Once high molecular weight (HMW) DNA has been prepared it must somehow be fragmented and DNA in the desired size range isolated. In general, as the desired DNA fragment size increases the fewer manipulations of the DNA can be tolerated. DNA fragmentation utilizes two general approaches: A) physical shearing and B) partial digestion with a restriction enzyme that cuts relatively frequently within the genome. Since physical shearing is not dependent upon the frequency and distrubution of particular restriction enzyme sites, this method should yield the most random distribution of DNA fragments (Ward and Jen, 1990). However, the ends of the sheared DNA fragments must be repaired and cloned directly or restriction enzyme sites added by the addition of synthetic linkers. These subsequent steps might damage the HMW DNA and lead to lower yields of clonable DNA. Because of the subsequent steps required to clone DNA fragmented by shearing, most researchers fragment DNA by partial restriction enzyme digestion. The advantage of partial restriction enzyme digestions is that no further enzymatic modifications of the ends of the restriction fragments are necessary. Four common techniques used to achieve reproducible partial digestions of HMW DNA are: 1) varying the concentration of the restriction enzyme (Burke and Olson, 1991); 2) varying the time of incubaton with the restriction enzyme (Anand, et al, 1989); 3) varying the concentration of an enzyme cofactor (e.g. Mg⁺⁺) (Albertsen, et al, 1989); and 4) varying the ratio of endonuclease to methylase (Larin, et al, 1991). 


1. Procedure

• The partial digestion of HMW DNA embedded in LMP agarose microbeads is conducted and analyzed according to Zhang et al (1995) with modifications. It is normally necessary to perform a preliminary experiment for each preparation of HMW DNA to determine the optimal conditions for partial digestion. The digestion reaction of HMW DNA in microbeads is similar to that for conventional aqueous phase DNA digestion. The following procedure describes the partial digestion of HMW DNA embedded in agarose microbeads by limiting the concentrations of restriction enzyme Bam HI for rice and Hind III for sorghum.

 



2. Sorghum

• Set up the prereaction as follows:

a. Label four 1.5 ml tubes and pipet 50 ul of the beads with a cut off pipette tip into each tube. Be sure to mix the microbeads well before pipeting.

b. Make a cocktail with 4.5 x 29 ul H2O, 6.5 x 10 ul 10 x reaction buffer, 4.5 x 1 ul 10 mg/ml BSA, and 4.5 x 5 ul 40 mM spermidine, and then aliquot 45 ul of the cocktail to each tube containing the microbeads. (A single restriction digestion has the following composition:

• Megabase-size DNA in microbeads     50 ul

• 10X Hind III reaction buffer     10 ul

• BSA (10 mg/ml)     1.0 ul

• SPD (40mM)    5 ul

• ddH2O     29 ul




• (10 X Hind III reaction buffer: 60 mM Tris-HCl, 60 mM MgCl2, 0.5 M NaCl, 10 mM DTT, pH 7.5)

c. Incubate the microbeads without the restriction enzyme on ice for 20 minutes.

d. Dilute the Hind III enzyme serially ranging from 0 to 20 units in 1X Hind III reaction buffer.

• tube 1-0      u/5ul;

• tube 2-1      u/5ul;

• tube 3-2      u/5ul;

• tube 4-4      u/5ul




• e. Add 5 ul of Hind III to each reaction and incubate on ice for additional 30 minutes to allow the enzyme to diffuse into the microbeads.

f. Incubate the reactions in a 37°C water bath for 5 minutes and stop the reaction by addition of 1/10 volume of 0.5 M EDTA, pH 8.0 on ice.

g. Analyze the digests on a 1.0% agarose CHEF gel at 6.0 V/cm, with a 50 second pulse, for 20 hours, at 11°C in 0.5x TBE buffer.

h. Select the amount of Hind III giving the best partial digest and set up the digestion reaction on a large scale by setting up the number of reactions you need as above (do not increase the total volume of a reaction more than 100 ul).

3. Rice

• Set up pre-partial digestion as follows:

a. Label five 1.5 ml tubes, mix the microbeads well in the storage tube, and pipet 100 ul of the beads with a cut off pipette tip into each tube. Be sure to mix the microbeads well before pipeting.

b. Make a cocktail with 5.5 x 66 ul H2O, 5.5 x 20 ul 10 x reaction buffer, 5.5 x 2 ul 10 mg/ml BSA, and 5.5 x 10 ul 40 mM spermidine, and then aliquot 98 ul of the cocktail to each tube containing the microbeads. A single restriction digestion has the following composition:

• Megabase-size DNA in microbeads     100 ul (5-7 ug)

• 10X Bam HI reaction buffer      20 ul

• SPD (40mM)      10 ul

• ddH2O      68 ul

• BSA (10mg/ml)      2 ul




c. Incubate the mixture on ice for one hour and then add 2 ul dilutions of Bam HI enzyme in 1 X reaction buffer:

• tube 1-0 u/ml;

• tube 2-1 u/ml;

• tube 3-2 u/ml;

• tube 4-4 u/ml,

• tube 5-8 u/ml.




d. Incubate on ice for additional 30 minutes, then transfer into a 37 C water bath, and incubate for 5 minutes.

e. After digestion, transfer the reactions onto ice immediately and then add 20 ul of 0.5 M EDTA, pH 8.0 to each tube to stop the reaction. Keep the digests on ice before analyzing by pulsed-field gel electrophoresis (PFGE).

4. Electrophoretic analysis of partial digestions

• a. Analyze the digests on a 1.0% agarose CHEF gel at 6.0 V/cm, with a 50 second pulse, for 20 hours, at 11°C running in 0.5x TBE buffer.

• (a). Prepare 2 L 0.5 x TBE and pour into the CHEF buffer chamber and start the cooling system to cool the TBE down to 11 °C.

• (b). Prepare an 1% pulsed-field gel (120 ml) in 0.5 x TBE and keep 1-5 ml of the molten agarose in a 65°C water bath for later use.

• (c). When the gel is completely solidified, remove the comb carefully, disassemble the gel mold, and load the gel as follows:

(1) During storage of the digests on ice, the beads sink to the bottom of the tube. Carefully remove the supernatant with a pipet, and load the beads into a well of the gel using a cut off pipette tip.

(2) Seal the beads in the well with the molten agarose kept in the 65 °C water bath in step b.

(3) Insert a small slice of an agarose plug that contains lambda DNA as a concatemer for a molecular weight standard. Seal the marker in place with the remaining molten agarose.

• (d). When the TBE in the CHEF buffer chamber cools down to 11°C, place the loaded gel with the gel-pouring plate in the CHEF buffer chamber and leave at least 15 minutes to equilibrate the temperature of the gel for at least 15 minutes.

• (e). Run the gel at 11°C, 50 sec., and 150 V for 20 hours.

• (f). Stain the gel with ethidium bromide, photograph, and determine the optimal partial digestion condition for BAC cloning. In this experiment, the partial digestion optimal conditions that show a majority of restricted DNA fragments from 200 to 400 kb in s ize on the pulsed-field gel will be selected for large scale partial digestion of HMW DNA to be used for the construction of the BAC libraries. They may include the optimal condition, and one-step above and below the optimal condition.

b. As above, perform a 10-fold scale partial digestions with Bam HI or 6-fold scale partial digestion with Hind III under conditions: one step above, optimal, and one-step below. After the large scale digestions are stopped, the reactions can be checked by PFGE using a small portion of each reaction while majority of the reaction is stored at 4°C or directly used for the first size selection.

B. Size selection of HMW DNA

After partial digestion, the DNA must be size-selected to remove the smaller DNA fragments that can compete more effectively than the larger DNA fragments for vector ends. The two most common size-selection techniques are sucrose gradient centrifugation (Burke and Olson, 1991) and pulsed-field gel electrophoresis (Albersten, et al, 1990, Birren and Lai, 1993). Most cosmid libraries have been constructed with DNA selected on sucrose gradients. All but one of the plant YAC and BAC libraries have used pulsed-field gel electrophoresis for size selection. The notable exception is one of the A.thaliana YAC library constructed by Ward and Jen (1990) who used sucrose gradients. Pulsed-field gel electrophoresis is primarily used for size selection because the DNA is usually more concentrated than DNA isolated on sucrose gradients; therefore, it is easier to concentrate DNA from agarose gels without substantial loss in yield and size. Additionally, 5-20% gradients do not efficiently resolve DNA above 100 kbs. Cosmid libraries can be constructed with DNA size-selected on sucrose gradients because the yields are good in the size range of 25 to 40 kb.

1. Procedure

a. First size selection

• i. Prepare a 1% LMP agarose CHEF gel using 1x TAE as the electrophoresis buffer.

ii. Prepare 2 liters of 1X TAE electrophoresis buffer and place in CHEF gel apparatus and cool to 11 °C.

iii. Load partially digested DNA and size markers (lambda concatemers and/or yeast chromosomes) on either side of the digests into a 1% LMP agarose gel using a cut off pipet tip. Seal the wells with 1% melten agarose.

iv. Run the CHEF gel at 6.0 V/cm, with a 90 second pulse, for 20 hours, at 11 °C.

v. Cut both sides of the gel including the size markers and part of the DNA lanes with a clean microscope coverslip. Remove the center of the gel and keep in a CHEF gel box at 11 °C.

vi. Stain, destain, and photograph the gel sides with a ruler, side-by-side.

vii. Cut DNA ranging from 300 to 500 kb from the unstained portion of the gel by measured migration for direct ligation or for a second size selection (see below).

b. Second size selection

• i. Melt the gel piece cut from first size selection gel at 67°C for 10 minutes, pipet the solution into the wells of a second size selection gel prepared as above (1% LMP agarose in 1xTAE), with a cut off pipet tip, and wait for 5 minutes until it solidifies.

ii. Load size markers on the either side of the digests with a known quantity of uncut lambda DNA for estimating the concentration of size selected DNA.

iii. Remove the small trapped DNA from the first size selection by PFGE with the following settings: 4.0 V/cm, 5 second pulse, 10-13 hours, at 11°C, in 1x TAE buffer.

iv. Stain and photograph the sides of the gel as above and excise the compressed DNA band from the unstained portion of the CHEF gel.

v. Wash the gel piece containing DNA with TE (1 ml), 2-3 times, on ice in a 1.5 ml microfuge tube, and store at 4 °C.

C. Ligation of the size-selected DNA fragments into pBeloBAC11

1. Wash the LMP agarose gel slices containing the size-selected DNA fragments on ice with 1.0 ul ice-cold TE three times, 10 minutes each time.

2. After the last wash, remove the solution completely, melt the gel slice at 68°C for 10-15 minutes, and transfer to a 45°C water bath to equilibrate the temperature of the molten agarose to 45°C (5-10 minutes).

3.Estimate the volume of the molten agarose and digest the agarose with 1 unit of GELase (Epicentre, USA) per 100 ug of gel slice. Incubate at 45 C for 1 hour.

Alternatively, add 1/10 volume of prewarmed 10 x beta-agarase reaction buffer (supplied by the manufacturer) at 40°C and 1 unit of b-agarase per 150 ul molten agarose, invert the tube several times very gently, and incubate at 40°C for 1 hour.

Note: you should always pipet the naked HMW DNA with a cut off pipet tip very gently or the DNA will be physically sheared.

4. After digestion, estimate concentration of selected DNA by the following procedure:

• a. Prepare a 1% agarose/TBE gel.

• b. Load lambda DNA of known concentration as standard.

• c. Load 10 ul aliquots of each sample into the gel.

• d. Electrophorese at 50 volts for 30-50 minutes.

5. When the agarose is completely digested, add the vector DNA at a molar ratio of 5-10 vector DNA : 1 source DNA and incubate at 55°C for 10 minutes.

6. Cool the mixture to room temperature (10-15 minutes), and add 1/10 volume of 10 x ligation buffer supplied by the manufacturer and 2 units of T4 DNA ligase per 100 ul reaction containing approximately 100 ng insert DNA.




• Size selected DNA (approx. 100 ng)     x ul

• Dephosphorylated pBeloBAC 11      x ul
       (in a molar ratio of 1 (insert DNA) to 5 (vector DNA))

• 10X T4 ligase buffer     10 ul

• T4 DNA ligase (1 unit/ml)      2 ul

• ddH2O          x ul         

• Total         100 ul

• (10X T4 DNA ligase buffer: 660 mM Tris-HCl, pH 7.6, 66 mM MgCl2, 100 mM DTT, 660 mM ATP)

7. Remove 20 ul of the reaction very gently with a cut-off pipet tip and mix with 1 ul 0.5 ug/ ul Hind III-digested lambda DNA for ligation tests.

8. Incubated the reaction and the test ligation reaction at 16°C overnight.

9.Analyze the test ligation reaction on an 1% agarose gel using the unligated Hind III-digested lambda DNA as a control. Ligation of a majority of the smaller fragments of Hind III-digested lambda DNA into larger fragments as demonstrated on the gel indicates a successful ligation of the source DNA into the BAC vector. This ligation is now ready for E. coli transformation by electroporation