- Methodology article
- Open Access
Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays
© Watson et al; licensee BioMed Central Ltd. 2004
- Received: 17 October 2003
- Accepted: 14 January 2004
- Published: 14 January 2004
The recent development of array based comparative genomic hybridization (CGH) technology provides improved resolution for detection of genomic DNA copy number alterations. In array CGH, generating spotting solution is a multi-step process where bacterial artificial chromosome (BAC) clones are converted to replenishable PCR amplified fragments pools (AFP) for use as spotting solution in a microarray format on glass substrate. With completion of the human and mouse genome sequencing, large BAC clone sets providing complete genome coverage are available for construction of whole genome BAC arrays. Currently, Southern hybridization, fluorescent in-situ hybridization (FISH), and BAC end sequencing methods are commonly used to identify the initial BAC clone but not the end product used for spotting arrays. The AFP sequencing technique described in this study is a novel method designed to verify the identity of array spotting solution in a high throughput manner.
We show here that Southern hybridization, FISH, and AFP sequencing can be used to verify the identity of final spotting solutions using less than 10% of the AFP product. Single pass AFP sequencing identified over half of the 960 AFPs analyzed. Moreover, using two vector primers approximately 90% of the AFP spotting solutions can be identified.
In this feasibility study we demonstrate that current methods for identifying initial BAC clones can be adapted to verify the identity of AFP spotting solutions used in printing arrays. Of these methods, AFP sequencing proves to be the most efficient for large scale identification of spotting solution in a high throughput manner.
- Bacterial Artificial Chromosome
- Comparative Genomic Hybridization
- Bacterial Artificial Chromosome Clone
- Bacterial Artificial Chromosome Array
- Bacterial Artificial Chromosome Clone Sequence
Comparative genomic hybridization (CGH) is a technique used to determine regional DNA copy number changes across an entire genome . This is accomplished by co-hybridizing differentially labeled genomic sample and reference DNA to a metaphase chromosome spread of cultured cells. Analysis of the metaphase chromosomes will reveal regions of amplification or deletion in the sample DNA . This technique is limited to the resolution at which the amplifications and deletions can be detected of approximately 10–20 Mb . The recent development of array based CGH technology has improved the resolution of genomic profiling . This involves the substitution of the target DNA from metaphase chromosomes to selected DNA segments spotted onto a microarray, where the distance between target segments determines the resolution. Current methods for creating CGH arrays include spotting whole bacterial artificial chromosomes (BAC) DNA, degenerate oligonucleotide primer (DOP) PCR derivatives of BAC DNA, and amplified fragment pools (AFP) of BAC DNA generated by linker mediated (LM) PCR [4–6]. These procedures aim at producing large quantities of DNA from a library of clones, generating spotting solutions with high DNA concentration.
Currently the highest density genome wide CGH array consist of 2460 LMPCR synthesized AFP spaced at 1.4 Mb intervals throughout the human genome . However, with the completion of the human and mouse genome sequencing, large clone sets (tens of thousands of BAC clones) providing complete genome coverage are available for construction of higher resolution arrays [11–14]. Since generating spotting solution from the initial BAC DNA requires multiple liquid transfer steps it is necessary to verify that the final spotting solution is representative of the initial clone. The construction of whole genome arrays necessitates the development of high throughput methods suitable for verification of AFPs prior to spotting arrays.
DNA restriction digest fingerprint analysis, fluorescent in-situ hybridization (FISH) mapping, and BAC end sequencing are commonly used to verify the identity and genomic location of BAC clones [7–9]. However, these clone verification procedures are applied to the BAC DNA prior to multi-step spotting solution synthesis.
Here we demonstrate that these commonly used methods applicable for identification of the initial BAC clone DNA can be adapted for use in verifying AFP just prior to spotting the array.
Comparison of three techniques for AFP identification.
BAC END SEQUENCING
Verification of AFP to original BAC
Representation of original BAC in AFP
Relative cost per assay
Requires normal cell line for metaphase
Requires 200 ng of digested BAC DNA
Can be automated for high throughput analysis
These methods are suitable for sampling AFPs derived from individual BAC clones. Although multiple FISH or Southern analysis can be performed in parallel, these approaches are not easily adapted for high throughput analysis (Table 1).
BAC end sequencing can be processed in a 96 well format but requires purified DNA template. AFPs are typically precipitated with ethanol and resuspended directly in spotting solvent (i.e., 20% DMSO, 50% formamide), which will inhibit the sequencing reaction. In this study we demonstrate that modifications to the Applied Biosystems sequencing protocol allow unpurified AFPs to serve as templates for sequence identification (Fig. 1B). To compensate for sub-optimal conditions due to carry over of unpurified material we increased the template quantity to 20 fmol from the minimum recommended of 2 fmol, and increased the number of sequencing cycles from the typical 35 to 85. Reactions performed using less than 20 fmol or fewer than 85 cycles did not yield sufficient signal for analysis (data not shown). These modifications may have been necessary due to the carry over of primers and reagents from the previous PCR reactions (Fig. 1A) and the complexity of the DNA mixture in the AFP.
To demonstrate the utility of this method we randomly selected 960 clones from the RPCI-11 or RPCI-13 human BAC libraries [15, 16] After LMPCR amplification (see methods), 4% of the total unpurified AFP were sequenced using the T7 primer. Half (468) of the AFP yielded sequences and 448 of these were matched to specific BAC clone sequences. Twenty matched repetitive sequences, representing multiple GeneBank entries.
To determine if the probability of identifying the LMPCR product increased with use of the Sp6 primer, 83 AFPs were sequenced. Of the 83 AFP sequenced, 64 returned usable sequences and 60 of these were matched to a specific BAC. Four matched repetitive sequences, representing multiple GeneBank entries. Combining the results from the Sp6 and T7 sequence reads, it was possible to identify 76 of the 83 AFPs (91%).
The ability to sequence unrefined PCR products and the requirement of only 4% of the AFP makes direct end sequencing of AFP an effective means of verifying array spotting solution.
Linker mediated PCR amplification of BAC DNA
Fifty nanograms of each BAC DNA sample was transferred to a 96 well plate and digested for eight hours with 5 U of Mse I (New England Biolabs) in a 40 μl reaction. The reaction mixture was inactivated at 65°C for 10 min. Ten percent of the product was transferred to a new plate and ligated to linkers. The ligation mixture consisted of the digested DNA, 0.2 μM primers each of Mse I long (5' AGTGGGATTCCGCATGCTAGT 3') and Mse I short (5' TAACTAGCATCG 3') (Alpha DNA, Quebec) and 80 U of T4 DNA ligase in NEB ligase buffer (New England Biolabs). The primers were allowed to anneal for 5 min at room temperature before addition to the ligation mix. The ligation was performed overnight (12–16 h) at 16°C.
A 2.5 μl aliquot of the 40 μl ligation mixture was amplified in a 50 μl PCR reaction. The reaction mixture contained the linker-ligated DNA template, 8 mM MgCl2, 1 mM each dNTP's (Promega), 0.4 μM Mse I longprimer, and 5 U of Taq polymerase (Promega, storage buffer B) in Promega PCR buffer. After a 3 min 95°C denaturation step, the PCR cycled at 95°C for 1 min, 55°C for 1 min, and 72°C for 3 min, for 30 cycles. A 10 min extension at 72°C completed the protocol. The second round of PCR was initiated using 0.25 μl of the PCR product under the same conditions for 35 cycles. After ethanol precipitation, the final concentration of DNA was quantified using a ND-1000 spectrophotometer (Nanodrop, Delaware). Typical yield for LMPCR was 40–50 μg.
Sequencing of AFP
To determine the sequence of each amplified fragment pool, 2 μl of AFP was combined with 4 μl Big Dye (Perkin Elmer), 0.32 pmol T7 primer (5' TAATACGACTCACTATAGG 3') or SP6 (5' ATTTAGGTGACACTATAG 3') (Alpha DNA) in a 10 μl final volume. After a 1 min initial denaturation step at 95°C, the reaction mixture was subjected to 85 cycles of 95°C 15 s, 50°C for 5 s, and 72°C for 4 min. All steps were ramped at 1°C/s using a MJ Research Peltier thermocycler. The big dye sequencing reaction product was either ethanol precipitated or purified via PCR Min-elute (Qiagen). Sequencing reaction products were resolved using an ABI Model 377 or ABI Model 3700 sequencer (Applied Biosystems).
Sequences were analyzed using NCBI BLAST to query the non-redundant (nr) and high throughput genomic sequences (htgs) database of GeneBank v.2.2.5. The FTP version of BLAST  was downloaded and a script written to allow all 960 sequences to query automatically. Expect values (E values) of 0.001 and bit scores of 30 were used as the minimum allowed cut off.
The use of Southern analysis to verify BAC clones for array construction has previously been described . DNA was prepared from overnight cultures of BAC clones. Two hundred nanograms of Hind III digested BAC DNA fragments were separated by electrophoresis on a 1% agarose gel. The separated fragments were transferred to a Hybond-N+ membrane as recommended by the manufacturer (Amersham). One microlitre of AFP (~1 μg) was labeled with α32P-dATP using the RadPrime random priming system (Invitrogen). The labeled probes were precipitated in ethanol with (or without) 50 μg Cot-1 DNA (Invitrogen) and redissolved in 15 μl of hybridization solution (50% formamide, 2X SSC, 10% dextran sulfate, 4% SDS). The probe was denatured at 80°C for 10 min and allowed to cool to 37°C for 2 h before addition to the prehybridized membrane. Hybridization was performed at 65°C overnight in the presence of 0.5 μg/μl of sheared herring sperm DNA (Invitrogen). Washes were performed at 65°C with Buffer 1 (5 mg/ml BSA, 0.5 mM EDTA, 40 mM Na2HPO4 (pH 7.2), 5% SDS) followed by Buffer 2 (2 mM EDTA, 80 mM Na2HPO4 (pH 7.2), 2% SDS). Autoradiographs were generated from phosphoimager plates and analyzed using the STORM 860 system (Amersham).
Fluorescence in situ hybridization
Selected AFPs were mapped by FISH using metaphase chromosomes. Two microlitres of AFP (~2 μg) were labeled by random priming overnight in the presence of 2 nmol of Cy3-dCTP, Cy5-dCTP (Perkin Elmer), FITC-dUTP, or Texas Red-dUTP using the BioPrime kit (Invitrogen) as per manufactures directions. The labeled probe was purified using a Sephadex G-50 column, combined with 21 μg of Cot-1 DNA and precipitated with ethanol. The labeled probe was then resuspended in 80 μl of hybridization buffer (50% formamide, 2X SSC, 10% dextran sulfate, 0.1% Tween-20, 10 mM Tris pH 7.4) and denatured for 5 min at 100°C. The metaphase slide was dehydrated through a series of 70%, 80%, and 100% ethanol washes for 2 min each, denatured in 70% formamide in 0.6X SSC for 2 min at 70°C and processed through the same ethanol series at -20°C and allowed to dry. Thirty-five microlitres of probe was then added to the slide and hybridized overnight at 37°C. Images were processed with Qcapture (Q-imaging, Vancouver) with a Zeiss Axioscope microscope.
We would like to thank Homa Azad of the BC Cancer Research Centre Sequencing Service and the Michael Smith Genome Sciences Centre at the BC Cancer Agency for performing sequencing reactions. We would like to acknowledge Drs. Donna Albertson and Daniel Pinkel at the University of California at San Francisco for useful discussion on LMPCR methodologies. Also, we would like to thank Bryan Chi for assistance in bioinformatics analysis, Baljit Kamoh for FISH analysis, and Kim Lonergan for manuscript preparation.
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