Human Molecular Genetics, 2001, Vol. 10, No. 16 1657-1664
© 2001 Oxford University Press
Mismatch repair detection (MRD): high-throughput scanning for DNA variations
Neurogenetics Laboratory, Department of Psychiatry, University of California San Francisco, San Francisco, CA 94143, USA
Received March 29, 2001; Revised and Accepted June 8, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Although there are several methods for genotyping previously identified single nucleotide polymorphisms (SNPs), there is a paucity of approaches for high-throughput scanning for unknown variations. Mismatch repair detection (MRD) utilizes a bacterial mismatch repair system in vivo to detect sequence variants in human DNA samples. We describe modifications in MRD that allow a high degree of parallel processing, and use this modified version to accurately scan for variations in 35 different human DNA fragments simultaneously. MRD’s potential for high-throughput scanning can be used to identify new SNPs and to comprehensively compare sequences between patients and controls for identifying disease susceptibility alleles.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The identification of single nucleotide polymorphisms (SNPs) covering the entire genome will lead to numerous association studies of complex traits. Most scenarios for such studies assume a universal set of relatively frequent SNPs, distributed in all or most ethnic populations. One widely considered approach is to identify susceptibility alleles through direct association studies using SNPs located in coding or regulatory sequences. The main alternative strategy is to search for linkage disequilibrium (LD) between disease susceptibility alleles and SNPs from a dense genome-wide map (1–4). Either of the above approaches requires efficient genotyping to score for the presence or absence of previously identified SNPs (5–10). Both approaches, however, may be unrealistic when variant alleles, either those directly responsible for disease susceptibility or SNPs, are infrequent or are specific to a particular population (11,12). In such cases, identifying susceptibility alleles may require comprehensive sequence comparison between patients and controls (11). Accomplishing such sequence comparison requires a high-throughput DNA variation scanning technology to identify all possible variations in the tested fragments. The variant detection array (VDA) method is perhaps the only existing approach for DNA variant scanning with a high potential for parallel processing (13–17). However, VDA is expensive and may be sub-optimally specific and sensitive (18–20).
Mismatch repair detection (MRD) provides an alternative approach for high-throughput variant scanning. MRD detects variants using the mismatch repair system of Escherichia coli, which detects mismatches with high specificity and sensitivity (21). Escherichia coli detects single point mismatches as well as one-, two- and three-nucleotide loops, but does not detect loops of five nucleotides or more (22,23). The template for repair by E.coli is a hemimethylated double-stranded DNA (24). Mismatches in the hemimethylated DNA activate the mismatch repair pathway in E.coli and result in a large portion of the unmethylated strands being degraded and the methylated strands serving as a templates of repair (21,23,24). MRD utilizes the co-repair of a five-nucleotide loop in a marker gene as a signal of repair of a point mismatch in the tested DNA. A previously described version of MRD used LacZ
as the marker gene and the presence or absence of a variation in the fragment was assessed by visualization of bacterial colony color (21). By testing individual fragments for variations, MRD has been used to map genetically simple disorders (25), but the original form of the technology was not suitable for the high-throughput analysis needed to map complex traits. We now report modifications that permit use of MRD for high-throughput applications, most importantly through substitution of Cre recombinase for LacZ as the marker gene. We also provide initial evaluation of this modified version using human DNA samples.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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MRD procedure
MRD uses two vectors that are identical except for a 5 bp deletion in the gene coding for Cre recombinase on one of the two vectors. DNA fragments, such as exons, are cloned in the vector containing the active Cre, and grown in a bacterial strain that lacks the dam methylase. These clones, referred to as the standard panel, are made only once and serve as sequence comparison templates for exons from each person to be tested. The choice of the individual used as the source for the standard does not influence the results of MRD.
As depicted in Figure 1A, linearized DNA of the vector carrying the Cre gene with the 5 bp deletion, the single-stranded DNA from the pool of standards, and the pool of tested PCR products, are put in one tube. The three components are denatured and re-annealed to form heteroduplexes between the unmethylated single-stranded standard, its complementary PCR product, and the linearized vector carrying the inactive Cre. Two enzymatic steps are then performed to prepare closed circular heteroduplex molecules, which are transformed into an E.coli strain (mutation sorter, MS) engineered to carry on its episome (F' factor) two lox sites flanking tetracycline-resistant (TetR) and streptomycin-sensitive (StrS) genes. This strain carries a streptomycin-resistant (StrR) gene on its chromosome. In the absence of a variation in the test fragment, no repair occurs for the 5 bp loop in Cre (Fig. 1B). Subsequent replication generates a plasmid with an active Cre gene leading to the recombination of the TetR/StrS/lox cassette, rendering the cell tetracycline sensitive and streptomycin resistant (due to the StrR gene on the MS chromosome). However, the presence of a variation in the test fragment leads to its repair and the co-repair of the loop in Cre. As no active Cre is left in the cell it maintains its tetracycline-resistant and streptomycin-sensitive phenotype. Two pools, variant and non-variant, are separated by growth in two different media (Fig. 1C). As the multiple fragments being tested differ from each other in length, the fragment content of each pool can be identified using gel electrophoresis.
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Testing many different fragments simultaneously
To test MRD’s capacity for multiplexing, we performed MRD on a pool of 32 exons of genes involved in cancer pathogenesis or progression. We initially constructed a panel of 32 standard plasmids by cloning PCR products of the specific exons from one individual in the pMRD100 containing the active Cre gene. These plasmids were then pooled and served as a reference to compare with the test DNA. Thirty-two PCR reactions were performed in each of three independent tumor cell lines. The MRD procedure was performed with the PCR pools of each of the three tumors. After transformation, DNA was prepared from the two bacterial cultures grown in the presence of tetracycline or streptomycin. An agarose gel was run for a restriction digest that released the inserts of the DNA pools. The tetracycline pool, compared with the streptomycin pool, contained a higher proportion of variants between the standard and test DNA. We identified, in the three tumors, five such variant fragments in four distinct exons.
The exons predicted by MRD to carry variations were Patch exon 17, Cadherin exon 14, Ataxia Telangectasia Mutated (ATM) exon 31 and P53 exon 7. The Patch 17 exon was variant in two of the tumors. We sequenced Patch exon 17, Cadherin exon 14 and P53 exon 7 in all three tumors, and sequenced ATM exon 31 only in the tumor expected to carry the variation. The sequencing analyses confirmed all of the variants identified by MRD.
PCR products of the above experiment were run on an agarose gel before they were pooled. Some of these PCR products were quite impure, with the proper product representing minor species. This impurity did not interfere in the MRD results, showing that MRD can tolerate a high degree of PCR impurity.
Testing the specificity of MRD
We tested the specificity of MRD in a more complex background than in the above experiments by using an automated sequencing apparatus (ABI 377) to simultaneously scan 45 fragments derived from exons of genes involved in cancer pathogenesis and progression. Although we were able to identify multiple variations (data not shown), some fragments had the same size and therefore could not be analyzed.
We eliminated fragments that could not be resolved on the sequencing gels and tested only 35 fragments in subsequent experiments. We used MRD to scan for variations in 14 individuals with these 35 fragments which encompass >10 kb of DNA. Figure 2 represents the result of one such scan demonstrating a typical trace of the streptomycin and tetracycline pools of an MRD experiment run on an automated sequencing apparatus. One fragment was missing in both pools in all individuals and another was present in only a few individuals, presumably because of PCR failure. In these 14 individuals, we identified seven variations (Table 1). All seven variations were confirmed by subsequent sequencing, and consisted of five transitions, a transversion and a 1 bp deletion/insertion variant. We evaluated the consistency of MRD in detecting the same variation in different samples by sequencing all 14 individuals for three of these fragments. Only the individuals predicted by MRD to carry variations showed variants by sequencing. In the above two experiments we MRD-screened more than 400 fragments and obtained no false positives. Since not all the fragments were sequenced, this experiment did not address sensitivity.
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Testing the sensitivity of MRD
To test the sensitivity of MRD, we evaluated its ability to detect previously identified variations. We chose sequence tagged sites (STSs) known to carry SNPs, testing a total of five unrelated individuals using sequencing and MRD. These STSs included eight SNPs from published reports confirmed by us through direct sequencing, and two SNPs identified by us using sequencing. We made a standard for each of the STSs. All 10 of these SNPs were detected by MRD (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Our results show that MRD can scan for variations in multiple genomic fragments simultaneously with high specificity and sensitivity, is highly tolerant of PCR impurities, and that a single condition can be used for all the fragments tested without any need for optimization. All of these features are important for high-throughput applications.
The use of standards in this implementation of MRD provides several benefits for high-throughput variant detection. First, one is always comparing the test DNA with the same reference sequence. Secondly, by using standards one increases efficiency by avoiding any intermolecular ligation step. Finally, by analyzing the test DNA with two standards for two alternative common alleles, it is possible to distinguish the common variants from other alleles.
The number of fragments tested in the experiments described above is limited due to the relatively low resolution of sequencing gels. Methods with better power to distinguish between fragments are necessary to achieve the full potential of MRD. For example, microarray hybridization is now widely utilized for various quantitative assays using thousands of DNA fragments simultaneously (26,27). By adapting microarray hybridization technology as the last detection step in the MRD procedure, we anticipate that thousands of fragments can be scanned for variations simultaneously.
The different MRD steps can likely accommodate up to 5000 fragments simultaneously, including the hybridization step leading to heteroduplex formation and the transformation step. This hybridization is less complex than solution hybridizations that have previously been carried out successfully (28). At the end of the MRD procedure, we typically obtain a million transformants; this number should be sufficient for analyzing up to about 5000 fragments. The microarray hybridization step will be simpler than typical microarray expression applications that use more than 10 000 genes as a target and the whole cell RNA as a probe (29,30). The signal to noise ratio for MRD is likely to remain favorable as it scales up to applications involving many thousand fragments, as each fragment is detected independently. Our experience so far, showed that this ratio remained constant in the scale-up from two to 45 fragments.
Because of its potential for multiplexing, MRD is most suitable to those applications where many fragments in many individuals are scanned for variations. Currently, the only scanning technology that can accommodate a significant degree of multiplexing is VDA, which has been used to scan for variations in as much as 30 kb of human DNA, 
3-fold more DNA than covered in the work presented here. However, this application of VDA encountered problems with both sensitivity and specificity (18–20). In addition, the large number of probes needed limits the throughput and increases the cost of VDA. For example, at least 3000 oligos on the array are required using VDA to scan a 300 bp fragment for variations. In contrast, using MRD with array hybridization as the readout, as proposed above, a 300 bp fragment can be scanned for variations with one probe on the array. The difference in the number of probes needed to interrogate each fragment is reflected in a potentially much higher throughput and lower cost for MRD.
Given the absence of a high-throughput scanning technology and the relative wealth of SNP genotyping methodologies, proposals for identifying susceptibility alleles for common diseases have focused on genotyping a universal set of common SNPs. These approaches assume that disease susceptibility alleles are common. Only a few genes contributing to common disease have already been identified. The alleles involved in these diseases were common in some instances (31), but in several other instances relatively rare alleles are associated with, or causative of, disease (32–38). Recent studies have scanned for variants in numerous genes considered possible candidates for involvement with common diseases; these studies have shown that, in general, non-synonymous coding sequence variants are relatively infrequent, and are often specific to a particular ethnic population (20,39,40). It is likely that common disease is caused by a combination of common and rare alleles. A comprehensive sequence comparison of genes among patients and controls permits high-throughput detection of the rare or ethnically specific alleles and therefore provides a more comprehensive model than currently proposed for the elucidation of the genetic basis of common disease. Such comparisons can be carried out by direct sequencing; however, this is currently a prohibitively expensive undertaking.
We propose that MRD analysis of all the coding and regulatory regions of all genes can provide comprehensive sequence comparison of selected patients and controls to identify disease-associated variants. By testing 5000 fragments that are on average 300 bp in length, in a single MRD reaction, one could perform such sequence comparison on 1.5 Mb of genomic DNA. Therefore, in 100 MRD reactions, one could scan up to 150 Mb of DNA, an amount larger than the estimated total of the coding regions in an individual. Multiple developments need to occur before MRD can be applied on this scale, including the identification of the finished sequence for all genes and their regulatory regions, the construction of several hundred thousand standards, and either advancement in PCR multiplexing or alternative ways to capture many DNA fragments. Over the short run, we propose using MRD to identify SNPs that can be scored by MRD or other genotyping methods, and to scan for variations in panels of candidate genes in patients and controls.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of MRD vectors and strains
All the restriction and modifying enzymes used were from New England Biolabs (NEB). All the reactions were performed according to the enzyme manufacturer’s recommendations, except for the blunt-end ligation which was performed using ligation buffer (Gibco BRL). The bacterial strain used for all the transformations and growth was DH5
unless specified otherwise.
Construction of vectors
The Cre gene was PCR amplified using a construct carrying Cre as a template. The PCR left 13 bp 5' of the start ATG of the protein. The PCR product was cloned in PstI/BamHI-digested pBSK (Stratagene). The Cre gene was then used to replace the LacZ gene in pMF200 (21). To this end, the Cre fragment was released by an EcoRI/XbaI double digestion and the ends were filled using the Klenow fragment. PMF200 was partially digested with BglI and treated with T4 DNA polymerase to chew the 3' overhang followed by BamHI digest (the BamHI site is present in the leader sequence of LacZ of pMF100) and end filling by Klenow fragment. A blunt-end ligation produced pMRD100, which replaced the LacZ gene on the BglI–BamHI fragment of pMF100 by the Cre gene. In this construct, the plac promoter drives the expression of Cre.
PCR was performed using pMR100 as a template and the M13 reverse primer with a primer creating a 5 bp deletion in Cre. The deletion was for the sequence CTACA, 207 bp downstream from the ATG start site. The PCR product was digested with NruI and SphI. pMRD100 was also digested with NruI and SphI and the small fragment was substituted with the digest from the PCR product. The recombinant plasmid was pMRD200 carrying a 5 bp deletion in Cre but otherwise identical to pMRD100. Sequence analysis confirmed that no other change in Cre occurred except for the 5 bp deletion.
Construction of mutation sorter
The starting vector was pML11, a pBKS (Stratagene) derivative cloned in its multiple cloning sites, with the tetracycline gene flanked by two lox sites. In order to clone the wild-type StrS gene, we performed PCR using a bacterial strain carrying the wild-type gene as a template and the following primers: StrI, GAG GGT AAC CGC TAC CTT GAA AGT C; StrII, GCT GAA TTC GTT TGG CCT TAC TTA ACG GAG AAG.
The size of the StrS PCR product was 700 bp. T4 polymerase treatment was performed to trim the ends, followed by an EcoRI digest to produce a substrate for directional cloning. This fragment was cloned in EcoRI/SmaI-digested pML11, replacing the tetracycline gene and producing pMLstrp. In order to put the streptomycin and tetracycline genes in tandem, the large fragment of pML11 digested with KpnI/SmaI was ligated with the small fragment of pMLstrp, which was EcoRI digested, Klenow filled, with subsequent KpnI digestion. The recombinant produced was pMLST. pMLST was then digested with XhoI/SacI releasing the tetracycline/streptomycin/lox cassette. The fragment was cloned in pLitmus 29 (NEB) digested with XhoI/SacI. The resultant plasmid, pMLSTB, was digested with KpnI and a BglII linker was inserted.
In order to put the construct on the bacterial F' episome, we utilized the vector pNK2859 (41) carrying the transposase gene. This plasmid produces a stable transposition event, as the transposase gene carried on the plasmid does not get transferred itself. We ligated a BglII fragment of pMLSTB carrying the cassette of interest with a BamHI digest of pNK2859. The obtained plasmid, pNKMLST, carried the fragment of interest flanked by a transposition signal. The plasmid also carried the transposase gene that catalyzes the transposition event but does not get transposed itself.
We transformed pNKMLST into a DH5 strain carrying an F' episome. This strain was grown and in some cells transposition events to the episome presumably happened. In order to isolate those cells, the culture was mated to HB101 containing a plasmid with a temperature-sensitive replication and chloramphenicol (Cm) resistance gene (CmR). The transfer of an F' episome carrying the transposition product to HB101 should transmit the tetracycline resistance phenotype. The mating was performed as described previously (42). Selection for tetracycline and chloramphenicol resistance was done. Selected colonies were screened for carbenicillin (Carb) and streptomycin sensitivity (HB101 is streptomycin resistant). The desired conjugants had the following phenotype: TetR, CmR, CarbS and StrpS. Growth at 42°C was performed to get rid of the plasmid carrying the CmR gene. Different isolates were obtained that presumably carry the tet/str/lox cassette at different places on the F' episome. Through transformation with pMRD100, pMRD200 as well as the heteroduplex, the strain, named conj5, with the least background was selected.
Although conj5 was used for some MRD experiments, we preferred to use the DH5 background. Selection for a streptomycin-resistant mutant was carried out in DH5 by plating a culture on a plate containing (10 mg/ml) streptomycin. The new strain was called DH5S. The F' episome of conj5 was transferred to the DH5S strain by mating with a huge excess of DH5S. The conjugation was performed by mixing the two strains at 1:1000 ratio and leaving them at 37°C overnight before selection on tetracycline X-gal/IPTG plates. The DH5S that have acquired the F' episome were distinguished from conj5 by the colony color because the former appears white, whereas the latter is blue on ITPG/X-gal plates. The DH5S with the F' episome carrying the tet/str/lox cassette is called the MS strain.
Construction of standards
pMRD100 was grown in the dam– host, SCS110 (Stratagene), and unmethylated DNA was prepared. The vector DNA was digested with HincII and the linear molecule was purified by agarose gel electrophoresis (gel purification not necessary). PCR reactions from an individual were performed using pfu polymerase (Stratagene). In some cases, a few of the products were pooled. The PCR products were cleaned using DNA binding columns (Qiagen) according to the manufacturer’s protocol. Ligation reaction was set up between the linearized vector and 1–10 PCR products, with the latter being used in excess. After ligation overnight at room temperature, a column purification (Qiagen) was performed to get rid of the ligase and salts. SalI digestion reaction was then performed to linearize DNA vectors that do not contain inserts. Another column purification (Qiagen) was performed before electroporation into SCS110 cells using electroporation equipment (Bio-Rad) as described previously (43). In order to identify the colonies containing a recombinant molecule, PCR was performed from individual colonies using T3 and T7 as primers. PCR products that contain inserts were then subjected to sequencing using the dideoxy chemistry and T3 as a sequencing primer. Proper clones, as determined by sequencing, are the standards that are used as a reference for comparison with the test DNA. Alternatively, the ligation products were transformed into DH5 instead of SCS110. The proper clones were later transformed into SCS110. Single-stranded unmethylated DNA of the standards was prepared by using the helper phage M13K07 (NEB) according to the manufacturer’s recommendations. In later experiments, we deployed a more efficient process for the standard construction. 100 PCR fragments were pooled together and cloned to create a library for these clones. Sequencing of the clones identified those that can serve as standards. Another round of cloning was done for those clones not obtained in the first round.
MRD protocol
For each MRD reaction, a total of 1 µg of the pool of the unmethylated single-stranded standard DNA was digested with 5 U MboI for 30–60 min at 37°C to destroy any double-stranded contamination (in later experiments we used ScrFI instead of MboI). The enzyme and the buffer were removed using a column from Qiagen. About 2 µg of vector carrying the inactive Cre gene was digested with HincII and purified by gel electrophoresis (gel purification is not necessary). The above two preparatory steps were generally performed in big batches and aliquots were used in the subsequent MRD reaction.
PCR reactions from the tested individual were carried out using pfu polymerase. The template tumor cell line DNA used in the initial experiment to test many fragments simultaneously, was obtained from ATCC. The template DNA for the experiments testing the specificity and sensitivity of MRD were for individuals from the NIH DNA Polymorphism Discovery Resource (44). The PCR products from one individual were pooled and a purification was performed using a column supplied by Qiagen. The PCR pool was then phosphorylated (if primers did not have a 5' phosphate from the synthesis) with 10 U polynucleotide kinase (NEB) for 1–2 h at 37°C. The enzyme and the buffer were then removed by treatment with the SOPE resin/column from Edge Biosystems (EB). Dam methylation was then performed using dam methylase (NEB) at 37°C for 1–2 h according to the manufacturer’s recommended buffer. Column purification (Qiagen) was then performed to remove the enzyme and the buffer. The methylated PCR was mixed with 1 µg of the above-described unmethylated standards and 2 µg of the purified linear vector carrying the inactive Cre. An aliquot of 5 µl of 0.5 M EDTA and 1.5 µl of 3 M NaCl was added and the volume was adjusted to 100 µl with TE. An aliquot of 12.5 µl of freshly diluted 1 M NaOH was added, followed by incubation for 15 min at room temperature. Aliquots of 12.5 µl of 2 M Tris pH 7.2 and 125 µl formamide were then added and reannealing was allowed to occur at 30°C overnight. The heteroduplex was cleaned using a column (Qiagen) and resuspended in 30 µl H2O. Added next was 3.5 µl Taq ligase buffer, 1 µl of ethidium bromide (300 µg/µl) and 1 µl Taq ligase (NEB). The addition of the ethidium bromide was not essential but it may improve the signal to noise ratio. Nick closure reaction was performed at 65°C for 15 min. The SOPE resin/EB column were used to remove the enzyme and the buffer. Fifty units of exonuclease III (NEB) were then used with the appropriate buffer to convert the nicked DNA to single-stranded molecules. Another SOPE resin/EB column step was utilized to remove the enzyme, buffer and the single-stranded DNA. To ensure the removal of all the single-stranded DNA, 0.7 mg benzoylated naphthoylated DEAE cellulose (BNDC) was added and NaCl added to a final concentration of 1 M, and incubation proceeded for 15 min, shaking at room temperature. BNDC binds preferentially to single-stranded DNA. The mixture was run over an EB column to remove the salt and the BNDC with its bound single-stranded DNA. Although the results described in this work utilize the BNDC step, the deletion of this step did not affect results. After removing the single-stranded DNA, the closed circular heteroduplex molecule was concentrated by speed vacuum. The preparation of the closed circular heteroduplex molecules for the initial experiment testing 32 exons was performed employing an alternative protocol. We purified the closed circular molecules using agarose gel electrophoresis. To eliminate this step, the exonuclease III step was employed in later experiments as described above.
Transformation of the MS strain was done by electroporation. The electrocompetent MS cell preparation as well as the electroporation procedure was carried out as recommended (43). During the 1 h recovery phase, 1 µl of 1 M IPTG was added to the SOC medium. The culture was split into two parts that were plated onto two plates supplemented with Carb (75 µg/ml), streptomycin (10 µg/ml) and IPTG (64 µg/ml), or Carb (75 µg/ml), tetracycline (3.25 µg/ml) and IPTG (64 µg/ml). Typically, a total of about 1 million transformants was obtained. One milliliter of LB was put on each of the two plates and all the colonies from a plate were then collected into a tube. All of the experiments described used plates except the initial experiment with the 32 different fragments where the transformation mixture was grown in two cultures with the appropriate selective media. In both cases (plates or cultures) DNA from the cells obtained after the overnight growth with the selective media was miniprepped using the Qiagen columns as recommended by the manufacturer. Fifteen to thirty percent of the DNA obtained in the miniprep was digested with ClaI and XhoI (NEB) at 37°C for 1–2 h. When agarose gels were employed, the DNA was run on a 4% New Sieve gel. When ABI 377 gels were used, the digested DNA was passed over a filter column (EB) to get rid of the salt. The restriction fragments were labeled utilizing a fluorescent dUTP. This was achieved by adding 0.5 U of ampliTaq polymerase (Perkin Elmer), Taq buffer and MgCl2 at the recommended concentration, as well as R6G dUTP (Perkin Elmer) at a final concentration of 0.8 µM. The nucleotide extension reaction was performed at 72°C for 10 min. An EB column was then used to remove the excess fluorescence. The sample was concentrated by speed vacuum and then run on an ABI 377 sequencing gel. The data were analyzed by Gene Scan software (Perkin Elmer). Fragments were determined to carry a variation if the ratio in the tetracycline to the streptomycin pool is significantly higher than that of non-variant fragments. A typical trace is shown in Figure 2.
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ACKNOWLEDGEMENTS |
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We thank S.Service for helpful comments on the manuscript and help on the artwork, and Dr Ira Herskowitz for helpful comments on the manuscript. This work was supported by grants K02MH01375 and NS37484 from the National Institutes of Health to N.B.F.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: UCLA Center for Neurobehavioral Genetics, 695 Charles E. Young Drive South, Gonda Building, Room 3506, Los Angeles, CA 90095-1761, USA. Tel: +1 310 794 9571; Fax: +1 310 794 9613; Email: nfreimer@mednet.ucla.eduPresent addresses: Malek Faham, Stanford Genome Technology Center, 855 California Avenue, Palo Alto, CA 94304, USASiamak Baharloo, Double Twist Inc., Oakland, CA 94612, USAShinichiro Tomitaka, Department of Psychiatry, Tokyo Women’s Medical University, Kawada-cho 8-1, Shijuku-ku, Tokyo, JapanJoe De Young, Genomics Core Facility, University of California San Francisco, San Francisco, CA 94304, USANelson B. Freimer, Center for Neurobehavioral Genetics, Departments of Psychiatry and Human Genetics, University of California Los Angeles, Los Angeles, CA 90095, USA
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