Microarray

1. Microarray -DNA microarray hybridization is a method of choice for high-throughput RNA expression analysis.

2. -It’s a high throughput technology. • -In general array is an arrangment of points in rows and columns. • A microarray is an extension of the concept having a very small arrangement of many points in rows and columns. • This technique basically originated from Southern blotting and in this technique a series of high density DNA spots bound to some solid support. • Microarray bind DNA to a solid base (usually a glass), using robot automated processes (with high precision). • The precloned immobilized DNA molecules can be probed with labeled complementary sequences of DNA.

3. DNA microarrays have spots that contain known sequences of DNA to which labeled sample of unknown composition is hybridized and then detected. • There are two major types of DNA array used in expression analysis: • 1. Spotted DNA arrays • 2. Printed DNA arrays

4. Spotted DNA arrays -A spotted DNA array is made by transfering (spotting) actual DNA clones or PCR products derived therefrom) individually onto a solid support such as nitrocellulose membrane in a precise grid pattern. -Corresponding clones provide positive signal. -Comparative gene analysis requires the preparation of duplicate arrays or the sequential probing, stripping and re-probing of the same array with two different probes. -Nylon and glass microarrays are available. -Glass is an inert substrate and thus coated with negatively charged phosphate groups of DNA are exploited for immobilization on positively charged surface groups provided. eg. Poly-L-lysine and subsequently DNA cross linked to the surface. -Also, amino groups can be attached to DNA and immobilized on aldehydes or epoxy-derivatized surfaces.

5. -In case of membrane, probe is usually labeled with radioactive or enzymatic, which provide poor signal. While fluorescent probes having higher resolution, but becoz of autofluorescence of the substrate in case of nylon membranes has a high auto-fluorescence, generating low signal-to-noise ratio. -Interestingly, glass is a non-porous substance having little auto fluorescence, fluorescent probes can be used. -Different fluorophores can be used to lable different RNA populations. eg. Two different probes Cy3 and Cy5 which provide bright red and green fluorescence , respectively. So, if a particular cDNA is present only in Cy3 labeled population the spot on the array is green and red in case of Cy5, but if present in both population contain equal proportion spot will appear yellow.

6. -In case of bacteria and also in Saccharomyces cerevisiae, genomic array can be derived becoz either lack of introns or few numbers only. -Complete c-DNA sequence is not needed only the signature sequences or ESTs are sufficient to use. EST (expressed sequence tags): By using high throughput sequencing technology, thousand of clones picked randomly from cDNA libraries and subjected to single-pass sequencing to generate 2-300 bp cDNA signatures called expressed sequence tags (EST).

7. -Actually, the spotted arrays were having some differences in the features (spots), and to overcome this printed technologies were developed. -Spotting pins, piezoelectric devices similar to inkjet printers and bubblejet printheads that deposited DNA samples on the substrate as a bubble extended from the nozzle.

8. Oligonucleotide chips: principle -Sequences are obtained from public or private databases and synthesized on the chip. -Each gene is reprinted by 20 non-overlapping oligonucleotides each with a perfect match (PM) and mismatch (MM) feature.

9. Oligonucleotide chips are manufactured by in situ oligonucleotide synthesis -Affymetrix Inc. developed a light directed printing technology known as photolithography. -Glass or silicon wafer is hydroxylated and silanized so that DNA can be covalently attached to the surface in a simple chemical reaction. -Covalent binding sites are blocked by a photolabile protecting group. • A chromium mask is then applied to the surface of the chip which determines which areas are exposed to light. • Under illumination, the protecting groups in these areas are destroyed. Allowing the addition of a single nucleotide, which is also blocked with a photo labile protecting group. • This method allows the production of most accurate and densest arrays currently available up to 64,000 features over an area slightly larger than 1 cm2 (GeneChip).

10. -In contrast to DNA arrays, oligonucleotide chip arrays are not double stranded clones or PCR products, but single stranded targets ranging from 25-70 nucleotides. -Probes for chip hybridization are made from cleaved, biotinylated cRNA (RNA that has been transcribed in vitro from cDNA).

11. Properties of different types of DNA array forexpression analysis Property Spotted nylon spotted glass Affymetrix Target composition dsDNA (genomic, cDNA or PCR products) ss-oligonucleotides Target source derived from RNA, maintained clones Seq derived from public or private databases. Chemically synthesized Target size 100-300bp 20-25nt Array format Individual features represent non-redundant clones Single clones ~20 non overlapping oligos Density 1-10 >5000 64,000 to 10,00000 Manufacture Robotized or manual robotized On-chip photolithographic Substrate Nylon Glass Glass or silicon Probe labelling Radioactive or enzymatic Dual fluorescent Fluorescent Hybridization High vol (~50ml) 10 ul 200ul ~65 degree C ~65 degree C 40 degree C Data acquisition Autorad, phosphorimager Confocal Confocal for isotopic probes, flatbed scanner for enzymatic probes Cost Low Moderate High