Plant transformation BAC vectors

DNA transfer into plants has been accomplished by several methods including Agrobacterium-mediated transformation, biolistic bombardment, and microinjection. BAC vectors have been engineered for transformation of large DNA inserts into plant genomes. BIBAC (binary bacterial artificial chromosome) has been designed to replicate in both E. coli and A. tumefaciens and has all of the features required for transferring large inserts of DNA into plant chromosomes [15]. BIBAC test constructs containing 150 kb human DNA were introduced into several A. tumefaciens strains. Of these strains, those containing additional copies of VirG or VirG/VirE produced transgenic tobacco plants in which the entire 150 kb human DNA fragment integrated into the tobacco genome randomly. Recently, a pBACwich system has been developed to achieve site-directed integration of DNA into the genome (Choi et al., unpublished work). A 150 kb cotton BAC DNA was transferred into a specific lox site in tobacco by biolistic bombardment and Cre-lox site specific recombination. These results open up a number of new possibilities for plant molecular biology and genetic engineering of plants. These systems will streamline positional cloning and the transfer of desirable traits into plants.

Bacteria-yeast shuttle vectors

The bacteria-yeast shuttle vector, pClasper, combines the bacterial origin of replication from BAC vectors with the CEN6/ARS4 origin of yeast and includes a yeast LEU2 gene and the bacterial chloramphenicol resistance gene for selection in yeast or bacteria, respectively. Bradshaw et al. [17] also cloned homologous sequences flanking the region to be targeted into the polylinker of pClasper to isolate regions from within a YAC. After transformation of linearized plasmids into the LEU- strains carrying a specific YAC, yeast colonies were selected for the acquisition of the LEU+ phenotype. The recombinant was then shuttled to bacteria for preparation of plasmid DNA. Transformation-associated recombination (TAR: [18, 19]) cloning can be applied to the generation of circular YACs by using a single centromere vector containing various plant repeats. If the TAR vector contains an E. coli mini-F factor origin of replication and chloramphenicol resistance gene from the BAC vectors, circular YACs can be moved into bacterial cells and easily separated from yeast chromosomes. YACs are important tools for cloning large genome regions. However, it is laborious to isolate enough YAC DNA from yeast. The bacteria-yeast shuttle vectors are useful to clone specific regions from YACs, from hybrid cells, or directly from the genome, and advantageous for isolating DNA.

Megabase-size DNA isolation from plants

There are two general methods for preparing megabase-size DNA from plants. The protoplast method yields megabase-size DNA of high quality with minimal breakage, but the process is costly and labor-intensive. For example, to prepare protoplasts from tomato, young leaves are manually feathered with a razor blade before being incubated for four to five hours with cell-wall-degrading enzymes [20]. Furthermore, since each plant species requires a different set of conditions to generate protoplasts, the method will only work if a high-yielding protoplast method has been optimized for a given plant species.
Zhang et al. [21] developed a universal nuclei method that works well for several divergent plant taxa. Fresh or frozen tissue was homogenized with a blender or mortar and pestle, respectively. Nuclei were then isolated and embedded as described in the Materials and Methods section. The DNA was often more concentrated and was shown to contain lower amounts of chloroplast DNA. Although the nuclei method was universal, it produced a relatively high degree of sheared DNA. The primary advantage of the method is that it is economical and not as labor-intensive as the protoplast method.
Once protoplasts or nuclei are produced they are embedded in an agarose matrix as plugs or microbeads. The agarose provides a support matrix to prevent shearing of the DNA while allowing enzymes and buffers to diffuse in. Thus the DNA is purified and manipulated in the agrose and is stable for more that one year at 4 degrees C.

Generation and size selection of large DNA fragments for BAC cloning

Once high molecular weight (HMW) DNA has been prepared, it must somehow be fragmented and DNA in the desired size range isolated. In general, DNA fragmentation utilizes two general approaches: (1) physical shearing and (2) partial digestion with a restriction enzyme that cuts relatively frequently within the genome. Since physical shearing is not dependent upon the frequency and distribution of particular restriction enzyme sites, this method should yield the most random distribution of DNA fragments. 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 may 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 protocols fragment DNA by partial restriction enzyme digestion. The advantage of partial restriction enzyme digestion is that no further enzymatic modifications of the ends of the restriction fragments are necessary. Four common techniques that can be used to achieve reproducible partial digestion of megabase-size DNA are: (1) varying the concentration of the restriction enzyme, (2) varying the time of incubation with the restriction enzyme, (3) varying the concentration of an enzyme cofactor (e.g., Mg++), and (4) varying the ratio of endonuclease to methylase.
There are three cloning sites (HindIII, BamHI, and SphI) in pBeloBAC11, but only HindIII and BamHI produce 5' overhangs for easy vector dephosphorylation. These two restriction enzymes are primarily used to construct BAC libraries. The optimal partial digestion conditions for megabase-size DNA are determined by wide and narrow window digestions. To optimize the optimum amount of HindIII, 1, 2, 5, 10, and 50 units of enzyme are each added to 50 ml aliquots of microbeads and incubated at 37 degrees C for 20 min (wide window: Figure 2). Figure 2 shows that the optimal digestion conditions are between 5 and 10 units for rice. It also shows that the megabase-size DNA could be totally digested with 50 units of HindIII in 20 min. To determine a more narrow optimization window, 6, 8, and 10 units of HindIII are each added to 50 ml aliquots of microbeads and incubated at 37 degrees C for 20 min (narrow window: Figure 3). Six units of enzyme gave the best results in which the size of most of the DNA ranged between 300 to 500 kb. This size range was selected because the DNA in the fragments between 300 to 500 kb were usually revealed to be 50 to 300 kb. The exact conditions are used to reproduce the partial digestion. Several reactions of the same volume are combined after incubation to obtain the larger amount of DNA required for size selection of insert DNA for BAC cloning.
After partial digestion of megabase-size DNA, the DNA is run on a pulsed-field gel and DNA in a size range of 300-500 kb is excised from the gel. This DNA is ligated to the BAC vector or subjected to a second size selection on a pulsed-field gel under different running conditions. Studies have previously shown that two rounds of size selection can eliminate small DNA fragments comigrating with the selected range in the first pulsed-field fractionation [4, 5]. Such a strategy resulted in an increase in insert sizes and a more uniform insert size distribution.
A practical approach to performing size selections is to first test for the number of clones/ul of ligation and insert size from the first size selected material. If the numbers are good (500 to 2,000 white colony/ul of ligation) and the size range is also good (50 to 300 kb), then a second size selection is practical. When performing a second size selection expect a 80 to 95% decrease in the number of recombinant clones per transformation.

FIG. 2. Wide window: Partially digested rice, Oryza sativa L. (Lemont japonica), megabase-size DNA: rice DNA in 50 ul of agarose microbeads. (M): Saccharomyces cerevisiae (BIO-RAD), (C): rice DNA without enzyme, Lanes 3-7: rice DNA partially digested with HindIII (1) 1 U (2) 2 U (5) 5 U (10) 10 U (50) 50 U. The genomic DNA was subjected to CHEF on a 1% agarose gel in 0.5X TBE using a switch time of 90 s at 6 v/cm and 12 degrees C for 16 hours.

FIG. 3. Narrow window: Partially digested rice, Oryza sativa L. (Lemont japonica), megabase-size DNA: rice DNA in 50 ul of agarose microbeads. (m): Saccharomyces cerevisiae (BIO-RAD), (c): rice DNA without enzyme, Lanes 3-5: rice DNA partially digested with HindIII (6) 6 U (8) 8 U (12) 12 U. The genomic DNA was subjected to CHEF on a 1% agarose gel in 0.5X TBE using a switch time of 90 s at 6 v/cm and 12 degrees C for 16 hours.

Ligation

Twenty to two hundred nanograms of the size-selected DNA is ligated to dephosphorylated BAC vector (molar ratio of 10 to 1 in BAC vector excess). Table 3 shows a summary of BAC library ligation conditions for a number of BAC/PAC libraries. Most of the BAC or PAC libraries used a molar ratio of 5 to 15 : 1 (size-selected DNA : BAC vector).

TABLE 3
Summary of BAC Libraries Ligation Conditions

Transformation and arraying

Transformation is carried out by electroporation and the transformation efficiency for BACs is about 40 to 1,500 transformants from one ul of ligation product, or 20 to 1000 transformants/ng DNA. There is an inverse relationship between the insert size and the transformation efficiency. It has been demonstrated that a lower field strength (9-13 kV/cm) yields a higher average insert size but a lower number of clones [24].
The E. coli strain used for BAC library construction is DH10B (F- mcrA delta(mrr-hsdRMS-mcrBC) phi80dlacZ deltaM15 delta lacX74 deoR recA1 endA1 araD139 delta (ara, leu)7697 galU galK lambda- rpsL nupG), which includes mutations that: (1) block restriction of foreign DNA by endogenous restriction endonucleases (hsdRMS); (2) block restriction of DNA containing methylated DNA (mcrA, mcrB, mcrC and mrr); (3) block recombination (recA1); and (4) take up large DNA (deoR). Electroporation competent DH10B cell can be made in the laboratory or purchased commercially.
Recombinant clones are picked manually with toothpicks or robotically (e.g., Genetix Q-bot) into 384-well microtiter plates containing growth media. After incubation overnight at 37 degrees C duplicate copies of the library are produced and then the library is ready for screening using hybridization or PCR.

Library characterization

Several tests can be done to determine the quality of a BAC library. In our laboratory we perform three basic tests to evaluate the genome coverage of a BAC library - average insert size, average number of clones hybridizing with single copy probes and chloroplast DNA content.
The determination of the average insert size of the library is assessed in two ways. First, during library construction every ligation is tested to determined the average insert size by assaying 20-50 BAC clones per ligation. DNA is isolated from recombinant clones using a standard mini preparation protocol, digested with NotI to free the insert from the BAC vector and then sized on a CHEF gel. After the library is completed (3-20X genome coverage) the insert size from a single random clone from every 384-well plate is determined as above. Figure 4 shows a typical analysis of rice BAC clones from the second size selection ligation.
To determine the genome coverage of the library it is screened with single copy RFLP markers distributed randomly across the genome by hybridization. Microtiter plates containing BAC clones were spotted onto 22 cm x 22 cm Hybond N+ membranes by using a multitasking robot (Q-bot: Genetix, Inc., UK). Bacteria from 48 or 72 plates are spotted twice onto one membrane, resulting in 18,000 to 27,648 unique clones on each membrane in either a 4X4 or 5X5 orientation. Since each clone is present twice, false positives are easily eliminated and true positives are easily recognized and identified. With this system, the entire library screening process required only one to two membranes and one to three days of exposure to X-ray films. An example autoradiogram image of the a BAC high-density filter of the TAMU Arabidopsis BAC library hybridized with a cosmid RFLP probe is shown in Figure 5.
Finally, the chloroplast DNA content in the BAC library is estimated by hybridizing three chloroplast genes (ndhA, rbcL and psbA) spaced evenly across the chloroplast genome to the library on high-density hybridization filters. Figure 6 shows such a hybridization to part of our cotton BAC library and shows that approximately 1.1% of the 32,729 clones hybridized with the chloroplast probes.

FIG. 4. Analysis of random rice (Lemont: Oryza sativa subsp. japonica) BAC clones by CHEF electrophoresis. Lanes 1 and 20 are lambda concatemer. Lanes 2-19 are alkaline lysis minipreps of recombinant BAC clones digested with NotI. Note : The 6.7 kb band in each lane is pBeloBAC11.

FIG. 5. BAC library screening by colony hybridization : An autoradiogram image of BAC high-density replica filter hybridized with a cosmid DNA probe (m262). 12,288 TAMU Arabidopsis BAC clones are screened on double-spotted (4X4) high density filters with a cosmid RFLP marker. Positive hybridization addresses (20 clones): 1P4, 2H13, 5I18, 9J14, 11N16, 14G8, 14G16, 16A16, 17C10, 20B2, 20O22, 21B4, 26H10, 26M2, 27B4, 27L21, 29H19, 30H22, 30P22, 31O3

FIG. 6. BAC library screening by colony hybridization with chloroplast DNA. 16,896 cotton pBACwich clones (1,536 X 11) were screened on double-spotted high density filters with chloroplast DNA probes (Dr. J. Mullet, Texas A&M University). Each filter (8 cm X 12 cm) was inoculated by duplicating each colony on the same filter with a 384 prong high density replicating tool (HDRT) from four 384-well microtiter plates using the Biomek 2000 robot (Beckman, USA) for a total of 1,536 BAC clones per filter.

A mathematical analysis for the number of clones needed to anchor the target gene

An anchoring scheme refers to any method for determining which clones contain a given target gene. It is assumed that clones are distributed according to a homogeneous Poisson distribution along the genome. In practice, the collection of clones is often not randomly distributed because of unknown cloning bias and partial digestion rather than random shearing. However the homogenous distribution assumption is used in the mathematical analysis performed to determine the number of clones (Table 4) required to obtain a certain probability of having any DNA sequence represented in the genomic library [25].

The number of clones in the library is determined using the following equation :
N=ln(1-P)/ln(1-L/G) where,
N= number of clones in library
P= probability to get the target gene
L= length of average clone insert in basepairs
G= haploid genome length in basepairs

In general 99% coverage represents four to five haploid genome equivalents. However, if such a library is used for the construction of complete physical maps or whole genome sequencing, one needs to generate very deep libraries with a P value of 99.9 to 99.99%.

TABLE 4
The Number of Clones Required for a 99% Probability of Having any DNA Sequence Represented in a Genomic Library. Genome Sizes are Basically Referred to [26] except Arabidopsis [27].

In the following sections, we will discuss the procedures used to construct, analyze, and manipulate BAC libraries from plants, using examples for BAC libraries constructed in our laboratory.

Procedures

BAC vector preparation

Cell growth

1. Streak E. coli strain DH10B containing BAC vector (pBeloBAC11) onto an LB agar plate containing 12.5 ug/ml chloramphenicol and grow at 37 degrees C overnight.

2. Inoculate 5 ml of LB media containing 30 ug/ml chloramphenicol with a single colony from step 1 and grow for 8 hours at 37 degrees C.

3. Inoculate four 2.8 liter culture flasks containing 1 liter of LB chlorampenicol (30 ug/ml), prewarmed to 30 degrees C, with 1 ml/liter of culture from step 2. Grow at 30 degrees C with shaking (225 rpm) to a cell density of approximately 1 X 10^9 cells/ml (A600 = 1.0-1.2).

4. Harvest the cells by centrifugation at 4 degrees C for 15 minutes at 6,000 g (about 6,200 rpm in Beckman JA-14, Beckman, USA).

Solutions

• LB plates: 1% Tryptone, 0.5% Yeast extract, 1% NaCl, 1.5% Agar

• LB medium: 1% Tryptone, 0.5% Yeast extract, 1% NaCl

• Add chloramphenicol (stock solution: 20 mg/ml in ethanol) at below 70 degrees C after autoclaving.

Plasmid purification

1. Extract BAC vector using Qiagen Maxi Plasmid Purification Protocol (Five QIAGEN-tip 500 columns for a 4 l preparation).

2. Dissolve the final plasmid pellet in 3.6 ml of TE, add 0.4 ml of 10 mg/ml of ethidium bromide, and transfer the DNA solution to a 15 ml polypropylene Falcon tube.

3. Add about 4.0 g of solid CsCl and adjust the density of the solution to 1.59 g/ml with additional CsCl or TE. The final volume becomes about 5 ml.

4. Load solution into a Ultra-Clear centrifuge tube (Beckman order # 344057) and ultracentrifuge the mixture to density equilibrium in a Beckman NVT90.1 rotor at 70,000 rpm for 24 hours at 20 degrees C.

5. View the supercoiled plasmid and relaxed DNA bands with long-wavelength UV light and remove the lower band by puncturing the side of the tube with a 21-gauge syringe needle below the band and carefully draw the plasmid DNA into a 1 ml syringe.

6. Extract the ethidium bromide from the DNA solution with 1 volume of ddH2O-saturated isoamyl alcohol. Continue to extract with fresh ddH2O-saturated isoamyl alcohol until all visible color is removed (5-6 times).

7. Dilute the extracted DNA sample with 2 volume units of TE and precipitate with 95% ethanol and centrifugation. Wash the pellet gently with 70% ethanol, recentrifuge and air dry.

8. Resuspend the DNA pellet in 100 ul of TE and determine the DNA concentration by spectrophotometry and agarose gel electrophoresis using known DNA concentration standards.

Notes

7. The final yield is approximately 20-40 ug from 4 liters of media.

Solutions

• TE : 10 mM Tris-HCl, 1 mM EDTA, pH 8.0

• Water-saturated isoamyl alcohol

Digestion and dephosphorylation

1. Digest pBeloBAC11 (10 ug) to completion with 100 units of HindIII or BamHI (Gibco BRL, USA) and corresponding buffer with additional 4 mM spermidine at 37 degrees C for 2-4 hours.

2. Extract the digested DNA twice with phenol/chloroform (1:1), precipitate and wash with ethanol, and resuspend in 50 ul of 1X TA buffer (provided with HK phosphatase: Epicentre, USA) following the addition of 5 mM CaCl2.

3. Dephosphorylate the DNA by adding two units of HK Phosphatase (Epicentre) per ug of DNA and incubating at 30 degrees C for two hours.

4. Heat the reaction at 65 degrees C for 30 minutes to inactivate HK Phosphatase.

5. Ethanol-precipitate the dephosphorylated vector and dissolve in 100 ul of TE.

Notes

2. Restriction enzyme-incubation for an extended time period or with high salt may show exonuclease activity.
3. The digested BAC vector can be dephosphorylated with Shrimp Alkaline Phosphatase (SAP 1 unit/ug of DNA: USB, USA).

Solutions

• 10X HindIII buffer: 0.5 M Tris-HCl, pH 8.0, 0.1 M MgCl2, 0.5 M NaCl

• 10X BamHI buffer: 0.5 M Tris-HCl, pH 8.0, 0.1 M MgCl2, 1 M NaCl

• 10X Spermidine-trihydrochloride: 40 mM in sterilized ddH2O

• 10X TA buffer: 330 mM Tris-acetate pH 7.8, 660 mM K-acetate, 100 mM Mg-acetate, 5 mM DTT, and 1 mg/ml Bovine serum albumin

• Phenol/chloroform (1:1): Mix equal amounts of phenol and chloroform and equilibrate the mixture by extracting several times with 0.1 M Tris-HCl (pH 7.6)

Preparation of megabase-size DNA from plants

Nuclei preparation (This method was developed by [21] and modified by S. Choi)

1. Grind 30-50 g of the fresh or frozen tissue into powder in liquid nitrogen with a mortar and pestle (do not over grind) and immediately transfer into an ice cold 500 ml beaker containing 200 ml ice-cold wash buffer (HB) plus 0.15% beta-mercaptoethanol (add before use) and 0.5% Triton X-l00 (20% stock in HB).

2. Repeat step 1, 4-6 times in order to obtain enough nuclei to embed.

3. Swirl each mixture from step 1 with a magnetic stir bar for 20 minutes on ice.

4. Filter the mixture from step 3 into an ice-cold 250 ml centrifuge bottle through two layers of cheesecloth (American Fiber & Finishing, Inc., USA) and one layer of miracloth (Calbiochem order # 475855, USA) placed in a single large, wide mouth funnel. To obtain more nuclei, remove the cheesecloth and squeeze the remainder of the homogenate into the centrifuge bottle.

5. Pellet the filtered homogenate by centrifugation in a fixed-angle rotor at 1,800 g at 4 degrees C for 20 minutes (about 3,500 rpm in JA-14, Beckman).

6. Discard the supernatant fluid and gently resuspend the pellet in 1-5 ml of ice cold wash buffer with assistance of a small paint brush. After resuspension adjust the volume to 30 ml with wash buffer and transfer to a 50 ml round bottom oakridge tube.

7. Pellet the nuclei by centrifugation at 1,800 g, 4 degrees C for 15 minutes in a swinging bucket centrifuge (about 2,800 rpm in GH-3.8 Horizontal rotor, Beckman).

8. Wash the nuclei pellet 2 additional times by resuspension in wash buffer followed by centrifugation at 1,800 g, 4 degrees C for 15 minutes. During the last washing step the resuspended nuclei are filtered through one layer of miracloth by gravity into new oakridge tubes.

9. After the final wash, resuspend the pelleted nuclei in a small volume of HB (2 to 3 ml). Count the nuclei with a hemacytometer under a phase contrast microscope and adjust to approximately 4 x 10^7 nuclei/ml with addition of HB (for the plants with haploid genomic sizes of 500-1,000 Mb).

Notes

9. In practice, the optimum number of nuclei depends on the genomic size of the plant and for some plants species it can be hard to count the nuclei. When preparing nuclei for the first time we optimize the nuclei concentration empirically. We suggest preparing the nuclei as described above and in the final step embed 4 different concentrations of nuclei (e.g., 1 ml concentrated nuclei + 3 ml of HB, 2 ml concentrated nuclei + 2 ml of HB, and 4 ml concentrated nuclei + 0 ml of HB).

Solutions

• HB: 0.5 M Sucrose, 10 mM Trizma base, 80 mM KCl, 10 mM EDTA, 1 mM Spermidine, 1 mM Spermine, final pH 9.4-9.5 adjusted with NaOH Wash buffer: HB + 0.15% beta-mercaptoethanol (add before use) + 0.5% Triton X-100 (20% stock in HB)

Protoplast preparation (Tomato)
(This method was developed by [20] and modified by Wing et al. [28])

1. Harvest 30-50 g of the young leaves and cut from midvein 5 to 10 times to make strips of 1-2 mm. The leaf is still intact but feathered.

2. Transfer approximately 4 g of leaves into protoplast buffer plus cellulase 1% and pectolyase 0.05% in a large petri dish and shake gently for 4 hours at room temperature.

3. Check the release of protoplasts under an inverted microscope. If 70%-80% of lysed protoplasts are observed, remove the leaves and filter the liquid sequentially using 80 and 33 um sieves into a flask.

4. Pellet the protoplasts at 40 g for 10 minutes (about 500 rpm in JA-14) and resuspend in 50 ml of protoplast buffer.

5. Repeat filtering as step 3 and centrifuging as step 4, and resuspend in a small amount (about 2-3 ml) of protoplast buffer.

6. Count an aliquot under a microscope by using hemacytometer and adjust the final concentration to 4 X 10^7 protoplasts /ml of protoplast buffer.

Notes

• Making protoplast is specific for every plant and this needs to be optimized.

Solutions

• Protoplast buffer: 0.5 M D-Mannitol, after autoclaving add 20 mM 2-N-Morpholine ethanosulfonic acid, adjust pH 5.6 with KOH

Encapsulating in agarose microbeads (I) (This method was developed [28] for plant protoplasts and modified by Zhang et al. [21] for plant nuclei)

1. Prepare 1%-1.2% low melting point (LMP) agarose (Seaplaque GTG, FMC, USA) solution in HB for nuclei (or protoplast buffer for protoplasts) and store in a 45 degrees C water bath.

2. Warm 15 ml of light mineral oil in a 50 ml Falcon tube to 45 degrees C in a water bath.

3. Place 150 ml of ice cold HB for nuclei (or protoplast buffer for protoplasts) in a 500 ml beaker into an ice bath on top of a magnetic stir plate and swirl the solution vigorously using a magnetic stir bar (#5 in Corning stirrer PC-620, USA).

4. Place the nuclei or protoplast solution in a 500 ml flask and prewarm to 45 degrees C in a water bath.

5. Pipet an equal volume of 1%-1.2% LMP agarose in HB from step 1, kept in a 45 degrees C water bath, into the prewarmed nuclei or protoplast suspension and mix well but gently.

6. Add 20 ml of prewarmed light mineral oil at 45 degrees C from step 2 to the prewarmed nuclei or protoplast suspension, shake the mixture of the flask vigorously for 3-5 seconds, and immediately pour into the 500 ml beaker of step 3 containing the swirling 150 ml of ice cold HB.

7. Continue swirling for 5-10 minutes on ice to break up any clumps and allow for the agarose microbeads to be more uniform in size.

8. Harvest the agarose microbeads by centrifugation at 600 g, 4 degrees C for 15-20 minute in a swinging bucket centrifuge (about 1,800 rpm in Beckman GH-3.8).

9. Discard the supernatant fluid and resuspend all the pelleted microbeads in 5-10 volumes of ESP in a 50 ml Falcon Polypropylene tube.

   DNA purification in agarose
   

10. Incubate the beads in ESP for 24 hours at 50 degrees C with gentle shaking to degrade the proteins.

11. Pellet the microbeads by keeping at 50 degrees C without shaking for 1 hour and discard the supernatant.

12. Add new ESP for further 24 hour-incubation.

13. Wash the beads with six 1 hour equilibration with TE at 4 degrees C. The first three washes include PMSF (phenylmethyl sulfonyl fluoride) at 0.1 mM to inactivate the Proteinase-K.

Solutions

• ESP: 0.5 M EDTA, pH 9.0-9.3, 1% Sodium lauroyl sarcosine, add 0.1 mg/ml Proteinase K before use

• PMSF: 50 mM stock in isopropanol

Encapsulating in agarose plugs (II)

1. Mix equal volumes of nuclei and 1% LMP (Seaplaque GTG) agarose in HB (or protoplast buffer for protoplasts) and aliquot into plug molds (BIO-RAD catalog # 170-3622) on ice.

2. Immerse the casted plugs in a large volume of ESP (at least ten times the volume of the agarose plugs, 0.5 mg/ml Proteinase K for plugs) in a 50 ml Falcon polypropylene tube and incubate at 50 degrees C overnight with gentle agitation.

3. Change the ESP one time and incubate for 5 hours to overnight with agitation.

4. Equilibrate agarose-embedded DNA thoroughly with TE containing 1 mM PMSF to inactivate residual Proteinase K three times for 1 hour each.

5. Wash three times with only TE.

Notes

• The plugs can be chopped into fragments that are about the same size as beads without any appreciable DNA breakage, and then used for partial digestion [29].

Partial digestion

Wide window

1. Incubate 50 ul of microbeads or chopped pieces of half a plug with a 45 ul mixture of (HindIII or BamHI reaction buffer, 0.1 mg/ml Acetylated BSA, 4 mM Spermidine) on ice for 20 minutes.

2. Add 5 ul of HindIII or BamHI freshly diluted in distilled water (e.g., 0 U, 1 U, 2 U, 4U, 8 U, 16 U, and 50 U/5 ul) and allow to diffuse into the beads for 10 minutes on ice.

3. Transfer the reaction mixture to a 37 degrees C water bath for partial digestion and incubate for 20 minutes.

4. Stop the reaction by adding 1/10 volume of 0.5 M EDTA, pH 8.0 and placing the tubes on ice.

5. Load the partially digested DNA on a 1% agarose gel in 0.5X TBE with a wide-bore tip and seal the wells with the same molten agarose as the gel.

6. Perform pulsed-field gel electrophoresis on a CHEF Mapper (BIO-RAD, USA) under the condition of 6.0 V/cm, 90 s pulse, 0.5X TBE buffer, 12 degrees C, 18 hours.

7. After checking the EtBr-stained gel from step 6, choose the enzyme concentration giving a majority of DNA fragments ranging from about 300 to 600 kb. Due to DNA trapping during the step 6 running conditions, we expect the actual size of the DNA fragments obtained from the 300-600 kb fraction to be between 50-300 kb in size.

   Narrow window

8. From the information of the wide window, carry out another set of partial digestions to optimize the narrow window (e. g., 4 U, 5 U, 6 U, and 7 U/5 ml: Figure 2).

9. Repeat the steps 1-7.

Notes

2. The amount of restriction enzyme and the digestion time depend on the number of restriction sites in the plant genome: usually 0-50 units for 5 minutes-1 hour.
7. The position of the optimum DNA fragment is not constant but always depends on the concentration of the sample DNA and other cofactors such as running conditions, DNA types, etc. Therefore, sometimes the best region may be in the higher region or lower region.

Solutions

• 0.5X TBE: Trizma base 0.045 M, Boric acid 0.045 M, EDTA 1 mM, pH 8.3

Preparing the partially digested DNA for ligation-first size selection

1. Select the amount of enzyme giving the optimum digest from the narrow window and perform the digestion reaction in a large scale by carrying out a number of reactions (10-20 reactions) under exactly same conditions as the previously determined (same volumes, same tubes, same dilution of enzyme, same enzyme tube, etc.).

2. Load the partially digested DNA on a 1% LMP agarose gel (Seaplaque, =46MC) in 1X TAE with a wide-bore tip and seal with the same molten agarose.

3. Perform pulsed-field gel electrophoresis on a CHEF Mapper under the condition of 6.0 V/cm, 90 s pulse, 1X TAE buffer, 12 degrees C, 18 hours.

4. Cut DNA fragments ranging from about 300 to 600 kb from the gel and use for ligation or a second size selection.

Notes

4. When we use chopped DNA plugs (which have highly concentrated DNA) for a partial digestion we can use a shorter pulse time (20-40 s) to spread out the DNA. This condition can avoid the trapping of smaller DNA segments so that a second size selection may not be necessary.

Solutions

• 1X TAE: 40 mM Tris-acetate, 1 mM EDTA, pH 8.0

Preparing the partially digested DNA for ligation-second size selection

1. Dialyze the gel piece cut from the first size selection with TE + 50-100 mM NaCl.

2. Melt the gel piece cut from the first size selection at 65 degrees C for 5 minutes and pipet into the wells of a second size selection gel (1% LMP in 1X TAE) with a polyethylene transfer pipet (Fisher, USA).

3. Carry out a second size selection at 4.0 V/cm, 5 s pulse, 1X TAE buffer, 12 degrees C, 8 hours.

4. Excise the DNA band in the compression zone, and dialyze with TE + 50-100 mM NaCl.

Ligation

1. Melt the dialyzed LMP DNA piece from 1st or 2nd size selection at 65 degrees C for 5 min, and transfer to a 45 degrees C water bath.

2. Add 1 unit of GELase (Epicentre, USA) per 100 mg of gel with GELase buffer and incubate at 45 degrees C for one hour.

3. Check the concentration of the DNA solution by loading 10 ul on a 1% agarose minigel with EtBr and running a quick electrophoresis at 60 V, for 1 hour, in 0.5X TBE with uncut lambda DNA standard solutions (e.g., lambda 5 ng, 10 ng, 20 ng, 40ng, and 80 ng/well: Usually the concentration of the size selected DNA is between 0.5 and 2 ng/ul).

4. Ligate 50-200 ng of the size selected DNA to the dephosphorylated BAC vector in a molar ratio of 1 to 10-15 (size selected DNA to vector DNA; see Table 3 for details) in a total volume of 100 ul with 6 units of T4 DNA ligase (USB, USA) plus ligase buffer at 12 or 16 degrees C for 16 hours.

5. Drop-dialyze the ligation solution on Millipore filters (filter type VS, 0.025 um) with TE for 1 hour to remove the ligation buffer.

Notes

1. Strong et al. [30] described a electroelution method for purifying large, gel-fractionated DNA molecules that eliminates the need for melting of the agarose and subsequent enzymatic agarose digestion.

Transformation

1. Transform 1-2.5 ul of the ligation material into 20-25 ul of E. coli DH10B competent cells (Gibco BRL, USA) by using the BRL Cell-Porator system (Gibco BRL) according to its protocol (Voltage 400V, Capacitance 330 uF, Impedance low ohms, Charge rate fast, Voltage Booster resistance 4 Kohms).

2. Transfer the electroporated cells to 15 ml culture tubes with 0.4-1 ml SOC and shake at 220 rpm for 50 minutes at 37 degrees C.

3. Spread the SOC medium from step 2 on one or two LB plates containing 12.5 ug/ml chloramphenicol, 50 ug/ml X-Gal (stock: 20 mg/ml in dimethylformamide) and 25 ug/ml IPTG (stock: 200 mg/ml in ddH2O), and incubate at 37 degrees C for 20-36 hours.

4. Pick white colonies into 384 well microtiter plates containing LB freezing medium.

5. Incubate the microtiter plates at 37 degrees C overnight and store at -80 degrees C.

Notes

1. The Bio-Rad Gene Pulser II (BIO-RAD) can be used under the condition of 100 Ohms, 16 kV/cm, and 25 uFa. It may be advantageous to use a lower field strength in order to increase the average insert size. It has been demonstrated that a lower field strength (9-13 kV/cm) yields a higher average insert size but a lower number of clones [24].

Solutions

• SOC: 2% Bacto tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose, pH 7.0=B10.1

• LB freezing medium: LB, 36 mM K2HPO4, 13.2 mM KH2PO4, 1.7 mM Na Citrate, 0.4 mM MgSO4, 6.8 mM (NH4)2SO4, 4.4% v/v Glycerol, 12.5 ug/ml Chloramphenicol

BAC insert DNA isolation

1. Prepare 5 ml culture of LB medium containing 12.5 ug/ml chloramphenicol, inoculate a single colony, and incubate with shaking at 37 degrees C for 18-20 hours.

2. Centrifuge the culture at 2,000 g at 4 degrees C for 15 minutes using a table top centrifuge (about 3,000 rpm in GH-3.8).

3. Pour off the supernatant fluid and resuspend cell pellet in 0.2 ml of ice-cold GTE.

4. Transfer the suspension to a 1.5 ml microfuge tube, add 0.4 ml (freshly prepared) of 0.2 N NaOH+1% SDS solution, mix by inversion several times, and incubate on bench for 5 minutes.

5. Add 0.3 ml of the potassium acetate stock and invert gently.

6. Centrifuge the mixture for 15 minutes at 12,000 g in a micro-centrifuge (13,000 rpm in Biofuge 13, Baxter, USA).

7. Remove 0.75 ml of the supernatant fluid without disturbing the pellet and transfer to a clean microfuge tube.

8. Add 0.6 volumes of cold isopropanol (0.45 ml) and centrifuge at 12,000 g for 15 minutes to pellet the DNA.

9. Remove the supernatant, rinse the pellet with 1 ml of cold 70% ethanol, and dry upside down until the pellet becomes transparent.

10. Add 40 ul of TE and digest 10 ul of this DNA solution with NotI to free the insert from the BAC vector.

11. Run 1% agarose gels in 0.5X TBE at CHEF condition of 6 V/cm, initial switch time 5 s, final switch time 15 s and for 13-15 hours (Figure 4).

Solutions

• GTE: 50 mM Glucose, 25 mM Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0, 0.1 mg/ml RNase

• Potassium acetate stock: 60 ml 5 M Potassium acetate, 28.5 ml Glacial acetic acid, 11.5 ml ddH2O, pH 4.8-5.2

BAC library screening by hybridization

1. Inoculate Hybond N+ membranes (Amersham, UK) with a 384 prong High Density Replicating Tool (HDRT) from microtiter plates.

2. Place the membranes on LB agar plates containing 12.5 ug/ml chloramphenicol and incubate at 37 degrees C for 12 to 36 hours until colonies of 1 to 2 mm diameter are obtained.

3. Remove the membranes and place, colony side up, on a pad of absorbent filter paper (Whatman Cat. No. 3030 700) soaked in the following solutions and for the specified time: 1) Solution 1 (0.5 N NaOH, 1.5 M NaCl) for 7 minutes; 2) Solution 2 (1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0), 7 minutes; 3) Air dry for more than 1 hour; 4) Solution 3 (0.4 N NaOH), 20 minutes; 4) Solution 4 (5X SSPE), 7 minutes; 5) Air dry overnight.

4. Prehybridize the filters at 65 degrees C for at least 4 hours with hybridization buffer.

5. Exchange the prehybridization buffer with the fresh buffer and continue prehybridization for 2-4 hours at 65 degrees C.

6. Add probes and hybridize for 18 to 36 hours at 65 degrees C.

7. Wash the filters with 2X-0.1X SSC and 0.1% SDS 2-3 times for 20 minutes at 65 degrees C.

8. Blot with paper towels, wrap in plastic wrap, and expose for 24 to 72 hours with a intensifying screen.

Notes

1. Using a Q-bot (Genetix, Inc., UK), 384-well microtiter plates containing BAC clones are spotted onto 22 cm x 22 cm Hybond N+ membranes. Bacteria from 72 plates are spotted twice onto one membrane, resulting in 27,648 unique clones on each membrane. Alternatively, the nylon filters (12 X 8 cm) can be inoculated with a 384 prong HDRT from microtiter plates using the Biomek 2000 robot (Beckman).
3. The membranes can be stored at room temperature for months.

Solutions

• 5X SSPE: 0.9 M NaCl, 50 mM Sodium phosphate, 5 mM EDTA, pH7.7 Hybridization buffer: 0.5 M Sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA, 10 ug/ml Sheared denature salmon sperm DNA

• 2X SSC: 6M NaCl, 1M Citric acid-trisodium salt

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