Question

1. What is the principle of Genome-Wide Expression Patterns Analysis using PCR and how DNA chip can be used in this process.

Answer 1

Answer:

Genome-Wide Expression:

• Recently invented methods allow researchers to analyze the expression of thousands of genes simultaneously using DNA microarrays.
• Coupling these methods with the results from genome sequencing projects allows researchers to analyze the complete transcriptional program of an organism during specific physiological responses or developmental processes.
• DNA microarrays consist of thousands of individual gene sequences bound to closely spaced regions on the surface of a glass microscope slide. T
• methods have been developed for preparing DNA microarrays.
• In one method, an ≈1-kb portion of the coding region of each gene analyzed is individually amplified by PCR.
• A robotic device is used to apply each amplified DNA sample to closely spaced spots on the surface of a glass microscope slide, which then is processed by chemical and heat treatment to attach the DNA sequences to the glass surface and denature them.
• Typical arrays are 2 × 2 cm and contain ≈6000 spots of DNA.
• In an alternative method, multiple DNA oligonucleotides, usually 20 nucleotides in length, are synthesized from an initial nucleotide that is covalently bound to the surface of a glass slide.
• Tens of thousands of identical oligonucleotides are synthesized in a small square area on the surface of the slide.
• Several oligonucleotide sequences from a single gene are synthesized in neighboring regions of the slide to analyze expression of that gene.
• Thousands of genes can be represented on one glass slide.
• Because the methods for constructing these arrays of synthetic oligonucleotides were adapted from methods for manufacturing microscopic integrated circuits used in computers, these types of oligonucleotide microarrays are often called DNA chips.
• The DNA fragments that result from the PCR are generally checked on agarose gels, concomitantly obtaining an estimate of the DNA concentration.
• After purification, the DNA is taken up in a volume of spotting buffer that is smaller than the original volume during PCR, thereby increasing the DNA concentration.
• Usually, no accurate determination of the DNA concentration is performed, since on microarrays mostly relative measurements with two differently labelled targets are performed.
• In addition, a levelling in the DNA amounts present at the various spot positions can be obtained if the individual PCR products are at a concentration above the binding capacity of the array surface (4).
• Purification of the PCR products requires several steps as well as expensive consumables if, for example, purification is achieved by filter- or column-based techniques.
• Alternatively, mere precipitation avoids this specific cost factor but is work-intensive and inefficient.
• Here, we describe a procedure that avoids purification altogether; only the volume of the PCR products is reduced by evaporation to increase the DNA concentration.
• Additional pipetting steps are avoided and no DNA is lost.
• Because of the latter, more microarrays can be produced from a single PCR amplification and higher concentrations can be used during spotting, thereby reducing variation in the DNA amounts across a microarray.
• Additionally, the procedure simplifies the quality check of the PCR fragments on agarose gels and provides the means for an uncomplicated and thus routinely applicable analysis of the spotting quality on all microarrays.

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