1X SSC 1000 ml 0.5X SSC 1000 ml Low TE 150 ml
c) (>>Bead hybridisation protocol.)
14. PCR of single stranded hybridisation products. We want to do this immediately after the enrichment step so that we don't lose too much fragile SS DNA. My first attempt at this was with Vent exo-polymerase, which gives a blunt ended product. That didn't work (>>gel 5), so the second attempt was with TAQ, even though this leaves an overhanging A.
Volume (ml) GACAn enriched DNA 5 H2O 11.35 10X PCR buffer 2.5 dNTP (8 mM) 2.5 SNX F primer (10mM) 2 MgCl2 (25 mM) 1.5 TAQ polymerase 0.15 25 ml total reaction volume.
For the controls, the DNA was replaced with 5ml of: DS SNX linker, digested genomic DNA (result of step 9.), and water. A reaction was also run with 10 ml of the enriched DNA)
Thermocycler profile: 1X 94oC for 5 minutes, 40X 94oC for 45 s, 62oC for 1 minute, 72oC for 1 minute. (?? I think, but can't find any notes on it).
15. Run results of PCR on another gel (>>gel 6)
2% agarose (TBE), 100v for approx 30 minutes, stain with SyBr. Loading 4ml DNA + 2ml loading buffer. 3ml HiLo ladder
Result is a clear band in the 200 - 600 bp range for the sample. Nothing for the water control, a clear band at <50 for the DS SNX linker, a smear for the genomic DNA digest, and a strong band for the 10 ml elution. The only problem with this gel is that I thought that I had loaded things in a different order to that which they came out!.
16. What we now have is an enriched set of fragments containing GACA repeats, with the SNX linker on each end. The TAQ process will have added overhanging A's, so these need to be removed. For this we use T4 DNA polymerase (Wang, Koop & Hood, ??? Vol. 17, No. 2 (1994)). To do this we add 1 unit of the polymerase to the remaining PCR product, incubate at 25oC (room temperature) for 20 minutes, then 70oC for 10 minutes to kill the enzymes.
17. The resulting blunt ended fragments are cleaned up in a QiaQuick column to remove remaining primers and enzyme residues.
18. Cloning of fragments into plasmid and transformation of E-coli was achieved by the use of the Zero blunt TOPO PCR cloning kit (Invitrogen). For a change, the instructions were followed to the letter. Plates were incubated overnight at 37oC. This produced a large number of fast growing colonies. During longer incubations, some slower growing colonies seemed to show up. It is possible that these have very short inserts and so the lethal gene in the plasmid is not fully de-activated. We don't want short inserts anyway, so these were ignored.
19. Colonies which had grown during the overnight incubation were transferred individually onto known locations on new plates and (simultaneously) used as source DNA for another PCR to increase the amount of the fragment. The system used is to use a clean pipette tip to lift the colony from the original plate, touch the location on the new plate and grind it around in the bottom of a microfuge tube to disrupt some cells and release the plasmids to act as source for the PCR. The rest of the PCR ingredients are mixed together and 20 ml is added to each of these tubes. The M13 primer is included in the plasmid sequence for the purpose of using it as a primer in this PCR.
Volume (ml) DNA from colony - H2O 12.7 10X PCR buffer 2 dNTP (8 mM) 2 M13 F primer (10mM) 1 M13 R primer (10mM) 1 MgCl2 (25 mM) 1.2 TAQ polymerase 0.1 20 ml total reaction volume.
Only control is a blank tube (with no bugs added) Thermocycler profile: 3X 94oC for 1 minutes 53oC for 30 s, 72oC for 1 minute, 17X 90oC for 30 s, 53oC for 30s, 72oC for 1 minute.
20. Run results of PCR on another gel (>>gel nick224)
2% agarose (TBE), 100v for approx 30 minutes, stain with SyBr. Loading 5ml DNA + 2ml loading buffer. 3ml HiLo ladder
Results are bands in the 200 - 600 bp range for most of the samples. Nothing for the control (C). There are a few small inserts (3, 7, 10, 13, 18, 23) and a couple of blanks (19, 22) but the other 16 have products of about the right size. [The "right size" is 300 - 500 base pairs. The ends of the plasmid plus the SNX linker on each end of the insert adds up to about 180 bases. To be useful, inserts should be >150 bp]
N.B. It might have been better to stain this gel with EtBr. The SyBr gives no information of the amount of DNA present, just that it is there. It turns out that the yields from this PCR were rather low first time.
21. QiaQuick clean the things again to remove primers etc.
22. Run results of PCR on another gel (>>gel 303a (transilluminator image)) (>>gel 303b (Fluorimager image))
The aim of this is to quantify the amount of DNA present so that we can use close to 200 ng in the cycle sequencing reaction. It helps if this gel is thin and is stained well. Visualisation can be done on the UV transilluminator or on the scanner. We should be able to use the ImageQuant software to do the quantification. I have had a lot of trouble with this and ended up resorting to guesswork.
2% agarose (TBE), 100v for approx 30 minutes, stain with EtBr. Loading 5ml DNA + 2ml loading buffer. 3ml HiLo ladder. 5ml and 10ml HiLo ladder (for contrast)
Results should be the same as above, without the smears. Quantify by comparison of the strength of the bands in the ladder (with known amounts of DNA) to the equivalent sized bands from the samples.
23. Cycle sequencing reaction. This follows the instructions on the cycle sequencing kit (manufactured by ??). The amount of DNA and water added depends on the concentration of the DNA calculated from the previous step. The primer used is one of the M13 primers, used in the previous PCR. For my first attempt, the DNA was very dilute so I used the maximum possible volume and no water.
Volume (ml) DNA 15.5 (200 ng) H2O 0 Mix A 2 Mix B 2 Primer M13 F 0.5 20 ml total reaction volume.
There are no direct controls. A control with known sequence (PGEM) is used to check the whole process.
Thermocycler profile: 25 cycles of 96oC for 30 seconds, 50oC for 15 s, 60oC for 1 minute.
24. Autoseq columns to remove primers and unreacted junk. One for each sample according to the instructions on the box!.
25. Sequence [Black Box]
The result is a DNA sequence (>>sequence 1)
The first (and last) 90 or so bases of this consist of the plasmid residue and the SNX linker. 70 or so bases of plasmid and 20 or so of linker (see sequence) The rest is the insert. If we are lucky this contains a number of GACA repeats, flanked by nice big bits of sequence which we can use to develop our primer sites for assaying microsatellite alleles in lots of shrimp.
