This amplification is designed to (1) select fragments with linker on both sides of the insert DNA and to (2) generate more copies of these molecules prior to hybridization.

NOTE : Too much PCR at this step is very bad. We have evidence for recombination at the CA repeats which creates chimeric fragments. Some PCR at this step does seem to help get larger fragments with linkers on both ends and helps yields, but only amplify a few cycles (10 cycles seems to be enough).

We set up:

Tilapia (FK and FT)

Quantify spin cleaned DNA and used 50 ng in PCR. Used plenty of Dynazyme EXT enzyme and longer extension times to ensure no partially amplified fragments.

10x buffer 10
dNTP (10 mM each) 2
MgCl2 3
UF Primer A (10uM) 5
DNA (50 ng) 4
H2O 73
Dynazyme EXT 2.5
total 100

PCR 10 cycles of :
94 C 30 s
68 C 1 min
72 C 2 min 30 s

Assay PCR amplification (5-15 ul) on a gel to check product size and quantify. Spin clean on Millipore spin columns to remove primer molecules and unused dNTPs as well as to concentrate sample.

Spin clean 50 ul and then quantify
50 ul + 250 ul TE/10 on Millipore Ultrafree MC column Spin 5 min
Add 300 ul TE/10 Spin 5 min
Add 300 ul TE/10 Spin 5 min

Remove to clean tube and quantify

VI. Microsatellite library hybridization/enrichment and capture using a biotinylated msat probe and Dynal beads.

This procedure involves (a) denaturation of the linker-ligated, PCR amplified, genomic fragments; (b) hybridization of fragments to the biotinylated probe (5'-Biotin-ATAGAATAT[CA]16); (c) capture of the probe:target fragments with Dynal streptavidin beads; washing of non-specifically bound material and, hopefully (d) elution of the (GT)n:(CA)n - containing fragments. Note. We had initial discussions about whether to design our biotin probe labeled on the 5' or 3' end. We chose labeling on the 5' end so that the probe could also be used as a primer. However, since oligos are produced from the 3' end, our first batch of probe turned out to be poorly labeled. Subsequently, we ordered an HPLC purified probe which ensures the probe is full length (the other option is to label the 3' end).

Generally, the Tm or the temperature of dis-association (melting) of probe to target DNA increases 16.6 degrees C for each ten-fold increase in monovalent cations between .01 and .40 M NaCl. Tm can be calculated as 81.5 + 16.6(log[Na+]) + 41(#G + #C)/N - (500/N) and for our probe (assuming a [Na+] of .994; see below) is 85.5 degrees C. The maximum rate of hybridization occurs at 20-25 degrees C below Tm for DNA-DNA hybrids and 10-15 degrees C below Tm for DNA-RNA hybrids. The optimum hybridization rate occurs in a buffer of 1.5 M Na+. Hybridization rate is increased for repetitive sequences and is directly proportional to duplex length. It follows that to increase the stringency of a hybridization/wash, one can increase the temperature given the same salt concentration OR decrease the salt concentration given the same temperature. Hybridizations will be carried out in a 6X SSC salt solution (.9 M sodium chloride, .09 M sodium citrate) with 0.1% SDS [a sodium concentration of .994]. The biotin labeled duplex is then bound by streptavidin coated magnetic beads. Streptavidin is a protein with an approximate molecular weight of 66 kDa consisiting of four identical subunits, each having the same high binding affinity for biotin; it has the same biotin binding affinity as avidin, but less non-specific binding. The subsequent binding of biotin to the Dynal streptavidin beads is best accomplished (for fragments up to 1 kb) at 25 degrees C for 15 minutes in a 1.0 M salt buffer or (for fragments above 1 kb) at 43 degrees C for 60 minutes in a 1.0 M salt buffer. Binding capacity of the beads is fragment length dependent (twice as many copies of a 500 bp DNA fragment bind to the beads as a 1000 bp DNA fragment; Dynal says this may be caused by steric hindrance). Since free biotinylated oligonucleotides (not used during the hybridization step) bind to the beads much more rapidly than longer DNA fragments, it is important to ensure that the hybridization solution does not contain an excess of these fragments.

A. Mix double-stranded, size-selected, linker-ligated, PCR-amplified genomic fragments (100 - 400 ng) with the microsatellite probe (10-12 pmoles) and an excess of Sau3AI primer (primer B, 250 pmoles to prevent concatenation of the single strands) in 6X SSC plus 0.1% SDS. It would be good to assemble all ingredients except the genomic fragments - heating them [the genomic fragments] to 95 degrees C for 5 minutes to denature the strands before adding them to the mix. Preheat the mix to the hybridization temp and add the genomic DNA to it quickly so as to avoid cooling to RT.

We set up:

Mbuna
5 ul (340 ng) DNA
1.5 ul (10 uM) probe
5 ul (50uM) primer B
15 ul 20X SSC
.5 ul 10% SDS
HOH to 50 ul

Same for Tilapia except used 15 ul (250 ng) FKP (P=prePCR)
9 ul (250 ng) FTP
also did sample straight from ligation without any pre PCR
21 ul (250 ng) FTL (L=ligation)

B. Place the mixture at 68 degrees C for one hour with intermittent mixing. This is 17.5 degrees below the Tm of the probe and should allow sufficient specificity as well as yield.

During this time, prepare the beads by equilibrating in 6X SSC/0.1%SDS. Apply 1 mg of beads (100 ul of 10mg/ml) to a siliconized low binding 0.5 ml microfuge tube. Put the tube in the magnetic stand and wait a couple of minutes until the beads are bound into a ball against the the tube wall. Keeping the tube in the magnetic stand, pull off the 100 ul of buffer. (Do this slowly so that surface tension of solution doesn't pull beads from wall down into bottom of tube. Also put magnetic bar in stand so arrow points up. This locates magnet higher on tube wall and keeps beads away from bottom of tube). Add 200 ul of 6X SSC/0.1% SDS and remove from the magnet. Flick the tube or vortex gently. Apply the magnet, pull off the salt buffer and repeat. Let the beads sit in the 6X SSC/0.1% SDS solution until the hybridizations are finished. Just prior to mixing the hybridization reactions with the beads, apply the magnet and pull off the salt solution.

C. Following hybridization, add the probe/DNA mixture to 1 mg of streptavidin beads previously equilibrated in 6XSSC/0.1% SDS. For the 400-900 bp fragments, incubate with the beads at room temperature for 15 minutes. For the 900-1500 bp fragments, incubate at 43 degrees C for one hour. During this step, the CA probe bound to biotin, hybridized to microsatellite-containing DNA fragments, is now strongly bound to the streptavidin beads.

D. After the incubation times, apply the magnet, wait a few minutes and pull off the remaining solution.

E. Wash the beads

Wash the beads twice for 10 minutes at room temperature in 200 ul 6X SSC/0.1% SDS using the magnet to pull off the salt solution after each wash.

Wash two times for 15 minutes at 68 degrees in pre-warmed 3X SSC/ 0.1%.

Wash two more times for 10 minutes at room temperature in 6X SSC.

VII. PCR amplification of captured DNA fragments.

At this point, we should have streptavidin beads bound to the biotin-CA16 probe hybridized to single strands of DNA with microsatellite sequence flanked on one side by anonymous sequence and linker X and on the other by anonymous sequence and the reverse complement of linker X. We want to PCR amplify using the linker sequence as primer to increase the amount of DNA available for cloning. First, we want to get the hybridized DNA to separate from the beads and biotin probe. This can be accomplished in a couple of ways. DNA can be eluted (100 ul of 0.1 N NaOH at 60 C for 5 minutes followed by the addition of an equal volume of 1 M Tris (pH 7.5). Or it can be heated in TE/10 or water (95 C) to denature the strands. In both cases, after heating, place the tubes back in the magnetic stand (the magnet binds the beads:biotin) and pull off the buffer containing the DNA of interest. We used the heating protocol.

a. Wash the beads in TE/10 or water two times (use the magnet to separate the beads from the wash each time).

b. Denature DNA from the beads by heating in 50 ul TE/10 to 95 C for 5 minutes. Quickly, apply the magnet and pull off the DNA. Do this twice for each sample so that final volume is 100 ul. Use about 10 ul in subsequent PCR II.

c. PCR II.

NOTE : Again, don't do too many cycles. Just enough so you have enough to TA clone. You can run 10, 15, and 20 cycles for small amounts and run on gel to test what the minimum number of cycles you can get away with.

We set up:

For tilapia

10x Dynazyme buffer 10
dNTP (10 mM each) 2
Dyn MgCl2 3
Primer A (10 uM) 5
H2O 68
DNA 10
Dynazyme 2

total 100

PCR in 9600 : 1.5 min denature at 95 C
15 cycles of
94 C 20 s
68 C 45 s
72 C 2 min 30 s
10 min at 72 C for final extension to encourage A addition for TA cloning
Note : Dynazyme can be used for TA cloning.

d. Take PCR reactions and spin-clean (with 1/TE or water) in Millipore Ultra-free MC spin columns. This is to remove unused dNTPs, salts, primer and any biotin-conjugated probe that might have sneaked into solution above. Resuspend in a comfortable volume (once again can concentrate to 25-30 ul) and quantify. We're ready to TA clone.