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Sequencing by Hybridization

    One of the great benefits of the Human Genome Project and similar projects is the opportunity to study disease and other genetically determined characteristics via sequence comparison.  For instance, sickle-cell disease was linked to a base-pair substitution in the disease causing allele resulting in the alteration of the protein by a single amino acid from glutamic acid to valine.1  This discovery required the sequencing of both the normal and mutant alleles- a rather timely process when accomplished by standard techniques based on the manipulation of target DNA.  The publication of entire genomes opens the door for sequence comparison on a much larger scale, limited only by the speed of which DNA of interest can be analyzed.  Sequencing by hybridization (SBH) technology provides researches with a tool powerful enough to enlarge the scope of comparison to the genome level.

The Basic Idea

     Hybridization increases in specificity as primer size decreases.  For sequencing applications a large probe is uninformative at the single nucleotide level yet a small probe covers only a tiny portion of an average size gene.  SBH technology tackles this problem by controlling the synthesis and location of a large number of small probes of uniform length on a fixed array.  Different sections of target DNA bind very exclusively to only those primers that exactly compliment their sequence.  Target DNA does not have to be extensively manipulated and is examined simultaneously by all probes on an array because hybridization is a fundamentally parallel process.2 After exposure to a fluorescently labeled target sequence the array is scanned by a laser and hybridization at the discrete, square shaped locations detected.2 By creating an array of probes which overlap in sequence researchers can deduce the order along the target molecule of the hybridization events which occurred on the array.  In principle, sequencing of unknown genetic material might be accomplished with the entire set of 4^N probes of length N.  However, de novo SBH of large molecules has proved too complex to be successfully accomplished.3 SBH has instead been used for resequencing, sequence verification and analysis of single nucleotide polymorphisms.

Probe Array Synthesis

     Since arrays contain a very large number of different probes, synthesis must be accomplished in a parallel fashion or else manufacturing would be far too time consuming to be commercially feasible.  The company Affymetrix has developed a method to synthesize different primers on an array in parallel using light-directed combinatorial chemistry.2 First a synthetic linker is attached uniformly to the surface of a glass tile and subsequently blocked with a photoreactive blocker.  Next a photolithographic mask is used to expose to light only desired locations on the tile to the effects of light.  These locations are deprotected as the blocking compound disassociates with the linker upon exposure.  A particular deoxynucleotide is then applied to the tile and binds only to locations that have been photodeprotected.  One out of four nucleotides can be applied at a time so for a diverse array four cycles must be used for every position along the length of the probes.2 Production time, as a consequence, depends only on the length of the probes and not on the size of the array and is therefore well adapted to the production of larger tiles.  The end product of this process is a small tile with a pattern of tiny squares delineated by exposure to light which each represent a unique probe sequence.

Figure 1.     The figure depicts the stepwise synthesis of an Affymetrix (R) probe array
  using the light-directed combinatorial chemistry method.   
Accessed 2000 21 Feb.

Application: Single Nucleotide Polymorphism Analysis
Using a 4L tiled array

     One scheme, called a 4L tiled array, can be used to compare a target and known reference sequence and identify differences of a single nucleotide.  A set of four probes corresponding to every position along the length of a reference sequence is synthesized on a tiled array resulting in a total number of 4xL positions on the tile.  Each set of probes differs from the reference sequence by only a single nucleotide at a fixed position along the length of every probe in the array.  If an array consists of 10 nucleotide probes and the varied base is at position 4 relative to the 3í end then the array is termed a P10,4.  When the reference sequence, which matches one square in every probe set is hybridized with the array and read by laser, every set should produce one hybridization signal.4 This exclusiveness is critical to the functioning of the array and is produced by the requirement of an exact match for these small probes.  A suspected reference sample, therefore, can be verified directly by examining its hybridization to each probe set.  Each set identifies a single position along the reference sequence corresponding to the complement of the variable base of the one probe which hybridizes.4 Since each set of probes fails to overlap at only a single position relative to its neighbors, the array ensures that repeated sequences are differentiated.  For any repetition, probe hybridizations spanning the repetitionís ends will differentiate between different locations along the molecule.
     The overlap among neighboring probe sets in the 4L array also permits the identification of differences in sequence of a single nucleotide between reference and target molecules.  Since hybridization requires an exact match and only one nucleotide position varies for every probe, neighboring probes overlapping a substitution in a target sequence will fail to hybridize and produce a characteristic loss of signal.4 To positively identify these characteristic "footprints" reference and target sequence must be hybridized with identical arrays under identical conditions.  The footprint can then be detected as a reduction of the fluorescence intensity of the target relative to the reference sequence among the probes neighboring the substitution.4 Stephen P. A. Fodor and his lab at Affymetrix improve on this method by labeling reference and target molecules with different fluorescent markers to improve the fidelity of the signals.4
    When two or more single nucleotide substitutions occur in a span shorter than the length of the probes along the target sequence an enlarged footprint appears.  Multiple mismatches as well as other aberrant signals produce ambiguity that detracts from the accuracy of the method.  Therefore, Fodorís group developed computer algorithms for the 4L scheme which flags these regions for conventional sequence analysis.4


2000 Feb 21. GeneChip(R) Probe Array Synthesis.                                             <>  Accessed 2000 21 Feb..

1Campbell, Neil A.  Biology, fourth edition.  The Benjamin/Cummings Publishing Company, Inc. 1996. pp. 317f.

2Fodor, Stephen P. A. 1997.  Massively Parallel Genomics. Science 277: 393-395.

3Wallraff, G., J. Labadie, P. Brock, R. DiPietro, T. Nguyen, T. Huynh, W. Hinsberg, G. McGall.  DNA Sequencing on a Chip. Chemtech 27: 22-32. Feb.

4Fodor, Stephen P. A., M. S. Morris, D. J. Lockhart, J. Winkler, D. Stern, X. C. Huang, A. Berno, E. Hubbell, R. Yang, M. Chee. 1996. Accessing Genetic Information with High-Density DNA Arrays. Science 274: 610-614.

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