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Platform GPL28 Query DataSets for GPL28
Status Public on Feb 12, 2002
Title HumArray 1.14
Technology type spotted DNA/cDNA
Distribution non-commercial
Organism Homo sapiens
Description This array was designed for copy-number assessments across the human genome. We printed 2,460 BAC and P1 clones in triplicate (approx. 7,500 elements) in a 12mm X 12mm square. Each clone contains at least one STS, allowing linkage to the genome sequence. Cytogenetic mapping indicated that 2,298 of the arrayed clones are single copy; this array thus provides average resolution of approximately 1.4 Mb across the genome.

Protocol from Nat Genet 2001 Nov;29(3):263-4 web supplement:
<b>Genomic clones.</b> We selected the majority of the clones for the arrays from the set of cytogenetically mapped BACs reported previously4 and obtained these clones from the Roswell Park Cancer Institute. We obtained additional clones mapping near the telomeres of each chromosome5 and clones containing certain named genes from J. Flint, D. Ledbetter, C. Lese and Vysis, Inc. We included P1 clones used previously on arrays of chromosome 20 (ref. 1,2). We also used additional clones from the DuPont A library, including RMC01P052 (163D7), RMC01P057 (213C4), RMC07P014 (548H7), RMC07P025 (1128F4), RMC07P028 (252B4) and RMC07P038 (1429E1.c2), as well as RMC17P041 (75D7), RMC17P069 (88H6) and RMCXP002 (124A3) from the DuPont B library. The array provides only minimal coverage of the Y chromosome, since only one clone unique to the Y chromosome is included on the array in addition to the telomeric clones that are shared between the X and Y chromosomes.
<b>Isolation of BAC/P1 DNA.</b> We inoculated 0.1l of a glycerol stock of BAC or P1 containing bacteria into 5 ml LB media containing chloramphenicol (10 g/ml) or kanamycin (50 g/ml) for BACs or P1s, respectively and incubated the cultures overnight (~16 h) at 37 C with agitation at 225 rpm. We prepared larger cultures for DNA isolation, by inoculating 25 mlLB media, containing the appropriate antibiotic with 200 l of the previously grown overnight culture and incubating in a shaking incubator at 37 C at 225 rpm overnight (~16 h). We monitored bacterial growth by measuring the OD of a 1:10 dilution, which preferably ranged between 0.25 and 0.35 at a wavelength of 600 nm. We used the Qiagen Plasmid Mini kit to isolate DNA, following a modified version of the Qiagen Plasmid Mini Purification protocol. We transferred the 25 ml cultures into 50 ml tubes and centrifuged them for 15 min at 4000 rcf at 4 C to pellet the bacteria. We re-suspended the pellet in Qiagen buffer P1 (1.5 ml), containing RNase A at the manufacturer's recommended concentration and then added buffer P2 (1.5 ml). We mixed the tubes extremely gently by inversion and incubated at room temperature for 5 min. Then, we added buffer P3 (1.5 ml), again mixed very gently by inversion and incubated on ice for 10 min. We inverted each tube once and centrifuged at 4000 rpm at 4 C until the supernatant became clear (45 to 60 min). We filtered the supernatant through a 35-micron nylon mesh prior to loading onto Qiagen-tip 20 columns, which had been equilibrated following the manufacturer's protocol. We followed the manufacturer's protocol for the DNA isolation steps with the exception that the elution buffer QF was heated to 65 C before it was added to the column. After elution, we added 0.56 ml isopropanol to the DNA and incubated overnight at 4 C. We collected the DNA pellet by centrifugation for 45 min at 14000 rpm at 4 C. After aspirating the supernatant, we allowed the pellet to dry in air for at most 30 min before re-suspending the DNA in 50 l of H2O. We determined the DNA concentration using a fluorometer. The yield typically ranged between 60-100 ng/l (i.e. 3-5 g per isolation). To ascertain DNA purity, we digested each BAC (200 ng) with HindIII and electrophoresed the digest through a 0.75% agarose gel. We discarded DNA preparations that showed a significant contamination with host bacterial DNA, seen as a background smear of degraded DNA in the gel.
<b>Preparation of BAC/P1 DNA representations by ligation-mediated PCR.</b> We digested the DNA with MseI by incubating overnight at 37 C in a 5 l reaction containing 1.5 l DNA (20 to 600 ng), 0.4 U/l MseI (New England Biolabs), and 0.4x One-Phor-All-Buffer-Plus (Amersham). For ligation of adapters, we diluted 1 l of each digest to a final concentration of 1 ng/l with H2O and then mixed 1 ng of the digested DNA with 0.5 l One-Phor-All-Buffer-Plus (10x, Amersham), 0.5 l of 100 M Primer 1 (TAACTAGCATGC), 0.5 l of 100 M Primer 2 (5' aminolinker AGTGGGATTCCGCATGCTAGT) and 5.5 l H2O. We incubated the mixture at 65 C for 1 min after which we ramped the temperature down to 15 C with a ramp-speed of 1.3 C per minute. When the temperature reached 15 C, we added 1 l of 10 mM ATP and 1 l T4-DNA ligase (5 U/l, Gibco BRL) and continued incubation at 15 C overnight. To initiate the first round of PCR amplification, we added a 40 l mixture, consisting of 3 l PCR Buffer 1 (Expand Long Template PCR System, Roche), 2 l of a mixture of each nucleotide (10 mM) and 35 l H2O to the ligation reaction. Before we started the PCR program, we incubated the reaction at 68 C for 4 min and then added 1 l DNA polymerase (3.5 U/l, Expand Long Template PCR System, Roche). We carried out thermal cycling in a Perkin-Elmer Gene Amp PCR System 9700 block as follows: 94 C for 40 s, 57 C for 30 s, 68 C for 1.25 min for 14 cycles, followed by, 94 C for 40 s, 57 C for 30 s, 68 C for 1.75 min for 34 cycles and 94 C for 40 s, 57 C for 30 s and 68 C for 5 min for the final cycle. We electrophoresed 3.5 l of the PCR product through a 1% agarose gel to determine the size range of the amplified DNA, which ideally ranged from 100 to 2000 bp. To make the DNA for spotting on the arrays, we carried out a second round of amplification in a 100 l reaction containing 1 l of the primary PCR product, 4 M Primer 2, TAQ-buffer II (1x; Perkin Elmer), 0.2 mM dNTP mix, 5.5 mM MgCl2 (Perkin Elmer), 2.5 U Amplitaq Gold (Perkin Elmer) and H2O. We carried out an initial incubation at 95 C for 10 min in a MJ Research Peltier Thermal Cycler 225, followed by 95 C for 30 s, 50 C for 30 s and 72 C for 2 min for 45 cycles and finally 7 min at 72 C. This reaction yields ~10 g of DNA, with each fragment containing a 5' amino linker.
<b>Preparation of DNA spotting solutions.</b> We evaporated the amplification reaction (100 l) to a final volume of 50 l by incubation at 45 C in a hybridization oven (Techne, Hybridizer HB-1D) for approximately 75 min and then added 2.5 volumes of ice-cold ethanol and 0.1 volumes of 3 M NaOAc to precipitate the DNA. (The use of ethanol precipitation proved superior to isopropanol precipitation.) We inverted the tubes and chilled them at 20 C for 15 min before collecting the precipitate by centrifugation at 1699 rcf for 90 min. We washed the pellets with 70% ethanol (150 l) and collected them by centrifugation at 1699 rcf for 45 min. We air-dried the pellets for approximately 60 to 90 min and then re-suspended them in 12 l of 20% DMSO in H2O (~0.8 g/l). We transferred the DNA solutions into 864 well microtitre plates for robotic arraying. Previously, we prepared DNA for spotting in 80% DMSO and 0.3 g/l nitrocellulose. However, we subsequently evaluated spotting solutions containing various concentrations of dimethyl formamide, formamide or DMSO, with or without nitrocellulose. We determined that nitrocellulose was not required for spotting DNA prepared by PCR using primers with 5'amino linkers and that spotting solutions made with 20% DMSO performed well. We have also generated representations of large insert clones for arraying by using degenerate oligonucleotide primed PCR and by amplification of mixtures of subclones from BACs, but found the ligation-mediated PCR procedure to be superior.
<b>Array printing.</b> We used a custom built printer, employing a 4 x 4 array of quartz capillary tubes spaced on 3 mm centers to print ~70-100 µm diameter spots on 130 µm centers. We printed each DNA solution in triplicate to create an array of ~7500 elements in a 12 mm square area. We printed the arrays on chromium coated microscope slides (PTI or Nanofilm) for these studies, but also routinely print the arrays on glass slides (Corning GAPs). We allowed the printed slides to dry overnight, then exposed the slides to UV light (65 mJ) in a UV Stratalinker 2400 (Stratagene). We hybridized to these slides without additional treatment, except for the pre-hybridization slide blocking described below. We found no indication of DNA loss from the spots at any stage of the procedure when we performed hybridization in formamide buffers at 37 °C.
Keywords = comparative genomic hybridization, CGH, array
Contributor(s) Snijders AM, Nowak N, Segraves R, Blackwood S, Brown N, Conroy J, Hamilton G, Hindle AK, Huey B, Kimura K, Law S, Myambo K, Palmer J, Ylstra B, Yue JP, Gray JW, Jain AN, Pinkel D, Albertson DG
Citation(s) 11687795
Submission date Jan 10, 2002
Last update date Oct 28, 2005
Contact name Donna G Albertson
Phone 415 502-8463
Organization name University of California San Francisco
Department Comprehensive Cancer Center
Street address
City San Francisco
State/province CA
ZIP/Postal code 94143
Country USA
Samples (61) GSM794, GSM795, GSM796, GSM797, GSM798, GSM799 
Series (2)
GSE16 Microarray-based CGH in human cancer cell lines
GSE5051 Cross-Platform Array Comparative Genomic Hybridization (array CGH) Meta-Analysis

Data table header descriptions
ID Unique row identifier, genome position order
CLONE_ID Clone identifier
SPOT_ID Spot identifier
SRC_PLATE_POS Source plate position
CHR Chromosome
POS_GENOME_KB Genomic position in kilobases on September 2000 freeze of UCSC human genome sequence
SEQ_ASSIGN Sequence assignment
STS_NAME Sequence tag site name
STS_NAME Sequence tag site name
SPOT_COL Spot column
SPOT_ROW Spot row
SUBARRAY_COL Subarray column
SUBARRAY_ROW Subarray row

Data table
1 total_hum._M. HumArray1H3_L6 0 total_hum._M. 16 3 2 4
2 GS1-232B23 HumArray1S1_A18 1 0 INT 1 p tel 1 p tel 7 9 2 1
3 RP11-82d16 HumArray1H3_V36 1 468 1p36 12 14.16 0 D1S243 AFM214YG7 13 8 4 2
4 RP11-62m23 HumArray1H3_W1 1 2241 1p36 96 17.52 6.2 D1S468 AFM280WE5 10 7 1 3
5 RP11-60j11 HumArray1H3_W3 1 4504 1p36.2 290 26.29 16.9 D1S2663 AFMA210XG9 10 7 3 3
6 RP11-111O05 HumArray1H3_W36 1 5440 1p36 124 21.19 12.8 D1S2893 AFM123XC3 13 8 4 3
7 RP11-51b04 HumArray1H3_W2 1 7000 INT 1p36.3 195 23.89 16.4 D1S253 AFM254WB9 10 7 2 3
8 RP11-199o01 HumArray1H3_W4 1 9046 1p36.3 508 45.64 22.9 D1S244 AFM220YF4 10 7 4 3
9 RP11-813J05 HumArray1H5_M21 1 10000 INT 1p36.2 p110D 19 22 1 1
10 RP11-188f07 HumArray1H3_W5 1 12618 1p36 590 51.32 32.2 D1S434 AFM217YH8 13 7 1 3
11 RP11-178m15 HumArray1H3_W6 1 13000 INT 1p36.2-.3 782 32.4 D1S228 AFM196XB4 13 7 2 3
12 RP11-265f14 HumArray1H3_C1 1 14282 1p36 39.9 AFM217ZC3 1 1 1 3
13 RP11-219f04 HumArray1H3_W7 1 15137 1p36.2-.3 827 61.06 36.2 D1S507 AFMA127ZC9 13 7 3 3
14 RP11-145c04 HumArray1H3_W8 1 19277 1p36 46.2 AFMA153XE9 13 7 4 3
15 RP11-224f08 HumArray1H3_W9 1 20960 1p36.1 1197 75.41 48.8 D1S2843 AFM343ZA9 16 7 1 3
16 CTD-2128D14 HumArray1H5_F5 1 22484 1p35-1p36.1 74.12 RAP1GA1 M64788 16 19 1 2
17 RP11-139h05 HumArray1H3_G23 1 23266 1p36.1 50.9 AFM193XB12 1 3 3 3
18 CTD-2194B23 HumArray1H5_M22 1 23982 1p36.2 EPHB2 D1S3214 19 22 2 1
19 RP11-72i19 HumArray1H3_W11 1 24179 1p35-36 54.2 AFM113XC7 16 7 3 3
20 RP11-57f20 HumArray1H3_W10 1 24473 1p36.1 1391 81.92 54.2 D1S482 AFM294ZD1 16 7 2 3

Total number of rows: 2463

Table truncated, full table size 201 Kbytes.

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