T

Improved On-Chip Synthesis of Oligonucleotides with Virtual Masks

Th e original on-chip procedure for making microar-rays uses physical chrome and glass masks. A chip that uses oligonucleotides of length N needs 4N such masks. This results in both a high cost and lengthy construction time for the array. Avoiding physical masks greatly reduces fabrication cost and allows greatly increased flexibility in designing custom arrays. The NimbleGen corporation has recently introduced a proprietary maskless technology into microarray synthesis. The physical mask is replaced by a computer generated

"virtual mask" which controls a digital micromirror array. This is an array of tiny, individually addressable polished mirrors that can be positioned either to direct the UV light source onto a known position in the array or direct light away from the array. By coordinating the addition of protected phosphoramidite precursors and the sequence of illumination it is possible to make custom arrays with more than 200,000 separate oligonucleotide probes.

FIGURE 25.18 Virtual Mask Process for On-Chip Synthesis of Oligonucleotides

NimbleGen builds its arrays using photo deprotection chemistry with its Maskless Array Synthesizer (MAS) system. At the heart of the system is a Digital Micromirror Device (DMD; Texas Instruments, Inc.), employing a solid state array of miniature aluminum mirrors to pattern up to 786,000 individual pixels of light. The DMD creates "virtual masks" that replace the inflexible physical chromium masks used in traditional arrays.

in building computer chips. A glass slide is first covered with a reactive group. This is then covered with a photosensitive blocking group that can be removed by light. In each synthetic cycle, those sites where a nucleotide will not be attached are covered with a mask. Those sites where a particular nucleotide (say, A) is to be attached are illuminated to remove the blocking group. The nucleotide is then added and is chemically coupled to the exposed sites. Only one kind of nucleotide can be added at a time, as it will couple to all exposed sites. Also, the other end of the added nucleotide must be blocked before addition and coupling. The cycle is repeated with another nucleotide (say, T). This cycling process is repeated with different masking patterns and different nucleotides until the required oligonucleotides are finished.

Convert to cDna

Cut out small "tags" from each gene

FIGURE 25.19 Principle

SAGE—The

To analyze the total mRNA expressed in a cell, small sequences from each mRNA are converted to complementary DNA and linked together into one long concatemer, which is sequenced. Each of the segments represents a single mRNA; therefore, the number of repeats of each segments correlates with the level of expression of the corresponding gene in the cell.

Join to give concatemer

Sequence and count tags for each gene

If all the mRNA molecules in a cell were joined end to end and then sequenced, this would reveal how many copies of each mRNA were present— hence the level of gene expression.

Serial Analysis of Gene Expression (SAGE)

A DNA sequencing approach can also measure the expression level of multiple mRNA molecules simultaneously. The basic idea is to sequence all of the mRNA in a cell and then examine the accumulated sequence to see how many copies of each mRNA are represented. To actually do this the mRNA molecules must be joined end to end to give a single giant concatenated molecule, which is converted to DNA for sequencing. The term serial analysis of gene expression (SAGE) refers to this large concatemer, which contains every expressed gene. The number of copies of each repeat in the con-catemer indicates the level of gene expression. To make the approach feasible, only a short sequence from each mRNA is sequenced. The DNA concatemer thus contains many linked sequence tags of approximately 10 bases each (Fig. 25.19).

The first step in SAGE is to extract all the mRNA from a eukaryotic cell and convert it into cDNA using an oligo(dT) primer that hybridizes to the poly(A) tail of the mRNA (Fig. 25.20).The oligo(dT) primer also carries a biotin tag that can be bound by the protein streptavidin.The cDNA is cleaved by a restriction enzyme (the "anchor-

biotin Vitamin that is widely used to label or tag nucleic acids in molecular biology because it may be bound very tightly by avidin or strepta-vidin serial analysis of gene expression (SAGE) Method to monitor level of multiple mRNA molecules by sequencing a DNA concatemer that contains many serially linked sequence tags derived from the mRNAs streptavidin Protein from Streptomyces that binds biotin extremely tightly and specifically. Used in detection procedures for molecules labeled with biotin mRNA AAAAAAAA

TTTTTTT TBiotin

Make cDna using oligo dT primer attached to Biotin

Cut site cDNA

AAAAAAA^^ TTTTTTT TBiotin

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