The Genom of Homo sapiens.pdf

HUMAN GENOME SCANNING 325false-positive and -negative problem with BAC clones isto use unique single-copy sequence fragments such asPCR-amplified genomic fragments as hybridization substrateson the arrays. Currently, this is not practical becausethe reagent cost is high, and effort involved in amplifyinga large number of genomic fragments devoid ofrepeats is considerable.ARRAY PRODUCTION: A NOVEL APPROACHFOR MAKING DNA ARRAYSArray production and hybridization are the most criticalaspects of array CGH technology. Printing and subsequentlycrosslinking DNA fragments onto positivelycharged or reactive surfaces has now become a standardmethod for making cDNA arrays for gene expressionanalyses (Schena et al. 1995). However, this method doesnot work well for making CGH arrays because positivelycharged or reactive surfaces, even after extensive chemicalinactivation blocking, tend to generate high hybridizationbackground relative to the weak CGH signals.Entrapment of purified DNA in polymer matrix (Pinkel etal. 1998) does not require positively charged or reactivesurfaces for DNA attachment, and higher amounts of targetDNA can be confined in a spot area to increase hybridizationefficiency. However, this “gluing down”method requires a very high concentration of highly purifiedBAC DNA, and the attached DNA is barely accessiblefor hybridization to generate directly visible spot signals(Pinkel et al. 1998). The requirement for a highamount of highly purified DNA is a significant disadvantagefor this method for making high-density arrays.To effectively minimize the hybridization backgroundand maximize hybridization signals, we developed anovel approach for making DNA arrays (Cai et al. 2002)in which the BAC DNA is chemically modified using acrosslinker-like silane compound with an epoxide groupthat can covalently attach to DNA at slightly alkaline pH.The modified DNA retains normal hybridization specificityand could be purified after modification using standardprocedures. Since the silanol groups at the other endof the silane molecule are only specific to glass surfaces(Fig. 3), DNA arrays can be conveniently made frommodified DNA by simple deposition of the silanized DNAsamples onto clean natural glass surfaces. Once contact ismade with the glass surfaces, covalent attachment occursat neutral pH after curing treatment in ethanol at an elevatedtemperature (Cai et al. 2002). DNA arrays made thisway should have low background noise after hybridizationwashes because natural glass surfaces are slightlynegatively charged and thus repulsive to DNA.A distinct advantage of our DNA pre-modificationmethod over all other methods using activated surfaces isthat all activated surfaces are unstable, difficult to preparereproducibly, and sensitive to impurities in the samples.Because of limited DNA-binding capacity, activated surfacesrequire careful calibration of DNA concentrationsbefore printing to avoid overspill of excess DNA. In contrast,when DNA is modified before printing, it is not necessaryto calibrate DNA concentrations because modifiedDNA molecules can cross-link with each other after dryingto form a three-dimensional matrix, which has improvedhybridization efficiency due to more availableprobe targets within spots. Modified DNA can be printedat any concentration without saturating the surface andcausing spills, and even at concentrations of 2 µg/µl, allthe spots are perfectly confined. On the other hand, printingthe same DNA samples onto commercial slides withoutmodification with a crosslinker-like silane compoundat a concentration exceeding 300 ng/µl results in significantDNA spills. Because of this crosslinking property ofsilanized DNA, it can be arrayed on other surfaces suchas metal and plastics, which are difficult to activate forDNA binding.QUANTITATIVE HYBRIDIZATION IN aCGHOne major challenge in aCGH is to attain quantitativehybridization on BAC arrays with unfractionated totalgenomic probes. Because both the targets and probescontain repetitive or duplicated sequences, hybridizationsignals contributed from these sequences must be suppressedto a sufficiently low level. Hybridization inaCGH is much more challenging than in conventionalmetaphase CGH for two reasons. First, excess of targetDNA in spots demands highly effective suppression ofrepetitive sequence signals. On a BAC array, each spothas ~10 5 –10 6 copies of 100- to 250-kb target DNAFigure 3. Chemical modification of DNA for makingDNA microarrays. DNA can be covalentlymodified with a silane compound containing a reactiveepoxide group in slightly basic solution.Upon drying, silanized DNA readily adheres toglass surfaces forming stable spots that can withstandhybridization and high stringent posthybridizationwashes.