Rapid generation of long tandem DNA repeat arrays by homologous recombination in yeast to study their function in mammalian genomes

We describe here a method to rapidly convert any desirable DNA fragment, as small as 100 bp, into long tandem DNA arrays up to 140 kb in size that are inserted into a microbe vector. This method includes rolling-circle phi29 amplification (RCA) of the sequence in vitro and assembly of the RCA products in vivo by homologous recombination in the yeast Saccharomyces cerevisiae. The method was successfully used for a functional analysis of centromeric and pericentromeric repeats and construction of new vehicles for gene delivery to mammalian cells. The method may have general application in elucidating the role of tandem repeats in chromosome organization and dynamics. Each cycle of the protocol takes ~ two weeks to complete.


Introduction
Tandem repeats, also referred to as satellite DNA, represent a major class of repetitive DNA, whose function in the shaping of the human genome is only beginning to be explored. These repetitive sequences can be located in exons, introns, or intergenic regions, and their polymorphisms provide a unique source of genomic variability. Recent evidence also suggests that the repeat variants can influence the expression of entire loci and disease susceptibility [1,2]. Satellite sequences vary both in their repeat unit size and in their array length. Microsatellites are the smallest, with a repeat size of as little as few base pairs. The expansions of microsatellites associated with diseases have been referred to as dynamic mutations. Another class of repeat sequences is the classic satellites. They are much more abundant in the mammalian genome and consist of larger size repeat units ranging from 20-30 base pairs to a few kilobases. The array size of classic satellites may exceed several megabases.
The most commonly known regions enriched by classic satellites are centromeric and pericentromeric regions. In mammals and other multicellular eukaryotes, these regions are characterized by very large arrays of different tandem repeated DNA sequences. Although centromeric DNA repeat sequences are thought to be structurally and/or functionally important for forming a functional kinetochore, they are poorly conserved between species. For example, mouse chromosomes have two types of DNA repeat sequences, the major satellite repeat (~6 Mb array/234 bp per repeat unit) and the minor satellite repeat (~600 kb array/120 bp per repeat unit) [3,4]. The major mouse satellite is found in the pericentromeric region, and the minor mouse satellite is found in the centric constriction of the centromere [3,5]. The centromeres of human chromosomes are characterized by the presence of megabase-size alpha-satellite DNA arrays (also known as alphoid DNA), which are composed of a tandem array of a 171 bp repeat unit with no homology to mouse minor and major satellites. Moreover, alpha-satellite DNA arrays are diverged between different chromosomes. Nonalphoid DNA repeats have also been identified adjacent to alpha-satellite DNA in the pericentromeric regions of human chromosomes, for example, satellites I, II, and III [6][7][8], beta-satellite DNA [9], sn5 satellite DNA [3,10], and gamma-satellite DNA [11][12][13][14]. Alpha-satellite DNA is the only centromeric DNA sequence identified to date with a proposed role in kinetochore formation and maintenance. Long arrays of alpha-satellite DNA can form kinetochores de novo during transfection into human cells [15][16][17][18][19][20][21]. However, this process is poorly studied, and untill now the precise function of satellite DNA in centromeres remains mostly undetermined.
Because there is no approach to delete or mutate satellite DNAs in the genomic context, their function can be clarified, for example, by their insertion into ectopic chromosomal site. However, such an approach has limitations because chromosomal regions enriched by satellite DNA are underrepresented in the existing BAC and YAC libraries. Moreover, the monomers of centromeric and pericentromeric arrays in BAC/YAC clones are highly diverged, making their mutational analysis impossible. Several years ago, two groups suggested to study the function of centromeric repeats by the construction of synthetic alphoid arrays using repetitive directional ligation on the basis of a native higher-order 2-3 kb repeat fragment [22,23]. Although this approach allowed the construction of several large synthetic arrays, it has significant limitations. First of all, it is a slow, laborious strategy not easily scaled up for rapid generation of tandem repeats with engineered changes. Secondly, the method uses restriction sites that may not be available in an amplified repeat unit. In addition, artificially introduced restriction sites remain in multiple copies in the final constructs.
To overcome all these problems, we developed another strategy to rapidly assemble long arrays of classic satellite DNA with a size up to 140 kb from a monomer or oligomer. This technique is comprised of two steps: rolling-circle amplification (RCA) of a short DNA multimer (e.g., a dimer for alphoid DNA) into 2-15 kb DNA branched molecules (that are poorly clonable in bacterial vectors) and subsequent assembly of the amplified molecules into long arrays by transformation-associated recombination (TAR) in yeast [24]. As a result, amplified arrays are propagated as circular YAC/BACs in yeast cells and can be transferred into E. coli cells if needed. As any nucleotide can be easily changed in the original satellite unit before its amplification, this new technique is optimal for identifying the nucleotides sequence(s) in the satellite DNA critical for a specific function.
Using the RCA-TAR method, we constructed a set of different alphoid DNA arrays that have been used to elucidate the structural requirements for de novo kinetochore formation in human cells [24][25][26][27]. In addition, a synthetic alphoid DNA array with an embedded tetoperator sequence was used to construct a new generation of Human Artificial Chromosomes (HACs) with a conditional centromere for gene delivery and gene expression studies [25,28]. Being applied for pericentromeric repeats, this method identified the insulator activity of gamma-satellite DNA that prevents heterochromatin spreading beyond the pericentromeric region [29]. This barrier activity may be exploited in gene expression studies to protect transgenes from epigenetic gene silencing. In addition, amplified repeats are also important for experiments on epigenetic engineering. The principle of this approach is based on the insertion of an amplified sequence carrying multiple tet operator (tetO) or lac operator (lacO) sequences into an ectopic chromosomal site that allows the tethering of chromatin modifiers into the array as tet repressor (tetR) or lac repressor (lacR) fusions. Recently, the synthetic tetO-alphoid/tetR-fusion tethering system has been used to clarify the role of open and condensed chromatin in maintenance of the human kinetichore [27]. This system also allowed the induction of de novo kinetochore assembly on both newly introduced synthetic alphoid DNA arrays 25 and at the ectopic site [30]. Some examples of mammalian satellite DNAs amplified by RCA-TAR are shown in Table 1. This method may also have application for the construction of proteinpolymers derived from short peptide motifs found in some proteins to design novel drug delivery vehicles and for high-yield synthesis of peptide drugs and antigens [ [31] and references therein].
Collectively, the RCA-TAR-based strategy may be applied for the analysis of any type of repeats in mammals, whose functions are still unclear. Manipulation of the number of DNA repeats with different mutations can be the basis for experiments to elucidate the critical parameters that lead to heterochromatinization or

Yeast cells
The current protocol has been optimized for the strain VL6-48. The "recombinational cloning" part of this protocol has been optimized for processing 1.0 × 10 9 spheroplasts cell number from early stationary phase cultures grown in 50 ml of YEPD media.

Optimization of spheroplasts preparation
While there are no limitations in the choice of a yeast host strain, the time of the Zymolyase-20T treatment or its concentration for each new batch of zymolyase should be experimentally determined, if using different amounts of cells, different phases of cell culture growth, or different yeast strains, because different strains may exhibit a different sensitivity to the enzyme. Each spheroplasts transformation uses 2-4 μg of RCA product and 0.01-0.04 μg of the linearized vector. Typically, under such conditions, 200-5000 transformants are obtained. As a control for recombination, the RCA products are omitted from the transformation mix, resulting in a decreased yield of transformants, down to 5-20 colonies.

Confirmation of the repeat amplification
When the arrays are assembled, a control experiment should be carried out to confirm the correct amplification of the repeats in the BAC DNA arrays. For this purpose, several BACs with the largest arrays should be digested by an endonuclease that cuts the arrays until their original repeat units (i.e., a unique endonuclease site present only once in each monomer).

Successive size increase of the array
Should the analysis of a representative number of E. coli colonies (~60) fail to reveal an array with the desired size, an additional round of recombinational cloning is required to further increase the size of the array. For this purpose, the BAC vector with the largest insert found during the first round of amplification is digested with an appropriate endonuclease that cleaves at the insert/vector junctions. The vector DNA is eliminated with an additional endonuclease that cuts only the vector part into small fragments. The final digest is precipitated with ethanol/sodium acetate and dissolved in a small volume of water. For the second round of yeast spheroplast transformation, usually 2-4 μg of the released BAC-insert array and 0.01-0.02 μg of the TAR vector are required. The yield of clones with a 2-to 3fold larger insert size is typically 5-10%.
Amplification of the repeat unit without the RCA step Although the RCA reaction accelerates the assembly of the repeats into long arrays, this step may be omitted. Recombinational assembly may be performed using a synthesized oligonucleotide corresponding to a dimer or a tetramer of a repeat of interest, instead of the RCA products. Pyrex, Baffled Culture Flasks (Fisher Scientific Ltd., cat. no. 10-041-5B). This flask is sterilized and used to grow the 50 ml YEPD yeast culture for transformation.
Corning 15 ml polypropylene sterile screw-cap disposable graduated centrifuge tubes (Fisher Scientific Ltd., cat. no. 05-538-51). These tubes are used to mix yeast spheroplasts with melted SRB-TOP-His agar medium, which is then poured onto the plates with SORB-His regenerative agar.
Corning 50 ml sterile screw-cap disposable graduated centrifuge tubes (Fisher Scientific Ltd., cat. no. 05-538-55). These tubes are used to collect the yeast cells and spheroplasts during transformation.
Centrifuge (Thermo Scientific, Sorvall Legend RT Plus, Benchtop Centrifuge, cat. no. 75004377). This is used to pellet the yeast cells in 50 ml centrifuge tubes.
Forced-Air Incubator (Fisher Scientific Ltd., cat. no. 11-690637F). This is set to 30°C for growth of plate cultures.
Thermo Precision General-Purpose Water Baths for 50°C and 70°C (Fisher Scientific Ltd., cat. no. 15-460-2). The 50°C bath is used to keep SORB-TOP-agar medium melted, while the 70°C bath is used during yeast DNA isolation.
Sterile flat toothpicks. They may be purchased from a local grocery store. Place wide end down into a 100 ml beaker, cap with aluminum foil, and then autoclave. They can then be used by turning the beaker on its side and removing one at a time Spectrophotometer (Fisher Scientific Ltd., cat. no. S42475P). This is used at 660 nm for OD in order to determine yeast cell numbers in cultures and used with plastic cuvettes (Fisher Scientific Ltd., cat. no. 14-385-938).
Gene Pulser Xcell Total System (Bio-Rad Laboratories, cat. no. 165-2660). This is used to transform yeast recombinant molecules from yeast cells into E. coli cells. Pulser

Reagent setup
The basic TAR cloning vector The basic pNK-TAR vector contains a yeast selectable marker (HIS3), a yeast origin of replication ARSH4, a yeast centromeric sequence CEN6 from yeast chromosome VI, a BAC cassette with a bacterial selectable marker that allows the YAC clones to be transferred into E. coli cells, and a mammalian selectable marker, the Neo or BS gene. The vector contains targeting sequences (or hooks) homologous to a repeat of interest that are inserted into the polylinker. The amount of the linearized TAR vector needed for spheroplasts transformations is~100 ng diluted in 20-50 μl of water (keep at -20°C). The vector and its more detailed description are available upon request.

Sorbitol solution (1 M)
Add 182 g of sorbitol to about 700 ml of distilled/deionized water in a 1000 ml beaker. Stir until dissolved. Make the volume up to 1000 ml in a 1000 ml measuring cylinder and mix thoroughly. The solution is filter sterilized. Sorbitol solution can be stored at room temperature (RT).

SPE solution
(1M Sorbitol, 10 mM Na2EDTA, 0.01 M Na phosphate, pH 7.5). Add 91 g of sorbitol, 1.04 g of Na2HPO 4 × 7H 2 O, 0.16 g of NaH 2 PO 4 × 1H 2 O, and 10 ml of 0.5M EDTA, pH 7.5 to about 400 ml of distilled/deionized water in a 500 ml beaker. Stir until dissolved. Make the volume up to 500 ml in a 500 ml measuring cylinder and mix thoroughly. The solution is filter sterilized. SPE solution can be stored at RT.

SOS solution
(1M Sorbitol, 6.5 mM CaCl2, 0.25% yeast extract, 0.5% peptone). Add 91 g of sorbitol, 1.25 g of bacto yeast extract, 2.5 g of bacto peptone, and 3 ml of 1M CaCl 2 to about 400 ml of distilled/deionized water in a 500 ml beaker. Stir until dissolved. Make the volume up to 500 ml in a 500 ml measuring cylinder and mix thoroughly. The solution is filter sterilized. SOS solution can be stored at RT.

STC solution
(1M Sorbitol, 10 mM CaCl2, 10 mM Tris-HCl, pH 7.5). Add 91 g of sorbitol, 5 ml of 1M Tris-HCl pH 7.5 and 5 ml of 1M CaCl 2 to about 400 ml of distilled/deionized water in a 500 ml beaker. Stir until dissolved. Make the volume up to 500 ml in a 500 ml measuring cylinder and mix thoroughly. The solution is filter sterilized. STC solution can be stored at RT.

PEG MW 8000 solution
(20% (w/v) polyethylene glycol 8000, 10 mM CaCl2, 10 mM Tris-HCl, pH 7.5). Add 20 g of PEG 8000, 1 ml of 1M Tris-HCl pH 7.5 and 1 ml of 1M CaCl 2 to about 70 ml of distilled/deionized water in a 150 ml beaker. Stir until dissolved. Use a hot plate to gently warm the solution if necessary. Make the volume up to 100 ml in a 100 ml measuring cylinder and mix thoroughly. The solution is filter sterilized. Keep the PEG solution at RT. Make a new PEG solution every month to avoid reduction of the yield of transformants.

SDS solution (2%)
Add 2.0 g of sodium dodecyl sulfate to 80 ml of distilled/deionized water in a 100 ml beaker. Stir until dissolved. Make the volume up to 100 ml in a 100 ml measuring cylinder. SDS solution can be stored at RT.

SDS solution (10%)
Add 10.0 g of sodium dodecyl sulfate to 80 ml of distilled/deionized water in a 100 ml beaker. Stir until dissolved. Make the volume up to 100 ml in a 100 ml measuring cylinder. SDS solution can be stored at RT.
Ethidium Bromide (EtBr) (10 mg/ml) Add 1 g of ethidium bromide to 100 ml of distilled/ deionized water. Stir well for several hours to ensure that the dye has dissolved. Wrap the container in aluminum foil or transfer the solution to a dark bottle and store at RT.

KAc solution (5 M)
Dissolve 29.5 g of solid KAc (potassium acetate) in 75 ml of distilled/deionized water and add 11.5 ml of glacial acetic acid. Adjust volume to 100 ml with distilled/ deionized water and filter-sterilize. Store at RT.

SOC solution
(2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl). Add 2 g of bacto tryptone, 0.5 g of bacto yeast extract, 200 ml of 5M NaCl, and 50 ml of 5M KCl to about 100 ml of distilled/deionized water in a 250 ml beaker. Stir until dissolved. Make the volume up to 100 ml in a 100 ml measuring cylinder and mix thoroughly. Transfer the solution to a glass storage bottle and autoclave for 30 min. Alternatively, the solution can be filter sterilized. SOS solution can be stored at RT.

Chloramphenical solution (12 mg/ml in EtOH)
It is used as a selection agent for transformed cells containing chloramphenicol resistance genes. Add 1.2 g of chloramphenical to 100 ml of ethanol (EtOH) in a 250 ml beaker. Stir until dissolved. Transfer the solution into a glass storage bottle. Chloramphenical solution can be stored at -20°C.

YEPD liquid medium
(2% D-glucose, 1% Bacto yeast extract, 2% Bacto peptone, 2% Bacto agar, adenine hemisulfate 20 mg l -1 ). Add 20 g of D-glucose, 20 g of Bacto peptone, 10 g of Bacto yeast extract, and 5 ml of adenine solution (4 mg/ ml) to about 1000 ml of distilled/deionized water in a 1000 ml beaker. Stir until dissolved. Make the volume up to 1000 ml in a 1000 ml measuring cylinder and mix thoroughly. Transfer the solution into a glass storage bottle and autoclave for 21 min. It can be stored at RT.

YEPD medium with agar
(2% D-glucose, 1% Bacto yeast extract, 2% Bacto peptone, 2% Bacto agar, adenine hemisulfate 20 mg l -1 ). Add 20 g of D-glucose, 20 g of Bacto peptone, 10 g of Bacto yeast extract, and 5 ml of adenine solution (4 mg/ ml) to about 1000 ml of distilled/deionized water in a 1000 ml beaker. Stir until dissolved. Make the volume up to 1000 ml in a 1000 ml measuring cylinder and mix thoroughly. Transfer the solution into a 2000 ml glass flask, add 20 g of Bacto agar, and autoclave for 30 min. It can be stored at RT. Alternatively, YEPD medium can be purchased from Teknova, Inc. (http://www.teknova. com).

LB liquid medium
(1% Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl, 2 mM NaOH, pH 7.4). Add 10 g of Bacto tryptone, 5 g of Bacto yeast extract, 10 g of NaCl, and 200 ml of 1M NaOH to about 1000 ml of distilled/deionized water in a 1000 ml beaker. Stir until dissolved. Make the volume up to 1000 ml in a 1000 ml measuring cylinder and mix thoroughly. Transfer the solution to a glass storage bottle and autoclave for 15 min. Alternatively, the solution can be filter sterilized. The solution can be stored at RT.

LB medium with agar
(1% Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl, 2% agar, 2 mM NaOH, pH 7.4 ). Add 10 g of Bacto tryptone, 5 g of Bacto yeast extract, 10 g of NaCl, 20 g of agar, and 200 ml of 1M NaOH to about 1000 ml of distilled/deionized water in a 1000 ml beaker. Stir until dissolved. Make the volume up to 1000 ml in a 1000 ml measuring cylinder and mix thoroughly. Transfer the solution to a glass storage bottle and autoclave for 30 min. It can be stored at RT.
LB-Cm liquid medium (12.5 mg/ml chloramphenical). Add 1 ml of chloramphenical solution to 1000 ml of LB medium. Stir well. It can be stored at +4C°for at least six months.
LB-Cm plates (12.5 mg/ml chloramphenical). Add 1 ml of chloramphenical solution to 1000 ml of LB medium with agar. Mix well. Pour approximately 20 ml into each Petri dish. The plates can be stored at +4C°for at least six months.

Equipment setup Agarose gel electrophoresis
DNA is separated on a 1% (wt/vol) or 2% agarose/ 1XTBE electrophoresis gel by applying 4.5 V cm -1 for 3 h. After electrophoresis, the gel is stained with EtBr (1/ 10,000 dilution) for 15-30 min and detected by the Gel Doc System.

Clamped homogeneous electrical field (CHEF) electrophoresis
CHEF analysis is performed in a 1% (wt/vol) pulsed field certified agarose gel with 0.5XTBE buffer circulated through a cooler set at 14°C. Typical forward parameters are as follows: 9.0 V/cm, initial switch 0.11 s, and final switch 0.92 s, with linear ramp. Typical reverse parameters are: 6.0 V/cm, initial switch 0.11 s, final switch 0.92 s, with linear ramp for 20.5 h. After electrophoresis, the gel is stained with EtBr (1/10,000 dilution) for 15-30 min and detected by the Gel-Doc System.

Electroporation into E. coli cells
Electroporation is performed with DH10B competent cells using a Bio-Rad Gene Pulser MXcell Electroporation System with the settings 2.5 kV, 200 W, and 25 μF.

Procedure
Preparation of the circular DNA template for rolling circle amplification (RCA) TIMING 18 hr Before the RCA reaction, the repeat should be cloned into TOPO vector and sequenced. The sequenced repeat is isolated from the vector DNA using an appropriate endonuclease that cleaves at insert/vector junctions (see Figure 1a). CRITICAL STEP Take 3 μl from Step 5, load into the well of 2% agarose/EtBr gel, and run the gel to ensure that the DNA was not lost during isolation. 6. Take 60 μl from Step 5 and mix with 30 μl of x10 DNA ligase buffer, 10 μl of DNA ligase, and 200 μl of water. Total volume of the ligation reaction is 300 μl. Incubate the reaction overnight at 16°C.
CRITICAL STEP The ligation reaction should be performed with diluted DNA to prevent inter-molecular ligation. To ensure that the ligation reaction has worked, take 3 μl of the isolated fragment before the ligation reaction (from Step 5) and 30 μl of the ligation mixture after overnight incubation (from Step 6). Load into the wells of 1% agarose gel, run the gel, and stain the gel by ethidium bromide. The ligated fragment moves slower The RCA products were generated from a 340 bp alphoid dimer. Cleavage of RCA products with an enzyme results in restoration of the input repeat. (c) Recombinational assembly includes co-transformation of RCA products into yeast along with a TAR vector (YAC/BAC) containing repeat-specific targeting hooks. End-to-end recombination of DNA fragments, followed by interaction of the recombined fragments with the vector hooks, results in the rescue of arrays as circular YACs. His + transformants and pooled colonies are shown. (d) Transferring of YACs into bacterial cells. E. coli transformants and streaked colonies are shown. BAC DNAs from randomly picked up colonies were restricted by an endonuclease that releases the vector part (7 kb) and arrays. The size of arrays varies from 2 to 12 kb. BAC DNA from colony #9 with the largest array is marked by the red arrow. (e) CHEF analysis of BACs with the largest arrays chosen after screening 60 E. coli transformants. The size of the inserts varies from 9 to 25 kb. The tandem repeat structure of one array (clone #4 with the size~25 kb) is confirmed by EcoRI digestion (f) An additional round of recombinational assembly to further increase the size of the array. Representative CHEF analysis of 8 BACs is shown. Restriction of BAC DNAs was done by an endonuclease that cleaves the molecule at insert/vector junctions (arrays are between 40 and 60 kb) and by double digestion with an additional endonuclease that cuts the vector part completely. than the initial isolated fragment. CAUTION Ethidium bromide is highly toxic on contact with skin. 7. Load the ligation mixture (from Step 6) into the wells of 1% agarose/EtBr gel. Run for 1-2 hr. Cut off the circular supercoiled DNA from the gel. Isolate the DNA by QIAquick Gel Extraction Kit. Final volume is 150 μl (~1.5 μg DNA) (see Figure 1a).  CRITICAL STEP Take 12 μl of the overnight RCA reaction mixture for analysis. Digest 6 μl with an endonuclease that cleaves the RCA products into its original repeat unit. Leave the other 6 μl undigested. Use 1% agarose/EtBr gel to see the RCA reaction products and 2% agarose/EtBr gel to see the repeat unit (see Figure  1b).
12. Heat inactivate Phi29 DNA polymerase by incubation at 65°C for 10 min. 13. Precipitate the RCA products with 2.5 volume of EtOH. 14. Pellet the precipitate by centrifugation for 5 min at maximum Eppendorf minicentrifuge speed (20.000 × g). 15. Remove the supernatant and wash the pellet with 70% EtOH. 16. Resuspend the damp DNA pellet in 100 μl of water.
PAUSE POINT The RCA product may be kept at 4°C for several weeks.
Recombinational cloning of RCA products using a TAR vector in yeast (see Figure 1c) Preparation of the yeast culture TIMING overnight 17. One day before the TAR cloning experiment, inoculate three different size colonies of the host yeast strain VL6-48 freshly grown on a YEPD plate in three separate 50 ml aliquots of YEPD medium in three 250-ml Erlenmeyer flasks. Grow the cultures overnight at 30°C with vigorous shaking to assure good aeration.

Preparation of competent yeast spheroplasts TIMING 2-3 hr
18. In the morning, measure the optical density (OD) of the cultures with 20 min intervals until an OD 660 of~2.0 is achieved in the flask.
CRITICAL STEP For actual measurement, dilute the culture 1/10 in water; the density should be between 0.18-0.22. The culture with such an optical density is ready for the preparation of highly competent spheroplasts. This optical density corresponds to approximately 2 × 10 7 cells per ml. 19. Transfer the yeast culture into a 50-ml corning tube and pellet the cells by centrifugation for 5 min at 1,000 × g, 5°C. Remove and discard the supernatant. 20. Resuspend the cell pellet in 30 ml of sterile water by vortexing and centrifuge for 5 min at 3,000 × g, 5°C. Remove and discard the supernatant. Remove the supernatant and gently resuspend the spheroplasts in each tube with 800 μl of SOS solution using a Pipetman. 29. Incubate the spheroplasts for 40 min at 30°C without shaking. 30. Transfer the spheroplasts from each tube into a 15-ml corning tube containing 7.0 ml of melted SORB-TOP-His medium (equilibrated at 55°C) using a Pipetman. Gently mix and quickly pour agar onto SORB-His plates with selective medium (without histidine) containing 1 M Sorbitol.
PAUSE POINT Keep the plates at 30°C for 5 days until the transformants become visible (see Figure 1c).

DNA isolation from yeast transformants TIMING 5 hr
33. Wash the yeast cells out from each plate with 5 ml water into a 50-ml corning tube and pellet the cells by centrifugation for 5 min at 1,000 × g, 5°C.
Remove and discard the supernatant. CRITICAL STEP Growing of E. coli transformants at 30°C rather than 37°C is needed to keep the integrity of the assembled tandem repeats array. CRITICAL STEP Choose small size E. coli transformant colonies. It has been noted that large arrays are preferentially found amongst smaller colonies. When setting up the inoculation, use a toothpick to first pick up a transformant, then streak it onto LB-Cm plates before dropping that toothpick into the 2 ml culture medium. Streaked transformants are grown at 30°C and used for further analysis when needed (see Figure 1d). Step 62 in a total volume of 40 μl for 2 hours. 64. Take half of the sample from Step 63 (20 μl) and run the DNA digest using gel electrophoresis with a 1.0% 1xTBE agarose gel, 1xTBE buffer, and a constant voltage setting of 6V/cm for approximately 2 hours. 65. Stain the gel with ethidium bromide for 10 min and photograph the DNA bands using a Gel-Doc 2000 system. Typical results are shown in Figure 1d. 66. Choose several BACs with the slowest band mobility and thus carrying the largest DNA array for further analysis. The actual size of the BAC inserts is determined by CHEF gel electrophoresis. Run the remaining half of the digest sample from Step 63 (20 μl) on a CHEF Mapper XA Chiller System. 67. Stain the gel with ethidium bromide for 15-30 min and photograph the DNA bands using a Gel-Doc System. Typical results are shown in Figure 1e. CAUTION Ethidium bromide is highly toxic on contact with skin. 68. Choose the BAC with the biggest DNA array. Check the integrity of the amplified repeat by digesting the remaining 10 μl sample from Step 62 with an endonuclease that cleaves the array into its original repeat unit (see Figure 1e).

Miniprep
Successive increase of the DNA array length (optional) (see Figure 1f) In situations when the size of the array needs to be amplified further, a second round of recombinational cloning is carried out. To do this, make a large-scale BAC DNA purification from the sample with the largest array first (Steps 69-82), then followed by another round of recombinational cloning (Steps 83-89 86. Centrifuge the tubes at 14.000 × g for 10 min at 4°C in a micro-centrifuge. 87. Dissolve the pellet in 10-20 μl of water and leave for 20 min at 10°C. 88. Take 1 μl of the fragment (from Step 87) and run 1% gel to ensure that the DNA is not lost. 89. Use 2-4 μg of the digested BAC DNA fragment and 0.01-0.02 μg of the linearized TAR vector for the second round of recombinational cloning (i.e., repeat Steps from 17 to 68).
CRITICAL STEP It is worth noting that at this stage, the size of the BAC DNA arrays may be determined directly by CHEF gel electrophoresis without preliminary check on 1% gel (see Figure 1f).
TIMING Steps 1-7 Preparation of the circular DNA template for rolling circle amplification reaction (RCA): 18 hr Steps 8-16 DNA amplification reaction by RCA: 13 hr Step 17 Growth of the culture of the yeast strain: overnight Steps 18-25 Preparation of competent yeast spheroplasts: 2 hr Steps 26-30 Transformation of spheroplasts by RCA DNA products along with a TAR cloning vector: 2 hr Step 30 Colony formation of the His + transformants: 5 d Steps 31-32 Incubation of plates with the pooled His + yeast transformants: overnight Steps 33-41 DNA isolation from the yeast pools: 4-5 hr Steps 42-46 Electroporation of yeast DNA into E. coli cells: 1.5 hr Steps 47-62 Miniprep BAC DNA purification from E. coli transformants: 3 hr Steps 63-68 Restriction of BAC DNA, 1% agarose gel and CHEF analyses: 24 hr Steps 69-89 Further increase of length of the DNA array: two weeks TROUBLESHOOTING Troubleshooting advice can be found in Table 2.

Anticipated results
The whole procedure, from Steps 1-68, may produce arrays consisting of tandem repeats with a size up to 140 kb. Starting with an amount of template DNA as little as 5-10 ng and with a size of the repeat as small as 340 bp (e.g., alphoid-satellite dimer), it is possible to first amplify the repeat up to 2-5 kb by the RCA reaction (Steps 8-16). For further size increase by recombinational cloning in yeast, each spheroplasts transformation uses 2-4 μg of the RCA products and 0.01 μg of the linearized TAR vector. Typically, under such conditions,~200-5000 yeast transformants are obtained (Steps 17-30). Homologous recombination in yeast produces approximately 2% of transformants containing DNA inserts bigger than 15 kb. With less than 1%, the size of the arrays may be as high as 15-30 kb. Thus, an efficient end to end recombination of incoming DNA molecules during yeast transformation results in a recovery of clones with relatively long arrays. After electroporation of DNA isolated from yeast transformants into E. coli cells,~2% of BACs contain DNA inserts bigger than 15 kb and less than 1% of BACs, between 15-30 kb (Steps 42-68). An additional round of recombinational cloning produces approximately 5-10% of clones with a size of arrays 2-to 3-fold larger than the size of the largest array chosen after the first round of recombination (e.g., 20 kb × 3 = 60 kb) (Steps 69-89). Thus, the combination of RCA with a recombinational capture in yeast may increase the original size of a repeat up to 176 times (e.g., 0.34 kb alphoid dimer × 176 = 60 kb) (see Table 1 for more examples). Orientation of the hooks should correspond to that illustrated in Figure 1c  67 Unstable arrays Growth of E. coli transformants at 37°C Grow the cells at 30°C